{"gene":"PKN1","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":1996,"finding":"PKN (PKN1) directly binds to GTP-bound RhoA (but not GDP-RhoA) and is activated by this interaction both in vitro and in vivo, establishing PKN as a Rho effector serine/threonine protein kinase.","method":"Yeast two-hybrid cloning, in vitro binding assay, in vivo kinase activation assay","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal binding and kinase activation confirmed in vitro and in vivo, replicated across multiple independent labs in contemporaneous papers","pmids":["8571126"],"is_preprint":false},{"year":1999,"finding":"Crystal structure of human RhoA complexed with the effector domain of PKN/PRK1 at 2.2 Å resolution reveals that the PKN effector domain adopts an antiparallel coiled-coil finger (ACC finger) fold that binds to RhoA switch I, beta strands B2/B3, and helix A5, predominantly via specific hydrogen bonds.","method":"X-ray crystallography at 2.2 Å resolution","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structure with detailed mechanistic interpretation, foundational structural paper","pmids":["10619026"],"is_preprint":false},{"year":1994,"finding":"PKN kinase activity is activated several-fold by unsaturated fatty acids (arachidonic acid, linoleic acid, oleic acid) and by limited proteolysis with trypsin, but not by Ca2+/phosphatidylserine/diolein (the classical PKC activators).","method":"In vitro kinase assay with peptide substrates, limited proteolysis","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct in vitro enzymatic characterization replicated in subsequent purification studies","pmids":["7945381"],"is_preprint":false},{"year":1995,"finding":"Purified native PKN from rat testis is a 120 kDa serine/threonine kinase activated by unsaturated fatty acids and detergents; autophosphorylation activity is partially inhibited by alkaline phosphatase pretreatment, suggesting autophosphorylation contributes to activity.","method":"Protein purification (~8000-fold), in vitro kinase assay, alkaline phosphatase treatment","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — purified native enzyme characterized by multiple enzymatic assays","pmids":["7654208"],"is_preprint":false},{"year":1996,"finding":"The RhoA-binding region of PKN1 maps to amino acid residues 33–111 (with residues 74–113 critical for association); PKN1 binding to RhoA inhibits both endogenous and GAP-stimulated GTPase activity of RhoA, suggesting PKN1 can sustain the active GTP-bound form of RhoA.","method":"Yeast two-hybrid system, in vitro binding assay with truncation mutants, synthetic peptide competition, GTPase activity assay","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro binding with deletion mapping plus functional GTPase inhibition assay, single lab with multiple orthogonal methods","pmids":["8647255"],"is_preprint":false},{"year":1996,"finding":"PKN1 associates with alpha-actinin via its N-terminal region (outside the RhoA-binding domain) binding to the third spectrin-like repeats of alpha-actinin; this interaction is regulated by Ca2+ for non-skeletal muscle type and is stimulated by phosphatidylinositol 4,5-bisphosphate for full-length alpha-actinin.","method":"Yeast two-hybrid, in vitro binding with truncation mutants, co-immunoprecipitation from COS7 cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast two-hybrid plus in vitro binding domain mapping plus co-IP in mammalian cells, multiple orthogonal methods","pmids":["9030526"],"is_preprint":false},{"year":1996,"finding":"PKN1 associates with and phosphorylates the head-rod domain of all three neurofilament subunits (NF-L, NF-M, NF-H); phosphorylation of NF-L by PKN inhibits neurofilament polymerization in vitro.","method":"Yeast two-hybrid, in vitro binding, in vitro kinase assay with purified neurofilaments, polymerization assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro binding domain mapping, direct kinase assay with purified substrates, and functional polymerization assay, single lab with multiple orthogonal methods","pmids":["8621664"],"is_preprint":false},{"year":1997,"finding":"PKN1 phosphorylates vimentin and GFAP in their head domains in vitro, resulting in nearly complete inhibition of filament formation; the regulatory domain of PKN1 interacts with vimentin.","method":"Yeast two-hybrid, in vitro binding assay, in vitro kinase assay, filament formation assay","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro kinase assay with functional filament formation readout, yeast two-hybrid and binding confirmation, multiple orthogonal methods","pmids":["9175763"],"is_preprint":false},{"year":1998,"finding":"PKN is cleaved by caspase-3 (or a related caspase) at specific aspartate residues during apoptosis, generating a constitutively active catalytic fragment; site-directed mutagenesis of the cleavage sites prevented fragment generation, and the cleavage was inhibited by the caspase inhibitor DEVD-CHO.","method":"In vitro cleavage with recombinant caspase-3, site-directed mutagenesis, immunoblot in apoptotic cells","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with recombinant caspase-3, mutagenesis validation, pharmacological inhibition, confirmed in multiple cell lines","pmids":["9751706"],"is_preprint":false},{"year":1999,"finding":"The regulatory region of PKN1 (residues 455–511) contains an autoinhibitory domain that inhibits kinase activity in a substrate-competitive manner (Ki = 0.6 µM); arachidonic acid relieves autoinhibition by this segment. Phosphorylation at Thr64, Ser374, or Thr531 in the regulatory region and Thr774 in the activation loop are required for full kinase activity.","method":"Deletion mutagenesis in insect cells, in vitro kinase inhibition assay with recombinant peptide fragments, site-directed mutagenesis of phosphorylation sites","journal":"Journal of biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstituted inhibition assay with defined peptide fragments, mutagenesis of key residues, multiple orthogonal approaches in single study","pmids":["10467162"],"is_preprint":false},{"year":1996,"finding":"PKN1 translocates from the cytosol to the nucleus in response to heat shock (42°C), sodium arsenite, and serum starvation, and returns to the cytoplasm upon recovery from heat shock.","method":"Confocal immunofluorescence microscopy, subcellular fractionation, immunoblotting","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct localization by confocal microscopy corroborated by biochemical fractionation, multiple stress conditions tested","pmids":["8816775"],"is_preprint":false},{"year":2000,"finding":"PKN1 phosphorylates tau protein at specific sites in microtubule-binding domains (Ser258, Ser320, Ser352), with Ser320 being PKN1-specific (not phosphorylated by PKC isoforms); PKN1 activation disrupts microtubule arrays in vitro and in vivo.","method":"In vitro kinase assay with tau peptides, site-directed mutagenesis, phospho-specific antibodies, transfection of active/inactive PKN in CHO cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro kinase assay, site-directed mutagenesis, phospho-specific antibody validation in cells, multiple methods","pmids":["11104762"],"is_preprint":false},{"year":2001,"finding":"PKN1 delays mitotic timing by directly phosphorylating and inhibiting Cdc25C phosphatase activity, thereby delaying Tyr15 dephosphorylation of Cdc2 and entry into mitosis; active PKN causes cleavage arrest in Xenopus embryos and delayed mitosis in Xenopus cycling extracts.","method":"Xenopus embryo microinjection, Xenopus egg extract cell cycle assay, in vitro Cdc25C kinase/phosphatase assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay on Cdc25C, multiple orthogonal functional assays (embryo, extract), kinase-dead control","pmids":["11134534"],"is_preprint":false},{"year":2001,"finding":"PKN1 directly interacts with and stimulates the activity of phospholipase D1 (PLD1) in vitro and in vivo; PKNα binds residues 228–598 of PLD1 and stimulates PLD1 activity in the presence of PIP2.","method":"Co-immunoprecipitation from COS7 cells, in vitro binding with PLD1 deletion mutants, in vitro PLD1 activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — co-IP of endogenous proteins, domain mapping with deletion mutants, direct functional enzyme assay","pmids":["11259428"],"is_preprint":false},{"year":2003,"finding":"PKNα (PKN1) phosphorylates MLTKα (a MAPKKK) and enhances its kinase activity in vitro; PKNα associates with all components of the p38γ MAPK cascade (p38γ, MKK6, MLTKα), functioning as both an upstream activator and a scaffold for p38γ MAPK signaling.","method":"In vitro kinase assay, co-immunoprecipitation, SDS-PAGE mobility shift assay, dominant-negative expression","journal":"Journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase and co-IP data with multiple pathway components, single lab","pmids":["12761180"],"is_preprint":false},{"year":2005,"finding":"PKN1 (activated by TGF-β1 via RhoA) phosphorylates Cdc25C at Ser216, promoting 14-3-3 binding to Cdc25C and inactivating it, thereby delaying G2/M transition; PKN1 and Cdc25C coimmunoprecipitate and colocalize to the nucleus prior to mitosis.","method":"RNAi knockdown, constitutively active PKN1 expression, in vitro kinase assay on Cdc25C, phospho-specific Ser216 detection, co-immunoprecipitation, immunofluorescence colocalization","journal":"Cell cycle","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro phosphorylation of Cdc25C, RNAi rescue, co-IP, and site-specific mutagenesis (S216A) evidence, multiple orthogonal methods in single lab","pmids":["17374997"],"is_preprint":false},{"year":2005,"finding":"PKN1 involvement in arsenite-induced G2/M delay is mediated by direct phosphorylation of Cdc25C at Ser216, which facilitates 14-3-3 association; a Cdc25C S216A phospho-mutant partially abrogated arsenite-induced cell cycle arrest.","method":"Constitutively active PKN1 expression in HeLa cells, in vitro kinase assay, Cdc25C S216A mutant expression, co-IP","journal":"Molecular carcinogenesis","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro kinase assay, site mutagenesis rescue, co-IP, functional cell cycle readout, consistent with the Xenopus findings","pmids":["15791647"],"is_preprint":false},{"year":2006,"finding":"Salmonella effector SspH1 interacts with PKN1 via its leucine-rich repeat (LRR) domain, and PKN1 expression decreases NF-κB-dependent reporter gene activity while PKN1 depletion by RNAi increases it, indicating PKN1 negatively modulates NF-κB signaling.","method":"Yeast two-hybrid screen, domain mapping of LRR-PKN1 interaction, RNAi knockdown, NF-κB reporter assay","journal":"Cellular microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid and RNAi with functional reporter readout, single lab","pmids":["16611232"],"is_preprint":false},{"year":2013,"finding":"X-ray structure of SspH1–PKN1 complex reveals that the SspH1 LRR domain binds specifically to the HR1b coiled-coil subdomain of PKN1, sterically displacing the SspH1 catalytic domain to activate its E3 ubiquitin ligase activity; SspH1 catalyzes ubiquitination and proteasome-dependent degradation of PKN1 in cells, attenuating androgen receptor responsiveness.","method":"X-ray crystallography of SspH1–PKN1 complex, ubiquitination assay in cells, proteasome inhibitor experiments, androgen receptor reporter assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus functional ubiquitination/degradation assay, mechanistic coupling of substrate recognition to enzyme activation demonstrated","pmids":["24248594"],"is_preprint":false},{"year":2008,"finding":"PKN1 phosphorylates TRAF1 in vitro and in vivo; this phosphorylation recruits TRAF1 to TNFR2 and is required for attenuation of constitutive NF-κB and JNK signaling; mutagenesis of the phospho-acceptor residue in TRAF1 abrogates PKN1-dependent TNFR2 recruitment.","method":"In vitro kinase assay, co-immunoprecipitation, RNAi, phospho-acceptor mutagenesis, NF-κB/JNK reporter assays","journal":"Genes to cells","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro kinase assay plus phospho-site mutagenesis plus functional reporter assays, multiple orthogonal methods, single lab","pmids":["18429822"],"is_preprint":false},{"year":2004,"finding":"PKN1 directly binds the TRAF domain of TRAF2 through a PXQX(S/T) motif (residues 580–584); mutation of this motif abrogates co-immunoprecipitation with TRAF2; RNAi knockdown of PKN1 reduces TRAF2-induced NF-κB activation.","method":"Yeast two-hybrid, in vitro binding with TRAF2 deletion mutants, co-immunoprecipitation, site-directed mutagenesis, RNAi, NF-κB reporter assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple binding assays plus RNAi functional readout, single lab","pmids":["14741690"],"is_preprint":false},{"year":2010,"finding":"PKN1 is activated by ischemia/reperfusion (phosphorylated at Thr774) in the heart; transgenic cardiac-specific overexpression of constitutively active PKN1 reduces infarct size and apoptosis, while dominant-negative PKN1 increases injury; PKN1 mediates phosphorylation of αB-crystallin and stimulates proteasome activity, partially accounting for cardioprotection.","method":"Transgenic mouse (cardiac-specific CA and DN PKN1), in vivo I/R model, TUNEL assay, immunoblot for αB-crystallin phosphorylation, proteasome activity assay, epoxomicin inhibition","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss- and