{"gene":"PRKX","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":1999,"finding":"PrKX is a catalytic subunit of cAMP-dependent protein kinase that phosphorylates the heptapeptide Kemptide, binds with high affinity to regulatory subunit type I (RIα, KD=10 nM) and heat-stable protein kinase inhibitor (KD=15 nM), but not to RIIα under physiological conditions (KD=2.3 µM). RIα inhibition of PrKX is reversed by nanomolar cAMP (Ka=40 nM). Microinjection data showed that RIα but not RIIα binds PrKX in vivo, preventing nuclear translocation in the absence of cAMP. RIIα is phosphorylated by PrKX in a cAMP-independent manner.","method":"Surface plasmon resonance, in vitro kinase assay (Kemptide phosphorylation), autophosphorylation assay, microinjection with fluorescent R subunits","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal in vitro methods (SPR, kinase assay, in vivo microinjection) in one rigorous study establishing substrate, binding partners, and regulation","pmids":["10026146"],"is_preprint":false},{"year":1998,"finding":"AAV type 2 Rep78 interacts with PRKX via a C-terminal zinc finger-like domain of Rep78 (Rep68, which lacks this domain, does not interact). This interaction inhibits PRKX autophosphorylation and kinase activity. PRKX was shown to activate CREB-dependent transcription when expressed in transfected cells, and this activation was suppressed by Rep78.","method":"Yeast two-hybrid screen, pMal-Rep pull-down assay, in vitro kinase assay, transfection/reporter assay","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid confirmed by pull-down and kinase assay; functional consequence demonstrated by reporter assay in single lab","pmids":["9733829"],"is_preprint":false},{"year":1998,"finding":"AAV Rep78 and Rep52 (but not Rep68) form stable complexes with PrKX and inhibit its kinase activity; Rep52 inhibits PrKX trans- and autophosphorylation with a Ki of ~167 nM, as determined by surface plasmon resonance. Rep52 also inhibits PKA with Ki ~300 nM. Interaction was confirmed by coimmunoprecipitation in vitro and in vivo.","method":"Yeast two-hybrid, coimmunoprecipitation (in vitro and in vivo), surface plasmon resonance, in vitro kinase assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reciprocal Co-IP, SPR with kinetics, in vitro kinase assay; replicated independently across two labs (PMID 9733829 and 9742109)","pmids":["9742109"],"is_preprint":false},{"year":2002,"finding":"PRKX activates migration of cultured renal epithelial cells in the presence of cAMP (blocked by H89); this effect was not observed with PKA. PRKX also activates branching morphogenesis of MDCK cells in collagen gels even without cAMP or HGF, an effect not seen with PKA or with a kinase-inactivated PRKX mutant, establishing kinase activity as essential.","method":"Transfection/overexpression in renal epithelial cells, migration assay, MDCK branching morphogenesis assay in collagen gels, kinase-dead mutant control","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — kinase-dead mutant controls, multiple cellular phenotype assays (migration and morphogenesis), PKA comparison in same study","pmids":["12082174"],"is_preprint":false},{"year":2002,"finding":"AAV2 Rep78 inhibition of PKA and PRKX is mapped to amino acids 526–561 (for PRKX) and 526–621 (for PKA). Steady-state kinetic analysis showed Rep78 increases the Km for the peptide substrate without affecting Vmax, indicating competitive (pseudosubstrate) inhibition. Homology was detected between the Rep78 inhibitory domain and the cellular pseudosubstrate inhibitor PKI.","method":"Deletion mapping, steady-state kinetic analysis of kinase inhibition, sequence homology analysis","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — kinetic mechanism established with deletion mapping in single lab; mechanistic interpretation supported by PKI homology","pmids":["11773379"],"is_preprint":false},{"year":2003,"finding":"AAV2 Rep78/Rep52 proteins contain a PKA/PrKX inhibitory domain homologous to the heat-stable PKA inhibitor PKI. Adenovirus replication was shown to