gain-of-function transgenic models, multiple mechanistic readouts, pharmacological rescue with proteasome inhibitor","pmids":["20595653"],"is_preprint":false},{"year":2011,"finding":"PKN1 (PKNα) serves as a component of an AKAP-Lbc–assembled signaling complex containing RhoA effector PKNα, MLTK, MKK3, and p38α; this complex is required for α1b-adrenergic receptor-mediated RhoA-dependent activation of p38α MAPK, as silencing AKAP-Lbc specifically reduces this pathway.","method":"Co-immunoprecipitation, siRNA knockdown of AKAP-Lbc, p38α kinase activity assay, selective pharmacological dissection","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP and RNAi with pathway-specific functional readouts, single lab","pmids":["21224381"],"is_preprint":false},{"year":2008,"finding":"A cleaved (caspase-activated) form of PKN1, but not wild-type PKN1, disrupts neurofilament organisation and axonal transport in neurons; PKN1 is cleaved and activated in SOD1G93A ALS model mice and in glutamate-treated neurons.","method":"Expression of cleaved vs. wild-type PKN1 in neurons, neurofilament transport assay, immunoblot for cleavage in transgenic mice and glutamate-treated cells","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct comparison of cleaved vs. wild-type overexpression with defined transport phenotype, corroborated in in vivo ALS model","pmids":["18519042"],"is_preprint":false},{"year":2011,"finding":"PKN1 and PKN3 share a phosphorylation consensus motif requiring an arginine at position −3 and intolerance of arginine at position +1 (PKN1) or −1 (PKN3); PKN1 phosphorylates EGFR at Thr654 in vitro and this site is constitutively phosphorylated in a PKN-dependent manner in vivo.","method":"Peptide library substrate screen, protein array, site mutagenesis, in vitro kinase assay, cellular phosphorylation assay","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — peptide library screen plus direct in vitro kinase assay with site mutagenesis and cellular validation, multiple methods in one study","pmids":["21749319"],"is_preprint":false},{"year":2013,"finding":"PKN1 inhibits Wnt/β-catenin signaling in melanoma cells; PKN1 is found in a protein complex with Frizzled 7 and co-purifying proteins by affinity purification/mass spectrometry; siRNA depletion of PKN1 enhances β-catenin reporter activity and increases WNT3A-induced apoptosis.","method":"siRNA screen, phosphoproteomics, affinity purification–mass spectrometry, β-catenin reporter assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — AP-MS for complex identification, RNAi with reporter readout, single lab with two orthogonal methods","pmids":["24114839"],"is_preprint":false},{"year":2017,"finding":"PKN1 phosphorylates RPH3A, which enhances binding of RPH3A to GTP-bound RAB21; this PKN1–RPH3A–RAB21 axis is required for polarized localization of RAB21 and PIP5K1C90 in neutrophils, integrin activation, and adhesion to endothelial cells; myeloid-specific loss of PKN1 decreases tissue injury in renal ischemia–reperfusion.","method":"In vitro kinase assay, co-immunoprecipitation, GTP-RAB21 pulldown, immunofluorescence of polarization, integrin activation assay, myeloid conditional KO mouse, renal I/R injury model","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vitro kinase assay, binding assays, conditional KO mouse with multiple cellular and in vivo phenotypes, multiple orthogonal methods","pmids":["28636945"],"is_preprint":false},{"year":2017,"finding":"PKN1 kinase activity is regulated by TORC2-dependent phosphorylation of the turn motif (TM); amino acid substitution in the TM reduces kinase activity; PKN1 contributes to cell motility in human prostate cancer cells.","method":"TORC2-specific inhibitor (torin), phospho-TM-specific antibody, TM mutagenesis, in vitro kinase assay, cell motility assay, depletion approaches","journal":"The Prostate","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological inhibition plus mutagenesis plus functional motility assay, single lab","pmids":["28875501"],"is_preprint":false},{"year":2018,"finding":"Loss of PKN1 in vivo doubles myocardial infarct size after I/R; PKN1 co-localizes to the sarcoplasmic reticulum during ischemia and interacts with CamKIIδ; PKN1 loss increases basal CamKIIδ activation and phospholamban Thr17 phosphorylation, suggesting PKN1 limits CamKIIδ-dependent SR calcium dysregulation.","method":"PKN1 knockout mouse, Langendorff I/R perfusion, GC-MS/MS and immunoblot co-IP with CamKIIδ, PhosTag gel, confocal immunofluorescence, siRNA in NRVM","journal":"Cardiovascular research","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse with infarct size readout, MS-validated protein interaction, siRNA rescue, multiple orthogonal methods","pmids":["29045568"],"is_preprint":false},{"year":2017,"finding":"PKN1 kinase activity is required for lymphocyte trafficking/egress from secondary lymphoid organs; knock-in mice with a PKN1 T778A kinase-dead mutation show lymphocyte sequestration in spleen and lymph nodes, reduced chemotaxis, and impaired S1P-directed migration in a cell-autonomous manner.","method":"Knock-in mouse (T778A mutation), cell transfer experiments, in vitro chemotaxis assay, flow cytometry","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell-autonomous kinase-dead knock-in, reciprocal cell transfer showing autonomy, multiple functional readouts","pmids":["28794483"],"is_preprint":false},{"year":2014,"finding":"PRK1/PKN1 regulates migration and gene expression in androgen-independent prostate cancer cells through its kinase activity; PKN1 interacts with scaffold protein SPAG9/JIP4 and this interaction is required for p38 phosphorylation and cell migration; a PKN1 inhibitor prevents metastases in mice.","method":"Sibling KD, transcriptome/interactome analysis, co-localization in tissue, p38 phosphorylation assay, cell migration assay, in vivo metastasis model with inhibitor","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-localization and KD with functional readouts, in vivo inhibitor experiment, single lab","pmids":["25504435"],"is_preprint":false},{"year":2020,"finding":"PKN1 (specifically the PKN1a splice variant) promotes synaptic maturation by upregulating neuronal glutamate transporter EAAT3 expression; PKN1a knockout unmasks aberrant mGluR-dependent LTD and AMPA receptor silencing, and reduces glutamate uptake through EAAT3.","method":"PKN1a-specific knockout mouse, mGluR-LTD electrophysiology, glutamate uptake assay, silent synapse analysis, spine morphology","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — specific knockout with electrophysiological, biochemical and morphological readouts, multiple orthogonal methods","pmids":["33244074"],"is_preprint":false},{"year":2023,"finding":"Overloading-activated RHOA–PKN (including PKN1) phosphorylates KRT8 (keratin 8) at Ser43, which impedes RAB33B trafficking from the Golgi, suppresses autophagosome initiation, and contributes to intervertebral disc degeneration; knockdown of Pkn1 and Pkn2 together ameliorates disc degeneration.","method":"Conditional Krt8 knockout, PKN1/2 knockdown, phospho-Ser43 KRT8 detection, RAB33B trafficking assay, autophagy initiation assay, in vivo disc degeneration models","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO and KD with defined phospho-substrate, trafficking readout, and in vivo disease model, multiple orthogonal methods","pmids":["36897022"],"is_preprint":false},{"year":2006,"finding":"The very C-terminus of PRK1/PKN1 (beyond the hydrophobic motif) is essential for activation by RhoA and for downstream signaling (neurite retraction); deletion of HR1 alone does not fully abolish RhoA binding or activation, indicating both the HR1 domain and C-terminus contribute to full RhoA-mediated activation.","method":"C-terminal deletion mutants, in vitro kinase activation assay with GTPγS-RhoA, in vivo RhoA activation assay, neurite retraction assay in neuronal cells","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — deletion mutagenesis with in vitro and in vivo functional readouts, single lab","pmids":["16427251"],"is_preprint":false},{"year":1999,"finding":"Drosophila Pkn (PKN ortholog) binds specifically to GTP-activated Rho1 and Rac1 through distinct binding sites, and both interactions increase kinase activity; genetic loss-of-function shows Pkn is required for epidermal cell shape changes during dorsal closure, acting in a Rho1-mediated pathway independent of the Rac-JNK pathway.","method":"Yeast two-hybrid, in vitro binding, in vitro kinase assay, Drosophila loss-of-function mutant, epistasis analysis","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — biochemical binding/kinase assays combined with in vivo genetic loss-of-function and epistasis, multiple methods","pmids":["10323867"],"is_preprint":false},{"year":2000,"finding":"PKN1 binds and phosphorylates high-risk HPV E6 oncoprotein; only high-risk (not low-risk) HPV E6 proteins bind PKN1; this is the first demonstration that HPV E6 is a phosphoprotein.","method":"Yeast two-hybrid, in vitro binding (wheat-germ lysate), co-immunoprecipitation in 293T cells, in vitro kinase assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid, in vitro binding, co-IP, and direct kinase assay, single lab, multiple orthogonal methods","pmids":["10809724"],"is_preprint":false},{"year":2000,"finding":"PKN (PRK1/PKN1) stimulates transcriptional activity of the ANF promoter via the serum response element (SRE) in cardiomyocytes; only PKN (not PRK2 or Rho kinase) generates robust ANF-SRE stimulation; this effect requires RhoA and is lost with SRE mutation.","method":"Luciferase reporter assays, constitutively active PKN1 transfection, dominant-negative Rho, SRE mutagenesis","journal":"American journal of physiology. Heart and circulatory physiology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — reporter assay with multiple constructs and controls, single lab, no direct substrate identification","pmids":["10843871"],"is_preprint":false},{"year":2005,"finding":"In PTEN-null MEFs, cortical actin formation is mediated through the PTEN/RhoA/PKN pathway, as dominant-negative RhoA or kinase-dead PKN inhibit cortical actin accumulation; however, this pathway does not mediate enhanced cell migration (which depends on Rac/PDK-1/Akt instead).","method":"Dominant-negative RhoA and kinase-dead PKN overexpression in Pten−/− MEFs, actin staining, cell migration assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis with DN/KD constructs and defined phenotypic readouts, single lab","pmids":["15531926"],"is_preprint":false},{"year":2010,"finding":"Hypotonic stress activates PKN1 via Thr774 phosphorylation in cardiac myocytes, with activation dependent on upstream PDK1 and Src-family kinase-mediated RhoA activation; active PKN1 promotes ERK phosphorylation via MEK to enhance cardiac myocyte survival.","method":"In vitro PKN1 activity assay, immunoblot for Thr774 phosphorylation, pharmacological inhibition (PP1, U-0126), Elk1-GAL4 transcriptional assay, siRNA knockdown, cell viability assay","journal":"American journal of physiology. Heart and circulatory physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — kinase activity assay with multiple pharmacological probes and siRNA, defined signaling cascade, single lab","pmids":["21037231"],"is_preprint":false},{"year":2023,"finding":"Downstream of Gαq-RhoA in uveal melanoma, PKN converges with ROCK to control FAK signaling; darovasertib inhibits both PKC and PKN/PRK kinases and synergizes with FAK inhibitors in vitro and in preclinical metastatic models.","method":"High-throughput chemogenetic drug screen, kinase inhibitor profiling, FAK phosphorylation assay, in vitro cell viability assay, preclinical mouse metastasis model","journal":"Cell reports. Medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological screen with mechanistic follow-up and in vivo validation, single study","pmids":["37858338"],"is_preprint":false},{"year":2021,"finding":"PKN1 negatively regulates hippocampal AKT activity and NeuroD2 levels; Pkn1 knockout elevates phospho-AKT and NeuroD2, leading to enhanced GluA1 (but not GluA2/3) protein levels and increased GluA1 membrane fraction, revealing a PKN1–AKT–NeuroD2–GluA1 regulatory axis.","method":"Pkn1 knockout mouse, immunoblot for phospho-AKT and NeuroD2, subcellular fractionation, flow cytometry for membrane GluA1","journal":"Frontiers in synaptic neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockout mouse with biochemical signaling readouts and fractionation, single lab","pmids":["33613259"],"is_preprint":false},{"year":2023,"finding":"PKN1 inhibition reduces AKT phosphorylation; PKN1 phosphorylation at S374 is functionally relevant for PKN1–AKT interaction and axonal outgrowth on inhibitory substrates in cerebellar granule cells; PKN1 pS374 decreases during cerebellar development.","method":"Pkn1 knockout cerebellar granule cells, hypoxia-ischemia in vitro model, AKT phosphorylation assay, axonal outgrowth assay, caspase-3 activation assay, site-specific mutagenesis of S374","journal":"Biomolecules","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockout cells with site mutagenesis and multiple functional readouts, single lab","pmids":["38002281"],"is_preprint":false},{"year":2026,"finding":"PKN1 HR1a forms a dimer, and HR1c drives further oligomerization; RhoA forms a 1:2 complex with HR1a and induces rearrangement of the HR1a dimer (supported by SAXS), whereas Rac1 binds monomeric HR1a, suggesting distinct activation mechanisms for RhoA vs. Rac1.","method":"Biophysical analysis (SEC, AUC), SAXS, integrative structural modeling of HR1-containing PKN1 dimers","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — SAXS and biophysical characterization with GTPase binding, multiple orthogonal biophysical methods, single study","pmids":["42036044"],"is_preprint":false},{"year":2026,"finding":"TDP-43 loss induces inclusion of a cryptic exon (PKN1-5a1) in PKN1 mRNA, generating a truncated peptide (PKN207) that escapes nonsense-mediated decay, is detectable in AD brains with TDP-43 pathology, and impairs cognition, memory, and synaptic plasticity in mice.","method":"RNA sequencing in ALS patient brains, NMD inhibition assay, mass spectrometry detection of PKN207 in AD brain, in vivo mouse cognitive/synaptic plasticity assays","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNA-seq, MS protein detection, and in vivo functional assays, single study, novel finding not yet replicated","pmids":["41720774"],"is_preprint":false},{"year":1998,"finding":"PKN1 regulates alphaB-crystallin expression under heat stress via cooperation with HSF1; the catalytically active fragment of PKN1, but not the inactive form, induces alphaB-crystallin accumulation in HeLa cells through both proximal and distal heat shock elements of the promoter.","method":"Transfection of active/inactive PKN1 fragment with HSF1, luciferase reporter assay, alphaB-crystallin immunoblot","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — reporter assay and immunoblot with kinase-active vs. inactive comparison, single lab","pmids":["9837746"],"is_preprint":false},{"year":1998,"finding":"PKN1 interacts with the paraneoplastic cerebellar degeneration antigen PCD17 via its N-terminal regulatory domain; PKN1 phosphorylates PCD17 in a fatty acid-stimulated manner; PCD17 functions as a transcriptional activator and is co-immunoprecipitated with PKN1.","method":"Yeast two-hybrid, in vitro binding, co-immunoprecipitation from COS7 cells, in vitro kinase assay, transcriptional reporter assay","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple binding methods plus direct kinase assay and functional transcription assay, single lab","pmids":["9637778"],"is_preprint":false},{"year":1999,"finding":"PKN1 interacts directly with the bHLH domain of the transcription factor NDRF/NeuroD2; co-expression of catalytically active PKN1 (but not kinase-deficient PKN1) further enhances NDRF/NeuroD2-dependent transcription of the insulin promoter element RIPE3.","method":"Yeast two-hybrid, in vitro binding with deletion mutants, co-immunoprecipitation from COS-7 cells, transient transfection reporter assay","journal":"Brain research. Molecular brain research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding confirmed by multiple methods plus kinase-activity-dependent transcription assay, single lab","pmids":["10640683"],"is_preprint":false},{"year":2006,"finding":"PKNα (PKN1) interacts with Cyclin T2a in a yeast two-hybrid screen and co-immunoprecipitation; co-overexpression of PKNα and Cyclin T2a strongly enhances expression of myogenic differentiation markers (Myogenin, Myosin Heavy Chain) in C2C12 cells during starvation-induced differentiation.","method":"Yeast two-hybrid, co-immunoprecipitation, in vitro pull-down, luciferase reporter assay, myogenic differentiation marker immunoblot","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal binding assays plus functional differentiation readout, single lab","pmids":["16331689"],"is_preprint":false},{"year":2024,"finding":"The C2-like domain of PKN1 contains amphipathic cardiolipin-binding motifs and binds cardiolipin and C18 fatty acids; two distinct types of cardiolipin/phosphatidic acid binding are observed; the pseudosubstrate sequence in the C2 domain overlaps with the arachidonic acid binding region, linking lipid binding to autoinhibitory regulation.","method":"Biophysical lipid-binding assays (SPR/ITC), CD spectroscopy, sequence analysis, kinetic assay of full-length PKN1","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — direct biophysical characterization with multiple lipid species and structural inference, single study, consistent with prior activation data","pmids":["38441874"],"is_preprint":false},{"year":2007,"finding":"Constitutively active PKN1 in mammary epithelium impairs tight junction sealing at parturition without disrupting tight junction formation (occludin and ZO1 localize correctly); dominant-negative PKN1 accelerates tight junction sealing in EpH4 cells, and active PKN1 impairs glucocorticoid-stimulated sealing.","method":"Transgenic mouse (mammary-specific CA-PKN1), intraductal [14C]sucrose injection for sealing assay, occludin/ZO1 immunofluorescence, EpH4 cell dominant-negative/active transfection","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo transgenic model with functional tight junction assay corroborated by in vitro dominant-negative results, single lab","pmids":["17591691"],"is_preprint":false},{"year":2023,"finding":"PKN1 controls adipocyte differentiation and glucose metabolism; PKN1-silenced adipocytes show decreased differentiation and glucose uptake with reduced PPARγ, FABP4, adiponectin, and CEBPα expression; insulin-resistant adipocytes show decreased PKN1 activation.","method":"siRNA knockdown in 3T3-L1 adipocytes and human VAT explants, glucose uptake assay, adipogenic marker immunoblot, PKN1 phosphorylation assay","journal":"Nutrients","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA KD with multiple functional metabolic readouts, single lab","pmids":["37242297"],"is_preprint":false},{"year":2019,"finding":"PKN1 kinase activity is required for protection of TRAF1 from cIAP-mediated degradation during constitutive CD40 signaling; the active phospho-Thr774 form of PKN1 is constitutively expressed in CLL but not in healthy B cells.","method":"PKN1 kinase inhibitor treatment (OTSSP167, XL-228), TRAF1 immunoblot, caspase-3 activation assay in primary CLL samples","journal":"Oncoimmunology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — pharmacological inhibitor approach with defined substrate (TRAF1) and functional readout, but no direct phosphorylation site mutagenesis, single lab","pmids":["34589290"],"is_preprint":false},{"year":2025,"finding":"Na+ and K+ ions directly and rapidly reduce PKN activation-loop phosphorylation in the absence of membranes; upon reduction of ion concentration, phosphorylation recovers without requiring PDK1 or ATP, via a 'reacquisition of phosphate' mechanism demonstrated by 32P tracing.","method":"Cell-free lysate PKN activation-loop phosphorylation assay with defined ion concentrations, PDK1-depleted lysate, 32P tracing experiment","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 1 / Weak — novel mechanism with 32P tracing but single preprint study, not yet peer-reviewed or replicated","pmids":["bio_10.1101_2025.09.04.674365"],"is_preprint":true}],"current_model":"PKN1 is a RhoA/Rac1-activated and fatty acid-activated AGC serine/threonine kinase whose N-terminal antiparallel coiled-coil (ACC/HR1) domains bind GTP-loaded Rho family GTPases (activating the kinase), while an internal autoinhibitory segment (residues 455–511) suppresses basal activity and is relieved by arachidonic acid or by caspase-3 cleavage; active PKN1 phosphorylates a broad substrate set including tau (Ser320), neurofilaments, vimentin/GFAP (head domains inhibiting filament assembly), Cdc25C (Ser216, delaying G2/M entry), TRAF1 (attenuating constitutive NF-κB/JNK signaling), αB-crystallin (promoting proteasome activity and cardioprotection), KRT8 (Ser43, suppressing autophagosome initiation), RPH3A (directing polarized RAB21 vesicle trafficking in neutrophils), and EGFR (Thr654); it also scaffolds p38γ MAPK cascades via MLTK and anchors signaling complexes through AKAP-Lbc, and its activity is further regulated by TORC2-dependent turn-motif phosphorylation and by alkali metal ion-dependent phosphate exchange on the activation loop."},"narrative":{"mechanistic_narrative":"PKN1 (PRK1) is a Rho-family GTPase-activated AGC serine/threonine protein kinase that couples RhoA/Rac1 signaling and lipid second messengers to cytoskeletal remodeling, cell-cycle timing, stress signaling, and membrane trafficking [PMID:8571126, PMID:10323867]. Its N-terminal antiparallel coiled-coil (ACC/HR1) finger binds GTP-loaded RhoA at switch I and stabilizes the active GTPase, defining PKN1 as a Rho effector [PMID:10619026, PMID:8647255]; HR1a dimerizes and engages RhoA in a 1:2 complex while binding Rac1 as a monomer, implying distinct activation geometries for the two GTPases [PMID:42036044]. Catalytic output is set by a substrate-competitive autoinhibitory segment (residues 455–511) that is relieved by unsaturated fatty acids such as arachidonic acid, by a C2-like lipid-binding region whose pseudosubstrate overlaps the arachidonic-acid site, and by caspase-3 cleavage that liberates a constitutively active fragment during apoptosis [PMID:10467162, PMID:38441874, PMID:9751706]; full activity additionally requires activation-loop (Thr774) and turn-motif phosphorylation, the latter TORC2-dependent [PMID:10467162, PMID:28875501]. Active PKN1 phosphorylates intermediate filament and microtubule substrates—neurofilament, vimentin/GFAP head domains, and tau (Ser320)—to inhibit polymer assembly and disrupt cytoskeletal arrays [PMID:8621664, PMID:9175763, PMID:11104762]. It enforces G2/M delay by phosphorylating Cdc25C at Ser216 to promote 14-3-3 sequestration, a checkpoint engaged downstream of TGF-β and arsenite stress [PMID:11134534, PMID:17374997, PMID:15791647]. PKN1 further phosphorylates RPH3A to direct polarized RAB21 trafficking and integrin activation in neutrophils [PMID:28636945], KRT8 (Ser43) to impede RAB33B Golgi trafficking and suppress autophagosome initiation [PMID:36897022], EGFR at Thr654 [PMID:21749319], and α B-crystallin to stimulate proteasome activity and confer cardioprotection during ischemia/reperfusion [PMID:20595653]. It scaffolds p38 MAPK cascades through MLTK and the AKAP-Lbc complex [PMID:12761180, PMID:21224381] and modulates TRAF1/TRAF2-dependent NF-κB and JNK signaling [PMID:18429822, PMID:14741690]. In vivo, PKN1 kinase activity is required for lymphocyte egress [PMID:28794483] and contributes to cardiac protection by limiting CamKIIδ-dependent SR calcium dysregulation [PMID:29045568].","teleology":[{"year":1994,"claim":"Before its regulators were known, PKN1 needed a defined activation input; this established that PKN1 is a distinct kinase activated by unsaturated fatty acids and proteolysis rather than by classical PKC cofactors.","evidence":"in vitro kinase assay with peptide substrates and limited trypsin proteolysis","pmids":["7945381"],"confidence":"High","gaps":["Did not identify the physiological activator or upstream signal","No structural basis for fatty-acid activation"]},{"year":1995,"claim":"Purification of the native enzyme confirmed PKN1 as a ~120 kDa fatty-acid-activated kinase whose activity depends on phosphorylation.","evidence":"~8000-fold purification from rat testis with kinase and alkaline-phosphatase assays","pmids":["7654208"],"confidence":"High","gaps":["Specific phosphosites not yet mapped","No upstream kinase identified"]},{"year":1996,"claim":"The central question of what links PKN1 to upstream signaling was answered by showing it is a direct GTP-RhoA effector, and that binding both activates PKN1 and stabilizes active RhoA.","evidence":"yeast two-hybrid cloning, in vitro binding, GTPase assays, and deletion mapping of the RhoA-binding region (residues 33–111)","pmids":["8571126","8647255"],"confidence":"High","gaps":["Structural mechanism of activation not yet resolved","Whether Rac1 also activates was unaddressed"]},{"year":1996,"claim":"To connect PKN1 to cellular structures, it was shown to bind the cytoskeletal crosslinker alpha-actinin and to phosphorylate neurofilament subunits, inhibiting filament polymerization—placing PKN1 at the cytoskeleton.","evidence":"yeast two-hybrid, in vitro binding/kinase assays, polymerization assays, co-IP","pmids":["9030526","8621664"],"confidence":"High","gaps":["In vivo relevance of filament phosphorylation not established","Cellular consequence on cytoskeletal architecture not directly shown"]},{"year":1996,"claim":"PKN1 was linked to stress responses by demonstrating stress-induced cytosol-to-nucleus translocation.","evidence":"confocal immunofluorescence and subcellular fractionation under heat shock, arsenite, serum starvation","pmids":["8816775"],"confidence":"High","gaps":["Nuclear substrates of translocated PKN1 not identified","Translocation signal/mechanism unknown"]},{"year":1999,"claim":"The structural basis of effector recognition was resolved: the PKN1 effector domain adopts an antiparallel coiled-coil (ACC) finger that contacts RhoA switch I.","evidence":"2.2 Å X-ray crystal structure of RhoA–PKN effector domain complex","pmids":["10619026"],"confidence":"High","gaps":["Did not capture how binding propagates to the catalytic domain","Full-length conformational change unresolved"]},{"year":1999,"claim":"How basal PKN1 activity is restrained was defined by mapping a substrate-competitive autoinhibitory segment (455–511) relieved by arachidonic acid, plus phosphosites required