be sensitive to PKA activity, and a mutant AAV2 lacking the Rep kinase-inhibitory domain failed to inhibit adenovirus replication, establishing that PKA/PrKX inhibition by Rep is required for AAV interference with adenovirus.","method":"Mutant virus construction, adenovirus replication assay, PKA activity modulation","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutant virus with defined domain deletion, functional replication assay; mechanistic link between PRKX/PKA inhibition and viral interference established in single lab","pmids":["12660177"],"is_preprint":false},{"year":2007,"finding":"PRKX binds and phosphorylates the C-terminal tail of polycystin-1 (PKD1 gene product) at Ser-4166 in vitro. Expression of constitutively active PRKX in human ADPKD epithelial cells rescued adhesion and migration defects caused by PKD1 deficiency. Co-injection of constitutively active PRKX with inhibitory PKD1 construct into mouse embryonic kidney ureteric buds restored normal branching morphogenesis.","method":"In vitro kinase assay with polycystin-1 C-terminal peptide, transfection/rescue assay in ADPKD cell lines, embryonic kidney organ culture injection","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro phosphorylation with specific site identified, cellular rescue experiments, and organ culture rescue; single lab with multiple assay types","pmids":["17980165"],"is_preprint":false},{"year":2010,"finding":"The αH-αI loop residue Arg-283 in PrKX is crucial for its preferential interaction with RI over RII regulatory subunits. A R283L mutant of PrKX was able to form holoenzyme complexes with wild-type RII subunits. Conversely, the reciprocal L277R mutation in PKA Cα destabilized RII-containing holoenzymes and facilitated cAMP-mediated activation. This RI preference is evolutionarily conserved across PrKX-like kinases from human to Trypanosoma brucei.","method":"Bioluminescence resonance energy transfer (BRET) in living cells, surface plasmon resonance, site-directed mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis combined with two orthogonal biophysical methods (BRET and SPR) in living cells and in vitro; evolutionarily conserved finding tested across multiple species","pmids":["20819953"],"is_preprint":false},{"year":2011,"finding":"PRKX stimulates endothelial cell proliferation, migration, and vascular-like structure formation (three essential processes for angiogenesis), whereas PKA had an inhibitory effect on vascular-like structure formation. PRKX was found to interact (bind) with Pin-1, Magi-1, and Bag-3.","method":"Overexpression in endothelial cells, proliferation assay, migration assay, tube formation assay, co-immunoprecipitation/binding assay for Pin-1/Magi-1/Bag-3","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — functional assays with PKA comparison, binding partners identified; single lab, limited mechanistic depth on binding partners","pmids":["21684272"],"is_preprint":false},{"year":2014,"finding":"PRKX (together with PKACα) phosphorylates TAK1 at Ser-412, which is required for full TAK1 activation in response to proinflammatory stimuli (TNF-α, LPS, IL-1β). shRNA-based knockdown of PRKX reduced TAK1 Ser-412 phosphorylation and impaired downstream NF-κB signaling and proinflammatory cytokine induction. The corresponding site in zebrafish TAK1 (Ser-391) was shown by morpholino knockdown and rescue to play a conserved role in NF-κB activation.","method":"In vitro kinase assay, shRNA knockdown, phospho-specific immunoblot, zebrafish morpholino knockdown and rescue","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro kinase assay identifies specific phosphorylation site, shRNA knockdown with signaling readout, and in vivo zebrafish morpholino rescue with multiple orthogonal approaches","pmids":["25028512"],"is_preprint":false},{"year":2023,"finding":"In black carp, PRKX (bcPRKX) interacts with TAK1 (bcTAK1), suppresses bcTAK1/bcIRF7/IFN signaling, and promotes lysosome-dependent (not proteasome-dependent) degradation of bcTAK1. Chloroquine (lysosome inhibitor) prevented bcTAK1 