for full activity.","evidence":"deletion mutagenesis in insect cells, in vitro inhibition assays with peptide fragments, phosphosite mutagenesis","pmids":["10467162"],"confidence":"High","gaps":["Did not identify the upstream activation-loop kinase","Coupling of GTPase binding to autoinhibition release not shown"]},{"year":1998,"claim":"An activation route independent of GTPase/lipid was established: caspase-3 cleaves PKN1 during apoptosis to release a constitutively active catalytic fragment.","evidence":"in vitro cleavage with recombinant caspase-3, cleavage-site mutagenesis, DEVD-CHO inhibition, immunoblot in apoptotic cells","pmids":["9751706"],"confidence":"High","gaps":["Substrates targeted by the cleaved fragment not defined here","Physiological apoptotic role unaddressed"]},{"year":2001,"claim":"PKN1 was assigned a cell-cycle role by showing it phosphorylates and inhibits Cdc25C to delay mitotic entry; later work mapped this to Ser216-driven 14-3-3 sequestration engaged by TGF-β and arsenite stress.","evidence":"Xenopus embryo/extract assays and in vitro Cdc25C kinase assays; later RNAi, S216A mutant rescue, co-IP, phospho-Ser216 detection","pmids":["11134534","17374997","15791647"],"confidence":"High","gaps":["Endogenous timing of PKN1-Cdc25C activity in unperturbed mitosis unclear","Relationship to nuclear translocation not integrated"]},{"year":2003,"claim":"PKN1's role in MAPK signaling was clarified as both an activating kinase and scaffold for the p38γ cascade, later extended to an AKAP-Lbc-anchored RhoA→p38 complex.","evidence":"in vitro kinase assays, co-IP of cascade components, dominant-negative expression; siRNA of AKAP-Lbc with p38 activity readouts","pmids":["12761180","21224381"],"confidence":"Medium","gaps":["Single-lab biochemistry without structural detail of the scaffold","Direct phosphorylation site on MLTK not mapped"]},{"year":2008,"claim":"PKN1 was placed in TNF-receptor signaling by showing it phosphorylates TRAF1 to drive TNFR2 recruitment and attenuate constitutive NF-κB/JNK, building on earlier TRAF2 binding.","evidence":"in vitro kinase assays, phospho-acceptor mutagenesis, RNAi, NF-κB/JNK reporter assays; earlier TRAF2 binding via PXQX(S/T) motif","pmids":["18429822","14741690","16611232"],"confidence":"High","gaps":["Net effect on NF-κB (positive vs negative) context-dependent across studies","Physiological TNFR2 context not fully defined"]},{"year":2010,"claim":"Genetic models established a cardioprotective role: activated PKN1 phosphorylates αB-crystallin to stimulate proteasome activity and limit ischemic injury; loss of PKN1 worsens infarct via CamKIIδ-dependent SR calcium dysregulation.","evidence":"cardiac transgenic CA/DN PKN1, in vivo I/R, proteasome assays; PKN1 KO with CamKIIδ co-IP and phospholamban phosphorylation","pmids":["20595653","29045568"],"confidence":"High","gaps":["Whether αB-crystallin and CamKIIδ pathways are mechanistically coupled unclear","Direct CamKIIδ substrate relationship not established"]},{"year":2011,"claim":"PKN1 substrate specificity and a growth-factor receptor target were defined: a basophilic consensus (Arg at −3) and direct EGFR Thr654 phosphorylation.","evidence":"peptide library/protein array, site mutagenesis, in vitro and cellular phosphorylation assays","pmids":["21749319"],"confidence":"High","gaps":["Functional consequence of EGFR Thr654 phosphorylation not detailed","In vivo substrate repertoire incompletely defined"]},{"year":2013,"claim":"A pathogen co-opts PKN1: the Salmonella SspH1 LRR binds the HR1b subdomain, activating its E3 ligase to ubiquitinate and degrade PKN1, with consequences for androgen receptor signaling.","evidence":"X-ray structure of SspH1–PKN1, in-cell ubiquitination and proteasome inhibitor experiments, AR reporter assay","pmids":["24248594"],"confidence":"High","gaps":["Host pathway controlled by PKN1 turnover during infection not fully mapped"]},{"year":2017,"claim":"PKN1's role in trafficking and immune-cell function was established: it phosphorylates RPH3A to direct polarized RAB21 vesicle trafficking and integrin activation in neutrophils, and its kinase activity is required for lymphocyte egress.","evidence":"in vitro kinase/binding assays, GTP-RAB21 pulldown, myeloid conditional KO with renal I/R; T778A kinase-dead knock-in with chemotaxis and cell-transfer assays","pmids":["28636945","28794483"],"confidence":"High","gaps":["Upstream GTPase driving the RPH3A axis not fully defined","S1P-receptor coupling to PKN1 activation unresolved"]},{"year":2017,"claim":"An additional layer of regulation was identified—TORC2-dependent turn-motif phosphorylation tunes PKN1 activity and supports prostate cancer cell motility.","evidence":"torin inhibition, phospho-TM antibody, TM mutagenesis, in vitro kinase and motility assays","pmids":["28875501"],"confidence":"Medium","gaps":["Direct TORC2-PKN1 contact not shown","Single-lab functional readout"]},{"year":2020,"claim":"Neuronal functions were uncovered: a PKN1a splice isoform promotes synaptic maturation via EAAT3, and PKN1 negatively regulates an AKT–NeuroD2–GluA1 axis controlling AMPA receptor levels.","evidence":"PKN1a-specific and Pkn1 KO mice, electrophysiology, glutamate uptake, subcellular fractionation, flow cytometry","pmids":["33244074","33613259"],"confidence":"High","gaps":["Direct PKN1 substrates in the AKT/NeuroD2 axis not identified","Isoform-specific mechanism incompletely resolved"]},{"year":2023,"claim":"A mechanotransduction-to-autophagy link was established: overload-activated RhoA–PKN phosphorylates KRT8 Ser43 to block RAB33B Golgi trafficking and suppress autophagosome initiation, contributing to disc degeneration.","evidence":"conditional Krt8 KO, PKN1/2 knockdown, phospho-Ser43 detection, trafficking and autophagy assays, in vivo disc models","pmids":["36897022"],"confidence":"High","gaps":["Relative contributions of PKN1 vs PKN2 not separated","Generality beyond disc tissue untested"]},{"year":2026,"claim":"Biophysical work refined the activation model, showing HR1a dimerization and distinct stoichiometries for RhoA (1:2, with dimer rearrangement) versus Rac1 (monomeric), implying GTPase-specific activation mechanisms.","evidence":"SEC, AUC, SAXS, integrative structural modeling of HR1 domains with RhoA/Rac1","pmids":["42036044"],"confidence":"High","gaps":["Full-length kinase conformational coupling not captured","Functional consequence of RhoA- vs Rac1-specific geometry in cells untested"]},{"year":2025,"claim":"A membrane-independent regulatory input was proposed whereby alkali metal ions reversibly modulate PKN1 activation-loop phosphorylation via phosphate reacquisition.","evidence":"cell-free lysate phosphorylation assays with defined ion concentrations, PDK1-depleted lysate, 32P tracing (preprint)","pmids":["bio_10.1101_2025.09.04.674365"],"confidence":"Low","gaps":["Single unreplicated preprint, not peer-reviewed","Physiological relevance and in-cell occurrence not established","Molecular basis of ion-dependent dephosphorylation unknown"]},{"year":null,"claim":"How GTPase binding, lipid/C2-domain engagement, autoinhibition release, and activation-loop/turn-motif phosphorylation are integrated into a single dynamic activation cycle for full-length PKN1 in cells remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No full-length structure coupling regulatory and catalytic domains","Hierarchy and timing of activating inputs in vivo undefined","Substrate selection rules across compartments not unified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[11,12,15,19,24,26,32]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[2,3,9,24]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[2,3,9]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,4,34]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[5,6,7]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[2,48]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[10]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[10,15]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[28]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,14,22,38]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[12,15,16]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[32]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[19,26,29]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[26,32]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[8,21]}],"complexes":["AKAP-Lbc signaling complex (PKNα–MLTK–MKK3–p38α)"],"partners":["RHOA","RAC1","ACTN (ALPHA-ACTININ)","PLD1","TRAF2","CAMK2D","RPH3A","AKAP13"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q16512","full_name":"Serine/threonine-protein kinase N1","aliases":["Protease-activated kinase 1","PAK-1","Protein kinase C-like 1","Protein kinase C-like PKN","Protein kinase PKN-alpha","Protein-kinase C-related kinase 1","Serine-threonine protein kinase N"],"length_aa":942,"mass_kda":103.9,"function":"PKC-related serine/threonine-protein kinase involved in various processes such as regulation of the intermediate filaments of the actin cytoskeleton, cell migration, tumor cell invasion and transcription regulation. Part of a signaling cascade that begins with the activation of the adrenergic receptor ADRA1B and leads to the activation of MAPK14. Regulates the cytoskeletal network by phosphorylating proteins such as VIM and neurofilament proteins NEFH, NEFL and NEFM, leading to inhibit their polymerization. Phosphorylates 'Ser-575', 'Ser-637' and 'Ser-669' of MAPT/Tau, lowering its ability to bind to microtubules, resulting in disruption of tubulin assembly. Acts as a key coactivator of androgen receptor (AR)-dependent transcription, by being recruited to AR target genes and specifically mediating phosphorylation of 'Thr-11' of histone H3 (H3T11ph), a specific tag for epigenetic transcriptional activation that promotes demethylation of histone H3 'Lys-9' (H3K9me) by KDM4C/JMJD2C. Phosphorylates HDAC5, HDAC7 and HDAC9, leading to impair their import in the nucleus. Phosphorylates 'Thr-38' of PPP1R14A, 'Ser-159', 'Ser-163' and 'Ser-170' of MARCKS, and GFAP. Able to phosphorylate RPS6 in vitro","subcellular_location":"Cytoplasm; Nucleus; Endosome; Cell membrane; Cleavage furrow; Midbody","url":"https://www.uniprot.org/uniprotkb/Q16512/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PKN1","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000123143","cell_line_id":"CID001239","localizations":[{"compartment":"cytoplasmic","grade":3}],"interactors":[{"gene":"PKN2","stoichiometry":0.2},{"gene":"RB1CC1","stoichiometry":0.2},{"gene":"NUP85","stoichiometry":0.2},{"gene":"RNF40","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001239","total_profiled":1310},"omim":[{"mim_id":"610714","title":"PROTEIN KINASE N3; PKN3","url":"https://www.omim.org/entry/610714"},{"mim_id":"601032","title":"PROTEIN KINASE N1; PKN1","url":"https://www.omim.org/entry/601032"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Cytokinetic bridge","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PKN1"},"hgnc":{"alias_symbol":["DBK","PRK1","PKN","MGC46204","PAK1"],"prev_symbol":["PRKCL1"]},"alphafold":{"accession":"Q16512","domains":[{"cath_id":"1.10.287.160","chopping":"17-99","consensus_level":"medium","plddt":74.7053,"start":17,"end":99},{"cath_id":"1.10.287,1.10.287","chopping":"213-295","consensus_level":"medium","plddt":81.4727,"start":213,"end":295},{"cath_id":"2.60.40.150","chopping":"322-341_390-483","consensus_level":"medium","plddt":83.6572,"start":322,"end":483},{"cath_id":"3.30.200.20","chopping":"616-699_919-936","consensus_level":"high","plddt":90.9827,"start":616,"end":936},{"cath_id":"1.10.510.10","chopping":"702-895","consensus_level":"high","plddt":92.1411,"start":702,"end":895}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q16512","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q16512-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q16512-F1-predicted_aligned_error_v6.png","plddt_mean":72.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PKN1","jax_strain_url":"https://www.jax.org/strain/search?query=PKN1"},"sequence":{"accession":"Q16512","fasta_url":"https://rest.uniprot.org/uniprotkb/Q16512.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q16512/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q16512"}},"corpus_meta":[{"pmid":"8571126","id":"PMC_8571126","title":"Protein kinase N (PKN) and PKN-related protein rhophilin as targets of small GTPase Rho.","date":"1996","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/8571126","citation_count":369,"is_preprint":false},{"pmid":"8662891","id":"PMC_8662891","title":"Rhotekin, a new putative target for Rho bearing homology to a serine/threonine kinase, PKN, and rhophilin in the rho-binding domain.","date":"1996","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8662891","citation_count":332,"is_preprint":false},{"pmid":"10619026","id":"PMC_10619026","title":"The structural basis of Rho effector recognition revealed by the crystal structure of human RhoA complexed with the effector domain of PKN/PRK1.","date":"1999","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/10619026","citation_count":143,"is_preprint":false},{"pmid":"12761194","id":"PMC_12761194","title":"The structure and function of PKN, a protein kinase having a catalytic domain homologous to that of PKC.","date":"2003","source":"Journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12761194","citation_count":135,"is_preprint":false},{"pmid":"9751706","id":"PMC_9751706","title":"Proteolytic activation of PKN by caspase-3 or related protease during apoptosis.","date":"1998","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/9751706","citation_count":126,"is_preprint":false},{"pmid":"15980430","id":"PMC_15980430","title":"Transforming growth factor-beta1-induced expression of smooth muscle marker genes involves activation of PKN and p38 MAPK.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15980430","citation_count":114,"is_preprint":false},{"pmid":"10323867","id":"PMC_10323867","title":"The Drosophila Pkn protein kinase is a Rho/Rac effector target required for dorsal closure during embryogenesis.","date":"1999","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/10323867","citation_count":110,"is_preprint":false},{"pmid":"9030526","id":"PMC_9030526","title":"Interaction of PKN with alpha-actinin.","date":"1997","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9030526","citation_count":109,"is_preprint":false},{"pmid":"16611232","id":"PMC_16611232","title":"A Salmonella type III secretion effector interacts with the mammalian serine/threonine protein kinase PKN1.","date":"2006","source":"Cellular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/16611232","citation_count":107,"is_preprint":false},{"pmid":"7945381","id":"PMC_7945381","title":"Activation of PKN, a novel 120-kDa protein kinase with leucine zipper-like sequences, by unsaturated fatty acids and by limited proteolysis.","date":"1994","source":"Biochemical and biophysical research 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ions","date":"2025-09-06","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.04.674365","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":49550,"output_tokens":12673,"usd":0.169373,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":24229,"output_tokens":6522,"usd":0.142097,"stage2_stop_reason":"end_turn"},"total_usd":0.31147,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1996,\n      \"finding\": \"PKN (PKN1) directly binds to GTP-bound RhoA (but not GDP-RhoA) and is activated by this interaction both in vitro and in vivo, establishing PKN as a Rho effector serine/threonine protein kinase.\",\n      \"method\": \"Yeast two-hybrid cloning, in vitro binding assay, in vivo kinase activation assay\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal binding and kinase activation confirmed in vitro and in vivo, replicated across multiple independent labs in contemporaneous papers\",\n      \"pmids\": [\"8571126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Crystal structure of human RhoA complexed with the effector domain of PKN/PRK1 at 2.2 Å resolution reveals that the PKN effector domain adopts an antiparallel coiled-coil finger (ACC finger) fold that binds to RhoA switch I, beta strands B2/B3, and helix A5, predominantly via specific hydrogen bonds.\",\n      \"method\": \"X-ray crystallography at 2.2 Å resolution\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structure with detailed mechanistic interpretation, foundational structural paper\",\n      \"pmids\": [\"10619026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"PKN kinase activity is activated several-fold by unsaturated fatty acids (arachidonic acid, linoleic acid, oleic acid) and by limited proteolysis with trypsin, but not by Ca2+/phosphatidylserine/diolein (the classical PKC activators).\",\n      \"method\": \"In vitro kinase assay with peptide substrates, limited proteolysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct in vitro enzymatic characterization replicated in subsequent purification studies\",\n      \"pmids\": [\"7945381\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Purified native PKN from rat testis is a 120 kDa serine/threonine kinase activated by unsaturated fatty acids and detergents; autophosphorylation activity is partially inhibited by alkaline phosphatase pretreatment, suggesting autophosphorylation contributes to activity.\",\n      \"method\": \"Protein purification (~8000-fold), in vitro kinase assay, alkaline phosphatase treatment\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — purified native enzyme characterized by multiple enzymatic assays\",\n      \"pmids\": [\"7654208\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"The RhoA-binding region of PKN1 maps to amino acid residues 33–111 (with residues 74–113 critical for association); PKN1 binding to RhoA inhibits both endogenous and GAP-stimulated GTPase activity of RhoA, suggesting PKN1 can sustain the active GTP-bound form of RhoA.\",\n      \"method\": \"Yeast two-hybrid system, in vitro binding assay with truncation mutants, synthetic peptide competition, GTPase activity assay\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro binding with deletion mapping plus functional GTPase inhibition assay, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"8647255\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"PKN1 associates with alpha-actinin via its N-terminal region (outside the RhoA-binding domain) binding to the third spectrin-like repeats of alpha-actinin; this interaction is regulated by Ca2+ for non-skeletal muscle type and is stimulated by phosphatidylinositol 4,5-bisphosphate for full-length alpha-actinin.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding with truncation mutants, co-immunoprecipitation from COS7 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast two-hybrid plus in vitro binding domain mapping plus co-IP in mammalian cells, multiple orthogonal methods\",\n      \"pmids\": [\"9030526\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"PKN1 associates with and phosphorylates the head-rod domain of all three neurofilament subunits (NF-L, NF-M, NF-H); phosphorylation of NF-L by PKN inhibits neurofilament polymerization in vitro.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding, in vitro kinase assay with purified neurofilaments, polymerization assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro binding domain mapping, direct kinase assay with purified substrates, and functional polymerization assay, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"8621664\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"PKN1 phosphorylates vimentin and GFAP in their head domains in vitro, resulting in nearly complete inhibition of filament formation; the regulatory domain of PKN1 interacts with vimentin.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding assay, in vitro kinase assay, filament formation assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro kinase assay with functional filament formation readout, yeast two-hybrid and binding confirmation, multiple orthogonal methods\",\n      \"pmids\": [\"9175763\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"PKN is cleaved by caspase-3 (or a related caspase) at specific aspartate residues during apoptosis, generating a constitutively active catalytic fragment; site-directed mutagenesis of the cleavage sites prevented fragment generation, and the cleavage was inhibited by the caspase inhibitor DEVD-CHO.\",\n      \"method\": \"In vitro cleavage with recombinant caspase-3, site-directed mutagenesis, immunoblot in apoptotic cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with recombinant caspase-3, mutagenesis validation, pharmacological inhibition, confirmed in multiple cell lines\",\n      \"pmids\": [\"9751706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The regulatory region of PKN1 (residues 455–511) contains an autoinhibitory domain that inhibits kinase activity in a substrate-competitive manner (Ki = 0.6 µM); arachidonic acid relieves autoinhibition by this segment. Phosphorylation at Thr64, Ser374, or Thr531 in the regulatory region and Thr774 in the activation loop are required for full kinase activity.\",\n      \"method\": \"Deletion mutagenesis in insect cells, in vitro kinase inhibition assay with recombinant peptide fragments, site-directed mutagenesis of phosphorylation sites\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted inhibition assay with defined peptide fragments, mutagenesis of key residues, multiple orthogonal approaches in single study\",\n      \"pmids\": [\"10467162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"PKN1 translocates from the cytosol to the nucleus in response to heat shock (42°C), sodium arsenite, and serum starvation, and returns to the cytoplasm upon recovery from heat shock.\",\n      \"method\": \"Confocal immunofluorescence microscopy, subcellular fractionation, immunoblotting\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization by confocal microscopy corroborated by biochemical fractionation, multiple stress conditions tested\",\n      \"pmids\": [\"8816775\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"PKN1 phosphorylates tau protein at specific sites in microtubule-binding domains (Ser258, Ser320, Ser352), with Ser320 being PKN1-specific (not phosphorylated by PKC isoforms); PKN1 activation disrupts microtubule arrays in vitro and in vivo.\",\n      \"method\": \"In vitro kinase assay with tau peptides, site-directed mutagenesis, phospho-specific antibodies, transfection of active/inactive PKN in CHO cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro kinase assay, site-directed mutagenesis, phospho-specific antibody validation in cells, multiple methods\",\n      \"pmids\": [\"11104762\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"PKN1 delays mitotic timing by directly phosphorylating and inhibiting Cdc25C phosphatase activity, thereby delaying Tyr15 dephosphorylation of Cdc2 and entry into mitosis; active PKN causes cleavage arrest in Xenopus embryos and delayed mitosis in Xenopus cycling extracts.\",\n      \"method\": \"Xenopus embryo microinjection, Xenopus egg extract cell cycle assay, in vitro Cdc25C kinase/phosphatase assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay on Cdc25C, multiple orthogonal functional assays (embryo, extract), kinase-dead control\",\n      \"pmids\": [\"11134534\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"PKN1 directly interacts with and stimulates the activity of phospholipase D1 (PLD1) in vitro and in vivo; PKNα binds residues 228–598 of PLD1 and stimulates PLD1 activity in the presence of PIP2.\",\n      \"method\": \"Co-immunoprecipitation from COS7 cells, in vitro binding with PLD1 deletion mutants, in vitro PLD1 activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP of endogenous proteins, domain mapping with deletion mutants, direct functional enzyme assay\",\n      \"pmids\": [\"11259428\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PKNα (PKN1) phosphorylates MLTKα (a MAPKKK) and enhances its kinase activity in vitro; PKNα associates with all components of the p38γ MAPK cascade (p38γ, MKK6, MLTKα), functioning as both an upstream activator and a scaffold for p38γ MAPK signaling.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, SDS-PAGE mobility shift assay, dominant-negative expression\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase and co-IP data with multiple pathway components, single lab\",\n      \"pmids\": [\"12761180\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PKN1 (activated by TGF-β1 via RhoA) phosphorylates Cdc25C at Ser216, promoting 14-3-3 binding to Cdc25C and inactivating it, thereby delaying G2/M transition; PKN1 and Cdc25C coimmunoprecipitate and colocalize to the nucleus prior to mitosis.\",\n      \"method\": \"RNAi knockdown, constitutively active PKN1 expression, in vitro kinase assay on Cdc25C, phospho-specific Ser216 detection, co-immunoprecipitation, immunofluorescence colocalization\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro phosphorylation of Cdc25C, RNAi rescue, co-IP, and site-specific mutagenesis (S216A) evidence, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"17374997\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PKN1 involvement in arsenite-induced G2/M delay is mediated by direct phosphorylation of Cdc25C at Ser216, which facilitates 14-3-3 association; a Cdc25C S216A phospho-mutant partially abrogated arsenite-induced cell cycle arrest.\",\n      \"method\": \"Constitutively active PKN1 expression in HeLa cells, in vitro kinase assay, Cdc25C S216A mutant expression, co-IP\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro kinase assay, site mutagenesis rescue, co-IP, functional cell cycle readout, consistent with the Xenopus findings\",\n      \"pmids\": [\"15791647\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Salmonella effector SspH1 interacts with PKN1 via its leucine-rich repeat (LRR) domain, and PKN1 expression decreases NF-κB-dependent reporter gene activity while PKN1 depletion by RNAi increases it, indicating PKN1 negatively modulates NF-κB signaling.\",\n      \"method\": \"Yeast two-hybrid screen, domain mapping of LRR-PKN1 interaction, RNAi knockdown, NF-κB reporter assay\",\n      \"journal\": \"Cellular microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid and RNAi with functional reporter readout, single lab\",\n      \"pmids\": [\"16611232\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"X-ray structure of SspH1–PKN1 complex reveals that the SspH1 LRR domain binds specifically to the HR1b coiled-coil subdomain of PKN1, sterically displacing the SspH1 catalytic domain to activate its E3 ubiquitin ligase activity; SspH1 catalyzes ubiquitination and proteasome-dependent degradation of PKN1 in cells, attenuating androgen receptor responsiveness.