degradation mediated by bcPRKX, whereas the proteasome inhibitor MG-132 did not.","method":"Co-immunoprecipitation, immunofluorescence, luciferase reporter assay, shRNA knockdown, inhibitor treatment (chloroquine vs MG-132), plaque assay","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and functional assays with specific inhibitors to distinguish lysosome vs proteasome pathway; fish ortholog, single lab","pmids":["36713382"],"is_preprint":false}],"current_model":"PRKX is a cAMP-dependent serine/threonine protein kinase (phylogenetically distinct from classical PKA) that is selectively inhibited by RI-type regulatory subunits through an αH-αI loop (Arg-283)-dependent pseudosubstrate mechanism, is activated by nanomolar cAMP, phosphorylates substrates including Kemptide, RIIα, polycystin-1 (Ser-4166), and TAK1 (Ser-412), and drives renal epithelial cell migration/morphogenesis and endothelial angiogenesis; its kinase activity is competitively inhibited by AAV Rep proteins (Rep52/Rep78) via a PKI-like pseudosubstrate domain, and it can promote lysosomal degradation of TAK1 to modulate innate immune signaling."},"narrative":{"mechanistic_narrative":"PRKX is a cAMP-dependent serine/threonine protein kinase that is structurally and regulatorily distinct from classical PKA and drives epithelial morphogenesis, cell migration, and angiogenesis [PMID:10026146, PMID:12082174, PMID:21684272]. It phosphorylates the model substrate Kemptide and is held inactive by high-affinity binding to type I regulatory subunit RIα and to the heat-stable protein kinase inhibitor PKI, while binding RIIα only weakly; this RIα-mediated inhibition is relieved by nanomolar cAMP, which also permits nuclear translocation [PMID:10026146]. Its selectivity for RI over RII regulatory subunits is dictated by the αH-αI loop residue Arg-283, since an R283L mutant gains the ability to form holoenzymes with RII, a determinant conserved across PrKX-like kinases [PMID:20819953]. Through its kinase activity PRKX promotes renal epithelial cell migration and branching morphogenesis of MDCK cells, effects that require an intact catalytic function and are not reproduced by PKA [PMID:12082174]. PRKX binds and phosphorylates the polycystin-1 C-terminal tail at Ser-4166, and constitutively active PRKX rescues adhesion, migration, and branching defects caused by PKD1 deficiency, linking the kinase to renal tubular morphogenesis [PMID:17980165]. In innate immunity, PRKX phosphorylates TAK1 at Ser-412 to enable full TAK1 activation and downstream NF-κB signaling in response to proinflammatory stimuli [PMID:25028512], and in a teleost ortholog it instead promotes lysosome-dependent degradation of TAK1 to suppress IFN signaling [PMID:36713382]. PRKX kinase activity is competitively inhibited by AAV Rep proteins (Rep78/Rep52) through a PKI-like pseudosubstrate domain that raises the substrate Km without altering Vmax, an inhibition required for AAV interference with adenovirus replication [PMID:9742109, PMID:11773379, PMID:12660177].","teleology":[{"year":1998,"claim":"Established that the viral AAV Rep proteins physically engage and inhibit PRKX, defining the first regulatory partners of the kinase and a route by which a virus could subvert cAMP signaling.","evidence":"Yeast two-hybrid, pull-down/Co-IP, in vitro kinase assay, and reporter assays with Rep78/Rep52 versus Rep68","pmids":["9733829","9742109"],"confidence":"High","gaps":["Did not resolve the precise inhibitory domain or kinetic mechanism","CREB-dependent transcriptional output not tied to specific endogenous targets"]},{"year":1999,"claim":"Defined PRKX as a bona fide cAMP-dependent kinase with distinct regulatory-subunit preference, answering how it is held inactive and activated.","evidence":"Surface plasmon resonance, in vitro Kemptide kinase assay, and microinjection of fluorescent R subunits","pmids":["10026146"],"confidence":"High","gaps":["Physiological substrates beyond Kemptide not identified","Mechanistic basis for RI versus RII selectivity not yet defined"]},{"year":2002,"claim":"Showed PRKX kinase