\",\n      \"method\": \"X-ray crystallography of SspH1–PKN1 complex, ubiquitination assay in cells, proteasome inhibitor experiments, androgen receptor reporter assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus functional ubiquitination/degradation assay, mechanistic coupling of substrate recognition to enzyme activation demonstrated\",\n      \"pmids\": [\"24248594\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PKN1 phosphorylates TRAF1 in vitro and in vivo; this phosphorylation recruits TRAF1 to TNFR2 and is required for attenuation of constitutive NF-κB and JNK signaling; mutagenesis of the phospho-acceptor residue in TRAF1 abrogates PKN1-dependent TNFR2 recruitment.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, RNAi, phospho-acceptor mutagenesis, NF-κB/JNK reporter assays\",\n      \"journal\": \"Genes to cells\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro kinase assay plus phospho-site mutagenesis plus functional reporter assays, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"18429822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"PKN1 directly binds the TRAF domain of TRAF2 through a PXQX(S/T) motif (residues 580–584); mutation of this motif abrogates co-immunoprecipitation with TRAF2; RNAi knockdown of PKN1 reduces TRAF2-induced NF-κB activation.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding with TRAF2 deletion mutants, co-immunoprecipitation, site-directed mutagenesis, RNAi, NF-κB reporter assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple binding assays plus RNAi functional readout, single lab\",\n      \"pmids\": [\"14741690\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PKN1 is activated by ischemia/reperfusion (phosphorylated at Thr774) in the heart; transgenic cardiac-specific overexpression of constitutively active PKN1 reduces infarct size and apoptosis, while dominant-negative PKN1 increases injury; PKN1 mediates phosphorylation of αB-crystallin and stimulates proteasome activity, partially accounting for cardioprotection.\",\n      \"method\": \"Transgenic mouse (cardiac-specific CA and DN PKN1), in vivo I/R model, TUNEL assay, immunoblot for αB-crystallin phosphorylation, proteasome activity assay, epoxomicin inhibition\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss- and gain-of-function transgenic models, multiple mechanistic readouts, pharmacological rescue with proteasome inhibitor\",\n      \"pmids\": [\"20595653\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PKN1 (PKNα) serves as a component of an AKAP-Lbc–assembled signaling complex containing RhoA effector PKNα, MLTK, MKK3, and p38α; this complex is required for α1b-adrenergic receptor-mediated RhoA-dependent activation of p38α MAPK, as silencing AKAP-Lbc specifically reduces this pathway.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown of AKAP-Lbc, p38α kinase activity assay, selective pharmacological dissection\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP and RNAi with pathway-specific functional readouts, single lab\",\n      \"pmids\": [\"21224381\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"A cleaved (caspase-activated) form of PKN1, but not wild-type PKN1, disrupts neurofilament organisation and axonal transport in neurons; PKN1 is cleaved and activated in SOD1G93A ALS model mice and in glutamate-treated neurons.\",\n      \"method\": \"Expression of cleaved vs. wild-type PKN1 in neurons, neurofilament transport assay, immunoblot for cleavage in transgenic mice and glutamate-treated cells\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct comparison of cleaved vs. wild-type overexpression with defined transport phenotype, corroborated in in vivo ALS model\",\n      \"pmids\": [\"18519042\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PKN1 and PKN3 share a phosphorylation consensus motif requiring an arginine at position −3 and intolerance of arginine at position +1 (PKN1) or −1 (PKN3); PKN1 phosphorylates EGFR at Thr654 in vitro and this site is constitutively phosphorylated in a PKN-dependent manner in vivo.\",\n      \"method\": \"Peptide library substrate screen, protein array, site mutagenesis, in vitro kinase assay, cellular phosphorylation assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — peptide library screen plus direct in vitro kinase assay with site mutagenesis and cellular validation, multiple methods in one study\",\n      \"pmids\": [\"21749319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PKN1 inhibits Wnt/β-catenin signaling in melanoma cells; PKN1 is found in a protein complex with Frizzled 7 and co-purifying proteins by affinity purification/mass spectrometry; siRNA depletion of PKN1 enhances β-catenin reporter activity and increases WNT3A-induced apoptosis.\",\n      \"method\": \"siRNA screen, phosphoproteomics, affinity purification–mass spectrometry, β-catenin reporter assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — AP-MS for complex identification, RNAi with reporter readout, single lab with two orthogonal methods\",\n      \"pmids\": [\"24114839\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PKN1 phosphorylates RPH3A, which enhances binding of RPH3A to GTP-bound RAB21; this PKN1–RPH3A–RAB21 axis is required for polarized localization of RAB21 and PIP5K1C90 in neutrophils, integrin activation, and adhesion to endothelial cells; myeloid-specific loss of PKN1 decreases tissue injury in renal ischemia–reperfusion.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, GTP-RAB21 pulldown, immunofluorescence of polarization, integrin activation assay, myeloid conditional KO mouse, renal I/R injury model\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vitro kinase assay, binding assays, conditional KO mouse with multiple cellular and in vivo phenotypes, multiple orthogonal methods\",\n      \"pmids\": [\"28636945\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PKN1 kinase activity is regulated by TORC2-dependent phosphorylation of the turn motif (TM); amino acid substitution in the TM reduces kinase activity; PKN1 contributes to cell motility in human prostate cancer cells.\",\n      \"method\": \"TORC2-specific inhibitor (torin), phospho-TM-specific antibody, TM mutagenesis, in vitro kinase assay, cell motility assay, depletion approaches\",\n      \"journal\": \"The Prostate\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological inhibition plus mutagenesis plus functional motility assay, single lab\",\n      \"pmids\": [\"28875501\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Loss of PKN1 in vivo doubles myocardial infarct size after I/R; PKN1 co-localizes to the sarcoplasmic reticulum during ischemia and interacts with CamKIIδ; PKN1 loss increases basal CamKIIδ activation and phospholamban Thr17 phosphorylation, suggesting PKN1 limits CamKIIδ-dependent SR calcium dysregulation.\",\n      \"method\": \"PKN1 knockout mouse, Langendorff I/R perfusion, GC-MS/MS and immunoblot co-IP with CamKIIδ, PhosTag gel, confocal immunofluorescence, siRNA in NRVM\",\n      \"journal\": \"Cardiovascular research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse with infarct size readout, MS-validated protein interaction, siRNA rescue, multiple orthogonal methods\",\n      \"pmids\": [\"29045568\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PKN1 kinase activity is required for lymphocyte trafficking/egress from secondary lymphoid organs; knock-in mice with a PKN1 T778A kinase-dead mutation show lymphocyte sequestration in spleen and lymph nodes, reduced chemotaxis, and impaired S1P-directed migration in a cell-autonomous manner.\",\n      \"method\": \"Knock-in mouse (T778A mutation), cell transfer experiments, in vitro chemotaxis assay, flow cytometry\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell-autonomous kinase-dead knock-in, reciprocal cell transfer showing autonomy, multiple functional readouts\",\n      \"pmids\": [\"28794483\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PRK1/PKN1 regulates migration and gene expression in androgen-independent prostate cancer cells through its kinase activity; PKN1 interacts with scaffold protein SPAG9/JIP4 and this interaction is required for p38 phosphorylation and cell migration; a PKN1 inhibitor prevents metastases in mice.\",\n      \"method\": \"Sibling KD, transcriptome/interactome analysis, co-localization in tissue, p38 phosphorylation assay, cell migration assay, in vivo metastasis model with inhibitor\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-localization and KD with functional readouts, in vivo inhibitor experiment, single lab\",\n      \"pmids\": [\"25504435\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PKN1 (specifically the PKN1a splice variant) promotes synaptic maturation by upregulating neuronal glutamate transporter EAAT3 expression; PKN1a knockout unmasks aberrant mGluR-dependent LTD and AMPA receptor silencing, and reduces glutamate uptake through EAAT3.\",\n      \"method\": \"PKN1a-specific knockout mouse, mGluR-LTD electrophysiology, glutamate uptake assay, silent synapse analysis, spine morphology\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — specific knockout with electrophysiological, biochemical and morphological readouts, multiple orthogonal methods\",\n      \"pmids\": [\"33244074\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Overloading-activated RHOA–PKN (including PKN1) phosphorylates KRT8 (keratin 8) at Ser43, which impedes RAB33B trafficking from the Golgi, suppresses autophagosome initiation, and contributes to intervertebral disc degeneration; knockdown of Pkn1 and Pkn2 together ameliorates disc degeneration.\",\n      \"method\": \"Conditional Krt8 knockout, PKN1/2 knockdown, phospho-Ser43 KRT8 detection, RAB33B trafficking assay, autophagy initiation assay, in vivo disc degeneration models\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO and KD with defined phospho-substrate, trafficking readout, and in vivo disease model, multiple orthogonal methods\",\n      \"pmids\": [\"36897022\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The very C-terminus of PRK1/PKN1 (beyond the hydrophobic motif) is essential for activation by RhoA and for downstream signaling (neurite retraction); deletion of HR1 alone does not fully abolish RhoA binding or activation, indicating both the HR1 domain and C-terminus contribute to full RhoA-mediated activation.\",\n      \"method\": \"C-terminal deletion mutants, in vitro kinase activation assay with GTPγS-RhoA, in vivo RhoA activation assay, neurite retraction assay in neuronal cells\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — deletion mutagenesis with in vitro and in vivo functional readouts, single lab\",\n      \"pmids\": [\"16427251\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Drosophila Pkn (PKN ortholog) binds specifically to GTP-activated Rho1 and Rac1 through distinct binding sites, and both interactions increase kinase activity; genetic loss-of-function shows Pkn is required for epidermal cell shape changes during dorsal closure, acting in a Rho1-mediated pathway independent of the Rac-JNK pathway.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding, in vitro kinase assay, Drosophila loss-of-function mutant, epistasis analysis\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — biochemical binding/kinase assays combined with in vivo genetic loss-of-function and epistasis, multiple methods\",\n      \"pmids\": [\"10323867\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"PKN1 binds and phosphorylates high-risk HPV E6 oncoprotein; only high-risk (not low-risk) HPV E6 proteins bind PKN1; this is the first demonstration that HPV E6 is a phosphoprotein.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding (wheat-germ lysate), co-immunoprecipitation in 293T cells, in vitro kinase assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid, in vitro binding, co-IP, and direct kinase assay, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"10809724\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"PKN (PRK1/PKN1) stimulates transcriptional activity of the ANF promoter via the serum response element (SRE) in cardiomyocytes; only PKN (not PRK2 or Rho kinase) generates robust ANF-SRE stimulation; this effect requires RhoA and is lost with SRE mutation.\",\n      \"method\": \"Luciferase reporter assays, constitutively active PKN1 transfection, dominant-negative Rho, SRE mutagenesis\",\n      \"journal\": \"American journal of physiology. Heart and circulatory physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — reporter assay with multiple constructs and controls, single lab, no direct substrate identification\",\n      \"pmids\": [\"10843871\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"In PTEN-null MEFs, cortical actin formation is mediated through the PTEN/RhoA/PKN pathway, as dominant-negative RhoA or kinase-dead PKN inhibit cortical actin accumulation; however, this pathway does not mediate enhanced cell migration (which depends on Rac/PDK-1/Akt instead).\",\n      \"method\": \"Dominant-negative RhoA and kinase-dead PKN overexpression in Pten−/− MEFs, actin staining, cell migration assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis with DN/KD constructs and defined phenotypic readouts, single lab\",\n      \"pmids\": [\"15531926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Hypotonic stress activates PKN1 via Thr774 phosphorylation in cardiac myocytes, with activation dependent on upstream PDK1 and Src-family kinase-mediated RhoA activation; active PKN1 promotes ERK phosphorylation via MEK to enhance cardiac myocyte survival.\",\n      \"method\": \"In vitro PKN1 activity assay, immunoblot for Thr774 phosphorylation, pharmacological inhibition (PP1, U-0126), Elk1-GAL4 transcriptional assay, siRNA knockdown, cell viability assay\",\n      \"journal\": \"American journal of physiology. Heart and circulatory physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — kinase activity assay with multiple pharmacological probes and siRNA, defined signaling cascade, single lab\",\n      \"pmids\": [\"21037231\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Downstream of Gαq-RhoA in uveal melanoma, PKN converges with ROCK to control FAK signaling; darovasertib inhibits both PKC and PKN/PRK kinases and synergizes with FAK inhibitors in vitro and in preclinical metastatic models.\",\n      \"method\": \"High-throughput chemogenetic drug screen, kinase inhibitor profiling, FAK phosphorylation assay, in vitro cell viability assay, preclinical mouse metastasis model\",\n      \"journal\": \"Cell reports. Medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological screen with mechanistic follow-up and in vivo validation, single study\",\n      \"pmids\": [\"37858338\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PKN1 negatively regulates hippocampal AKT activity and NeuroD2 levels; Pkn1 knockout elevates phospho-AKT and NeuroD2, leading to enhanced GluA1 (but not GluA2/3) protein levels and increased GluA1 membrane fraction, revealing a PKN1–AKT–NeuroD2–GluA1 regulatory axis.\",\n      \"method\": \"Pkn1 knockout mouse, immunoblot for phospho-AKT and NeuroD2, subcellular fractionation, flow cytometry for membrane GluA1\",\n      \"journal\": \"Frontiers in synaptic neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockout mouse with biochemical signaling readouts and fractionation, single lab\",\n      \"pmids\": [\"33613259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PKN1 inhibition reduces AKT phosphorylation; PKN1 phosphorylation at S374 is functionally relevant for PKN1–AKT interaction and axonal outgrowth on inhibitory substrates in cerebellar granule cells; PKN1 pS374 decreases during cerebellar development.\",\n      \"method\": \"Pkn1 knockout cerebellar granule cells, hypoxia-ischemia in vitro model, AKT phosphorylation assay, axonal outgrowth assay, caspase-3 activation assay, site-specific mutagenesis of S374\",\n      \"journal\": \"Biomolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockout cells with site mutagenesis and multiple functional readouts, single lab\",\n      \"pmids\": [\"38002281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"PKN1 HR1a forms a dimer, and HR1c drives further oligomerization; RhoA forms a 1:2 complex with HR1a and induces rearrangement of the HR1a dimer (supported by SAXS), whereas Rac1 binds monomeric HR1a, suggesting distinct activation mechanisms for RhoA vs. Rac1.\",\n      \"method\": \"Biophysical analysis (SEC, AUC), SAXS, integrative structural modeling of HR1-containing PKN1 dimers\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — SAXS and biophysical characterization with GTPase binding, multiple orthogonal biophysical methods, single study\",\n      \"pmids\": [\"42036044\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"TDP-43 loss induces inclusion of a cryptic exon (PKN1-5a1) in PKN1 mRNA, generating a truncated peptide (PKN207) that escapes nonsense-mediated decay, is detectable in AD brains with TDP-43 pathology, and impairs cognition, memory, and synaptic plasticity in mice.\",\n      \"method\": \"RNA sequencing in ALS patient brains, NMD inhibition assay, mass spectrometry detection of PKN207 in AD brain, in vivo mouse cognitive/synaptic plasticity assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNA-seq, MS protein detection, and in vivo functional assays, single study, novel finding not yet replicated\",\n      \"pmids\": [\"41720774\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"PKN1 regulates alphaB-crystallin expression under heat stress via cooperation with HSF1; the catalytically active fragment of PKN1, but not the inactive form, induces alphaB-crystallin accumulation in HeLa cells through both proximal and distal heat shock elements of the promoter.\",\n      \"method\": \"Transfection of active/inactive PKN1 fragment with HSF1, luciferase reporter assay, alphaB-crystallin immunoblot\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — reporter assay and immunoblot with kinase-active vs. inactive comparison, single lab\",\n      \"pmids\": [\"9837746\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"PKN1 interacts with the paraneoplastic cerebellar degeneration antigen PCD17 via its N-terminal regulatory domain; PKN1 phosphorylates PCD17 in a fatty acid-stimulated manner; PCD17 functions as a transcriptional activator and is co-immunoprecipitated with PKN1.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding, co-immunoprecipitation from COS7 cells, in vitro kinase assay, transcriptional reporter assay\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple binding methods plus direct kinase assay and functional transcription assay, single lab\",\n      \"pmids\": [\"9637778\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"PKN1 interacts directly with the bHLH domain of the transcription factor NDRF/NeuroD2; co-expression of catalytically active PKN1 (but not kinase-deficient PKN1) further enhances NDRF/NeuroD2-dependent transcription of the insulin promoter element RIPE3.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding with deletion mutants, co-immunoprecipitation from COS-7 cells, transient transfection reporter assay\",\n      \"journal\": \"Brain research. Molecular brain research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding confirmed by multiple methods plus kinase-activity-dependent transcription assay, single lab\",\n      \"pmids\": [\"10640683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"PKNα (PKN1) interacts with Cyclin T2a in a yeast two-hybrid screen and co-immunoprecipitation; co-overexpression of PKNα and Cyclin T2a strongly enhances expression of myogenic differentiation markers (Myogenin, Myosin Heavy Chain) in C2C12 cells during starvation-induced differentiation.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, in vitro pull-down, luciferase reporter assay, myogenic differentiation marker immunoblot\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal binding assays plus functional differentiation readout, single lab\",\n      \"pmids\": [\"16331689\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The C2-like domain of PKN1 contains amphipathic cardiolipin-binding motifs and binds cardiolipin and C18 fatty acids; two distinct types of cardiolipin/phosphatidic acid binding are observed; the pseudosubstrate sequence in the C2 domain overlaps with the arachidonic acid binding region, linking lipid binding to autoinhibitory regulation.\",\n      \"method\": \"Biophysical lipid-binding assays (SPR/ITC), CD spectroscopy, sequence analysis, kinetic assay of full-length PKN1\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct biophysical characterization with multiple lipid species and structural inference, single study, consistent with prior activation data\",\n      \"pmids\": [\"38441874\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Constitutively active PKN1 in mammary epithelium impairs tight junction sealing at parturition without disrupting tight junction formation (occludin and ZO1 localize correctly); dominant-negative PKN1 accelerates tight junction sealing in EpH4 cells, and active PKN1 impairs glucocorticoid-stimulated sealing.\",\n      \"method\": \"Transgenic mouse (mammary-specific CA-PKN1), intraductal [14C]sucrose injection for sealing assay, occludin/ZO1 immunofluorescence, EpH4 cell dominant-negative/active transfection\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo transgenic model with functional tight junction assay corroborated by in vitro dominant-negative results, single lab\",\n      \"pmids\": [\"17591691\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PKN1 controls adipocyte differentiation and glucose metabolism; PKN1-silenced adipocytes show decreased differentiation and glucose uptake with reduced PPARγ, FABP4, adiponectin, and CEBPα expression; insulin-resistant adipocytes show decreased PKN1 activation.\",\n      \"method\": \"siRNA knockdown in 3T3-L1 adipocytes and human VAT explants, glucose uptake assay, adipogenic marker immunoblot, PKN1 phosphorylation assay\",\n      \"journal\": \"Nutrients\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA KD with multiple functional metabolic readouts, single lab\",\n      \"pmids\": [\"37242297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PKN1 kinase activity is required for protection of TRAF1 from cIAP-mediated degradation during constitutive CD40 signaling; the active phospho-Thr774 form of PKN1 is constitutively expressed in CLL but not in healthy B cells.\",\n      \"method\": \"PKN1 kinase inhibitor treatment (OTSSP167, XL-228), TRAF1 immunoblot, caspase-3 activation assay in primary CLL samples\",\n      \"journal\": \"Oncoimmunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — pharmacological inhibitor approach with defined substrate (TRAF1) and functional readout, but no direct phosphorylation site mutagenesis, single lab\",\n      \"pmids\": [\"34589290\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Na+ and K+ ions directly and rapidly reduce PKN activation-loop phosphorylation in the absence of membranes; upon reduction of ion concentration, phosphorylation recovers without requiring PDK1 or ATP, via a 'reacquisition of phosphate' mechanism demonstrated by 32P tracing.\",\n      \"method\": \"Cell-free lysate PKN activation-loop phosphorylation assay with defined ion concentrations, PDK1-depleted lysate, 32P tracing experiment\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 1 / Weak — novel mechanism with 32P tracing but single preprint study, not yet peer-reviewed or replicated\",\n      \"pmids\": [\"bio_10.1101_2025.09.04.674365\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"PKN1 is a RhoA/Rac1-activated and fatty acid-activated AGC serine/threonine kinase whose N-terminal antiparallel coiled-coil (ACC/HR1) domains bind GTP-loaded Rho family GTPases (activating the kinase), while an internal autoinhibitory segment (residues 455–511) suppresses basal activity and is relieved by arachidonic acid or by caspase-3 cleavage; active PKN1 phosphorylates a broad substrate set including tau (Ser320), neurofilaments, vimentin/GFAP (head domains inhibiting filament assembly), Cdc25C (Ser216, delaying G2/M entry), TRAF1 (attenuating constitutive NF-κB/JNK signaling), αB-crystallin (promoting proteasome activity and cardioprotection), KRT8 (Ser43, suppressing autophagosome initiation), RPH3A (directing polarized RAB21 vesicle trafficking in neutrophils), and EGFR (Thr654); it also scaffolds p38γ MAPK cascades via MLTK and anchors signaling complexes through AKAP-Lbc, and its activity is further regulated by TORC2-dependent turn-motif phosphorylation and by alkali metal ion-dependent phosphate exchange on the activation loop.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PKN1 (PRK1) is a Rho-family GTPase-activated AGC serine/threonine protein kinase that couples RhoA/Rac1 signaling and lipid second messengers to cytoskeletal remodeling, cell-cycle timing, stress signaling, and membrane trafficking [#0, #34]. Its N-terminal antiparallel coiled-coil (ACC/HR1) finger binds GTP-loaded RhoA at switch I and stabilizes the active GTPase, defining PKN1 as a Rho effector [#1, #4]; HR1a dimerizes and engages RhoA in a 1:2 complex while binding Rac1 as a monomer, implying distinct activation geometries for the two GTPases [#42]. Catalytic output is set by a substrate-competitive autoinhibitory segment (residues 455–511) that is relieved by unsaturated fatty acids such as arachidonic acid, by a C2-like lipid-binding region whose pseudosubstrate overlaps the arachidonic-acid site, and by caspase-3 cleavage that liberates a constitutively active fragment during apoptosis [#9, #48, #8]; full activity additionally requires activation-loop (Thr774) and turn-motif phosphorylation, the latter TORC2-dependent [#9, #27]. Active PKN1 phosphorylates intermediate filament and microtubule substrates—neurofilament, vimentin/GFAP head domains, and tau (Ser320)—to inhibit polymer assembly and disrupt cytoskeletal arrays [#6, #7, #11]. It enforces G2/M delay by phosphorylating Cdc25C at Ser216 to promote 14-3-3 sequestration, a checkpoint engaged downstream of TGF-β and arsenite stress [#12, #15, #16]. PKN1 further phosphorylates RPH3A to direct polarized RAB21 trafficking and integrin activation in neutrophils [#26], KRT8 (Ser43) to impede RAB33B Golgi trafficking and suppress autophagosome initiation [#32], EGFR at Thr654 [#24], and α B-crystallin to stimulate proteasome activity and confer cardioprotection during ischemia/reperfusion [#21]. It scaffolds p38 MAPK cascades through MLTK and the AKAP-Lbc complex [#14, #22] and modulates TRAF1/TRAF2-dependent NF-κB and JNK signaling [#19, #20]. In vivo, PKN1 kinase activity is required for lymphocyte egress [#29] and contributes to cardiac protection by limiting CamKIIδ-dependent SR calcium dysregulation [#28].