activity drives renal epithelial migration and branching morphogenesis distinct from PKA, establishing a cellular role.","evidence":"Overexpression in renal epithelial and MDCK cells with migration and collagen-gel branching assays and a kinase-dead control","pmids":["12082174"],"confidence":"High","gaps":["Endogenous substrates mediating the morphogenetic effect not identified","Differences from PKA at the molecular level unexplained"]},{"year":2002,"claim":"Defined Rep78 inhibition as a competitive pseudosubstrate mechanism homologous to PKI, explaining how the virus blocks the kinase.","evidence":"Deletion mapping and steady-state kinetic analysis of inhibition with sequence homology analysis","pmids":["11773379"],"confidence":"Medium","gaps":["Single-lab kinetic characterization","Did not establish in vivo consequences of inhibition"]},{"year":2003,"claim":"Linked Rep-mediated PRKX/PKA inhibition to AAV interference with adenovirus replication, giving the inhibition a biological purpose.","evidence":"Mutant AAV2 lacking the Rep kinase-inhibitory domain and adenovirus replication assays","pmids":["12660177"],"confidence":"Medium","gaps":["Relative contribution of PRKX versus PKA inhibition not separated","Downstream effectors of the interference not mapped"]},{"year":2007,"claim":"Identified polycystin-1 Ser-4166 as a PRKX substrate and placed PRKX downstream/parallel to PKD1 in renal tubular morphogenesis.","evidence":"In vitro kinase assay on the polycystin-1 C-terminal peptide, ADPKD cell rescue, and embryonic kidney organ-culture injection","pmids":["17980165"],"confidence":"Medium","gaps":["In vivo phosphorylation of full-length polycystin-1 not demonstrated","Functional output of Ser-4166 phosphorylation not defined"]},{"year":2010,"claim":"Pinpointed Arg-283 in the αH-αI loop as the determinant of RI-over-RII selectivity, explaining the divergent regulation of PRKX from PKA.","evidence":"BRET in living cells, SPR, and reciprocal site-directed mutagenesis (R283L PrKX, L277R PKA Cα) tested across species","pmids":["20819953"],"confidence":"High","gaps":["Physiological significance of RI selectivity in cells not established","Structural model of the holoenzyme interface not resolved"]},{"year":2011,"claim":"Extended PRKX function to angiogenesis and named candidate protein partners, broadening its biological scope beyond renal cells.","evidence":"Endothelial proliferation, migration, and tube-formation assays with PKA comparison and binding assays for Pin-1, Magi-1, Bag-3","pmids":["21684272"],"confidence":"Medium","gaps":["Binding partners not validated by reciprocal or structural studies","No substrate connecting PRKX to the angiogenic phenotype identified"]},{"year":2014,"claim":"Identified TAK1 Ser-412 as a PRKX substrate required for NF-κB activation, embedding PRKX in proinflammatory signaling.","evidence":"In vitro kinase assay, shRNA knockdown with phospho-specific immunoblot, and zebrafish morpholino knockdown/rescue","pmids":["25028512"],"confidence":"High","gaps":["Stimulus-specific regulation of PRKX upstream of TAK1 not defined","Shared activity with PKACα not fully deconvoluted"]},{"year":2023,"claim":"Revealed an opposing, lysosome-dependent mechanism in which a PRKX ortholog degrades TAK1 to suppress IFN signaling, indicating context-dependent regulation of TAK1.","evidence":"Co-IP, immunofluorescence, luciferase reporter, shRNA knockdown, and chloroquine versus MG-132 inhibitor treatment in black carp","pmids":["36713382"],"confidence":"Medium","gaps":["Demonstrated in fish ortholog only","Mechanism reconciling activation versus degradation of TAK1 not resolved"]},{"year":null,"claim":"How PRKX selects among its substrates in different tissues and how its activation versus degradation of TAK1 is controlled remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of substrate recognition","Upstream signals controlling PRKX activation in vivo unknown","Reconciliation of pro-inflammatory and IFN-suppressive roles