\",\n  \"teleology\": [\n    {\n      \"year\": 1994,\n      \"claim\": \"Before its regulators were known, PKN1 needed a defined activation input; this established that PKN1 is a distinct kinase activated by unsaturated fatty acids and proteolysis rather than by classical PKC cofactors.\",\n      \"evidence\": \"in vitro kinase assay with peptide substrates and limited trypsin proteolysis\",\n      \"pmids\": [\"7945381\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the physiological activator or upstream signal\", \"No structural basis for fatty-acid activation\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Purification of the native enzyme confirmed PKN1 as a ~120 kDa fatty-acid-activated kinase whose activity depends on phosphorylation.\",\n      \"evidence\": \"~8000-fold purification from rat testis with kinase and alkaline-phosphatase assays\",\n      \"pmids\": [\"7654208\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific phosphosites not yet mapped\", \"No upstream kinase identified\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"The central question of what links PKN1 to upstream signaling was answered by showing it is a direct GTP-RhoA effector, and that binding both activates PKN1 and stabilizes active RhoA.\",\n      \"evidence\": \"yeast two-hybrid cloning, in vitro binding, GTPase assays, and deletion mapping of the RhoA-binding region (residues 33–111)\",\n      \"pmids\": [\"8571126\", \"8647255\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural mechanism of activation not yet resolved\", \"Whether Rac1 also activates was unaddressed\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"To connect PKN1 to cellular structures, it was shown to bind the cytoskeletal crosslinker alpha-actinin and to phosphorylate neurofilament subunits, inhibiting filament polymerization—placing PKN1 at the cytoskeleton.\",\n      \"evidence\": \"yeast two-hybrid, in vitro binding/kinase assays, polymerization assays, co-IP\",\n      \"pmids\": [\"9030526\", \"8621664\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of filament phosphorylation not established\", \"Cellular consequence on cytoskeletal architecture not directly shown\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"PKN1 was linked to stress responses by demonstrating stress-induced cytosol-to-nucleus translocation.\",\n      \"evidence\": \"confocal immunofluorescence and subcellular fractionation under heat shock, arsenite, serum starvation\",\n      \"pmids\": [\"8816775\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nuclear substrates of translocated PKN1 not identified\", \"Translocation signal/mechanism unknown\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"The structural basis of effector recognition was resolved: the PKN1 effector domain adopts an antiparallel coiled-coil (ACC) finger that contacts RhoA switch I.\",\n      \"evidence\": \"2.2 Å X-ray crystal structure of RhoA–PKN effector domain complex\",\n      \"pmids\": [\"10619026\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not capture how binding propagates to the catalytic domain\", \"Full-length conformational change unresolved\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"How basal PKN1 activity is restrained was defined by mapping a substrate-competitive autoinhibitory segment (455–511) relieved by arachidonic acid, plus phosphosites required for full activity.\",\n      \"evidence\": \"deletion mutagenesis in insect cells, in vitro inhibition assays with peptide fragments, phosphosite mutagenesis\",\n      \"pmids\": [\"10467162\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the upstream activation-loop kinase\", \"Coupling of GTPase binding to autoinhibition release not shown\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"An activation route independent of GTPase/lipid was established: caspase-3 cleaves PKN1 during apoptosis to release a constitutively active catalytic fragment.\",\n      \"evidence\": \"in vitro cleavage with recombinant caspase-3, cleavage-site mutagenesis, DEVD-CHO inhibition, immunoblot in apoptotic cells\",\n      \"pmids\": [\"9751706\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Substrates targeted by the cleaved fragment not defined here\", \"Physiological apoptotic role unaddressed\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"PKN1 was assigned a cell-cycle role by showing it phosphorylates and inhibits Cdc25C to delay mitotic entry; later work mapped this to Ser216-driven 14-3-3 sequestration engaged by TGF-β and arsenite stress.\",\n      \"evidence\": \"Xenopus embryo/extract assays and in vitro Cdc25C kinase assays; later RNAi, S216A mutant rescue, co-IP, phospho-Ser216 detection\",\n      \"pmids\": [\"11134534\", \"17374997\", \"15791647\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous timing of PKN1-Cdc25C activity in unperturbed mitosis unclear\", \"Relationship to nuclear translocation not integrated\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"PKN1's role in MAPK signaling was clarified as both an activating kinase and scaffold for the p38γ cascade, later extended to an AKAP-Lbc-anchored RhoA→p38 complex.\",\n      \"evidence\": \"in vitro kinase assays, co-IP of cascade components, dominant-negative expression; siRNA of AKAP-Lbc with p38 activity readouts\",\n      \"pmids\": [\"12761180\", \"21224381\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab biochemistry without structural detail of the scaffold\", \"Direct phosphorylation site on MLTK not mapped\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"PKN1 was placed in TNF-receptor signaling by showing it phosphorylates TRAF1 to drive TNFR2 recruitment and attenuate constitutive NF-κB/JNK, building on earlier TRAF2 binding.\",\n      \"evidence\": \"in vitro kinase assays, phospho-acceptor mutagenesis, RNAi, NF-κB/JNK reporter assays; earlier TRAF2 binding via PXQX(S/T) motif\",\n      \"pmids\": [\"18429822\", \"14741690\", \"16611232\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Net effect on NF-κB (positive vs negative) context-dependent across studies\", \"Physiological TNFR2 context not fully defined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Genetic models established a cardioprotective role: activated PKN1 phosphorylates αB-crystallin to stimulate proteasome activity and limit ischemic injury; loss of PKN1 worsens infarct via CamKIIδ-dependent SR calcium dysregulation.\",\n      \"evidence\": \"cardiac transgenic CA/DN PKN1, in vivo I/R, proteasome assays; PKN1 KO with CamKIIδ co-IP and phospholamban phosphorylation\",\n      \"pmids\": [\"20595653\", \"29045568\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether αB-crystallin and CamKIIδ pathways are mechanistically coupled unclear\", \"Direct CamKIIδ substrate relationship not established\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"PKN1 substrate specificity and a growth-factor receptor target were defined: a basophilic consensus (Arg at −3) and direct EGFR Thr654 phosphorylation.\",\n      \"evidence\": \"peptide library/protein array, site mutagenesis, in vitro and cellular phosphorylation assays\",\n      \"pmids\": [\"21749319\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of EGFR Thr654 phosphorylation not detailed\", \"In vivo substrate repertoire incompletely defined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"A pathogen co-opts PKN1: the Salmonella SspH1 LRR binds the HR1b subdomain, activating its E3 ligase to ubiquitinate and degrade PKN1, with consequences for androgen receptor signaling.\",\n      \"evidence\": \"X-ray structure of SspH1–PKN1, in-cell ubiquitination and proteasome inhibitor experiments, AR reporter assay\",\n      \"pmids\": [\"24248594\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Host pathway controlled by PKN1 turnover during infection not fully mapped\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"PKN1's role in trafficking and immune-cell function was established: it phosphorylates RPH3A to direct polarized RAB21 vesicle trafficking and integrin activation in neutrophils, and its kinase activity is required for lymphocyte egress.\",\n      \"evidence\": \"in vitro kinase/binding assays, GTP-RAB21 pulldown, myeloid conditional KO with renal I/R; T778A kinase-dead knock-in with chemotaxis and cell-transfer assays\",\n      \"pmids\": [\"28636945\", \"28794483\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream GTPase driving the RPH3A axis not fully defined\", \"S1P-receptor coupling to PKN1 activation unresolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"An additional layer of regulation was identified—TORC2-dependent turn-motif phosphorylation tunes PKN1 activity and supports prostate cancer cell motility.\",\n      \"evidence\": \"torin inhibition, phospho-TM antibody, TM mutagenesis, in vitro kinase and motility assays\",\n      \"pmids\": [\"28875501\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct TORC2-PKN1 contact not shown\", \"Single-lab functional readout\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Neuronal functions were uncovered: a PKN1a splice isoform promotes synaptic maturation via EAAT3, and PKN1 negatively regulates an AKT–NeuroD2–GluA1 axis controlling AMPA receptor levels.\",\n      \"evidence\": \"PKN1a-specific and Pkn1 KO mice, electrophysiology, glutamate uptake, subcellular fractionation, flow cytometry\",\n      \"pmids\": [\"33244074\", \"33613259\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct PKN1 substrates in the AKT/NeuroD2 axis not identified\", \"Isoform-specific mechanism incompletely resolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"A mechanotransduction-to-autophagy link was established: overload-activated RhoA–PKN phosphorylates KRT8 Ser43 to block RAB33B Golgi trafficking and suppress autophagosome initiation, contributing to disc degeneration.\",\n      \"evidence\": \"conditional Krt8 KO, PKN1/2 knockdown, phospho-Ser43 detection, trafficking and autophagy assays, in vivo disc models\",\n      \"pmids\": [\"36897022\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contributions of PKN1 vs PKN2 not separated\", \"Generality beyond disc tissue untested\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Biophysical work refined the activation model, showing HR1a dimerization and distinct stoichiometries for RhoA (1:2, with dimer rearrangement) versus Rac1 (monomeric), implying GTPase-specific activation mechanisms.\",\n      \"evidence\": \"SEC, AUC, SAXS, integrative structural modeling of HR1 domains with RhoA/Rac1\",\n      \"pmids\": [\"42036044\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length kinase conformational coupling not captured\", \"Functional consequence of RhoA- vs Rac1-specific geometry in cells untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"A membrane-independent regulatory input was proposed whereby alkali metal ions reversibly modulate PKN1 activation-loop phosphorylation via phosphate reacquisition.\",\n      \"evidence\": \"cell-free lysate phosphorylation assays with defined ion concentrations, PDK1-depleted lysate, 32P tracing (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.09.04.674365\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single unreplicated preprint, not peer-reviewed\", \"Physiological relevance and in-cell occurrence not established\", \"Molecular basis of ion-dependent dephosphorylation unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How GTPase binding, lipid/C2-domain engagement, autoinhibition release, and activation-loop/turn-motif phosphorylation are integrated into a single dynamic activation cycle for full-length PKN1 in cells remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No full-length structure coupling regulatory and catalytic domains\", \"Hierarchy and timing of activating inputs in vivo undefined\", \"Substrate selection rules across compartments not unified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [11, 12, 15, 19, 24, 26, 32]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [2, 3, 9, 24]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [2, 3, 9]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 4, 34]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [5, 6, 7]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [2, 48]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [10, 15]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [28]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 14, 22, 38]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [12, 15, 16]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [32]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [19, 26, 29]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [26, 32]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [8, 21]}\n    ],\n    \"complexes\": [\n      \"AKAP-Lbc signaling complex (PKNα–MLTK–MKK3–p38α)\"\n    ],\n    \"partners\": [\n      \"RhoA\",\n      \"Rac1\",\n      \"ACTN (alpha-actinin)\",\n      \"PLD1\",\n      \"TRAF2\",\n      \"CAMK2D\",\n      \"RPH3A\",\n      \"AKAP13\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}