unestablished"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,6,9]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,3,6,9]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,3]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[9,10]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[3,6,8]}],"complexes":[],"partners":["PRKAR1A","PKIA","REP78","REP52","PKD1","TAK1","PIN1","MAGI1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P51817","full_name":"cAMP-dependent protein kinase catalytic subunit PRKX","aliases":["Protein kinase PKX1"],"length_aa":358,"mass_kda":40.9,"function":"Serine/threonine protein kinase regulated by and mediating cAMP signaling in cells. Acts through phosphorylation of downstream targets that may include CREB, SMAD6 and PKD1 and has multiple functions in cellular differentiation and epithelial morphogenesis. Regulates myeloid cell differentiation through SMAD6 phosphorylation. Involved in nephrogenesis by stimulating renal epithelial cell migration and tubulogenesis. Also involved in angiogenesis through stimulation of endothelial cell proliferation, migration and vascular-like structure formation","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/P51817/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PRKX","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PRKX","total_profiled":1310},"omim":[{"mim_id":"400008","title":"PROTEIN KINASE, Y-LINKED; PRKY","url":"https://www.omim.org/entry/400008"},{"mim_id":"300406","title":"FG SYNDROME 3; FGS3","url":"https://www.omim.org/entry/300406"},{"mim_id":"300083","title":"PROTEIN KINASE, X-LINKED; PRKX","url":"https://www.omim.org/entry/300083"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"thyroid gland","ntpm":31.6}],"url":"https://www.proteinatlas.org/search/PRKX"},"hgnc":{"alias_symbol":["PKX1"],"prev_symbol":[]},"alphafold":{"accession":"P51817","domains":[{"cath_id":"3.30.200.20","chopping":"45-128_331-352","consensus_level":"high","plddt":93.3944,"start":45,"end":352},{"cath_id":"1.10.510.10","chopping":"133-312","consensus_level":"high","plddt":96.6404,"start":133,"end":312}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P51817","model_url":"https://alphafold.ebi.ac.uk/files/AF-P51817-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P51817-F1-predicted_aligned_error_v6.png","plddt_mean":88.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PRKX","jax_strain_url":"https://www.jax.org/strain/search?query=PRKX"},"sequence":{"accession":"P51817","fasta_url":"https://rest.uniprot.org/uniprotkb/P51817.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P51817/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P51817"}},"corpus_meta":[{"pmid":"9302280","id":"PMC_9302280","title":"Abnormal XY interchange between a novel isolated protein kinase gene, PRKY, and its homologue, PRKX, accounts for one third of all (Y+)XX males and (Y-)XY females.","date":"1997","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/9302280","citation_count":97,"is_preprint":false},{"pmid":"10026146","id":"PMC_10026146","title":"PrKX is a novel catalytic subunit of the cAMP-dependent protein kinase regulated by the regulatory subunit type I.","date":"1999","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10026146","citation_count":82,"is_preprint":false},{"pmid":"9733829","id":"PMC_9733829","title":"Adeno-associated virus Rep78 protein interacts with protein kinase A and its homolog PRKX and inhibits CREB-dependent transcriptional activation.","date":"1998","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/9733829","citation_count":56,"is_preprint":false},{"pmid":"25028512","id":"PMC_25028512","title":"Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) activation requires phosphorylation of serine 412 by protein kinase A catalytic subunit α (PKACα) and X-linked protein kinase (PRKX).","date":"2014","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/25028512","citation_count":53,"is_preprint":false},{"pmid":"22020623","id":"PMC_22020623","title":"PRKX, TTBK2 and RSK4 expression causes Sunitinib resistance in kidney carcinoma- and melanoma-cell lines.","date":"2012","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/22020623","citation_count":52,"is_preprint":false},{"pmid":"7633447","id":"PMC_7633447","title":"The human protein kinase gene PKX1 on Xp22.3 displays Xp/Yp homology and is a site of chromosomal instability.","date":"1995","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/7633447","citation_count":50,"is_preprint":false},{"pmid":"9742109","id":"PMC_9742109","title":"Inhibition of PrKX, a novel protein kinase, and the cyclic AMP-dependent protein kinase PKA by the regulatory proteins of adeno-associated virus type 2.","date":"1998","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/9742109","citation_count":48,"is_preprint":false},{"pmid":"12660177","id":"PMC_12660177","title":"PKA/PrKX activity is a modulator of AAV/adenovirus interaction.","date":"2003","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/12660177","citation_count":45,"is_preprint":false},{"pmid":"12082174","id":"PMC_12082174","title":"PRKX, a phylogenetically and functionally distinct cAMP-dependent protein kinase, activates renal epithelial cell migration and morphogenesis.","date":"2002","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/12082174","citation_count":43,"is_preprint":false},{"pmid":"26252946","id":"PMC_26252946","title":"PRKX, a Novel cAMP-Dependent Protein Kinase Member, Plays an Important Role in Development.","date":"2016","source":"Journal of cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/26252946","citation_count":23,"is_preprint":false},{"pmid":"21684272","id":"PMC_21684272","title":"PRKX critically regulates endothelial cell proliferation, migration, and vascular-like structure formation.","date":"2011","source":"Developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/21684272","citation_count":23,"is_preprint":false},{"pmid":"20819953","id":"PMC_20819953","title":"Regulation of cAMP-dependent protein kinases: the human protein kinase X (PrKX) reveals the role of the catalytic subunit alphaH-alphaI loop.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20819953","citation_count":21,"is_preprint":false},{"pmid":"17980165","id":"PMC_17980165","title":"Protein kinase X (PRKX) can rescue the effects of polycystic kidney disease-1 gene (PKD1) deficiency.","date":"2007","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/17980165","citation_count":18,"is_preprint":false},{"pmid":"15879576","id":"PMC_15879576","title":"Profiles of PrKX expression in developmental mouse embryo and human tissues.","date":"2005","source":"The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society","url":"https://pubmed.ncbi.nlm.nih.gov/15879576","citation_count":16,"is_preprint":false},{"pmid":"9154127","id":"PMC_9154127","title":"FISH localization of the human Y-homolog of protein kinase PRKX (PRKY) to Yp11.2 and two pseudogenes to 15q26 and Xq12-->q13.","date":"1997","source":"Cytogenetics and cell genetics","url":"https://pubmed.ncbi.nlm.nih.gov/9154127","citation_count":16,"is_preprint":false},{"pmid":"11773379","id":"PMC_11773379","title":"Adeno-associated virus type 2 Rep78 inhibition of PKA and PRKX: fine mapping and analysis of mechanism.","date":"2002","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/11773379","citation_count":14,"is_preprint":false},{"pmid":"34791059","id":"PMC_34791059","title":"Long non-coding RNA NRSN2-AS1 facilitates tumorigenesis and progression of ovarian cancer via miR-744-5p/PRKX axis.","date":"2022","source":"Biology of reproduction","url":"https://pubmed.ncbi.nlm.nih.gov/34791059","citation_count":13,"is_preprint":false},{"pmid":"27630294","id":"PMC_27630294","title":"Altered Expression of PRKX, WNT3 and WNT16 in Human Nodular Basal Cell Carcinoma.","date":"2016","source":"Anticancer research","url":"https://pubmed.ncbi.nlm.nih.gov/27630294","citation_count":11,"is_preprint":false},{"pmid":"24088663","id":"PMC_24088663","title":"Critical role of Yp inversion in PRKX/PRKY-mediated Xp;Yp translocation in a patient with 45,X testicular disorder of sex development.","date":"2013","source":"Endocrine journal","url":"https://pubmed.ncbi.nlm.nih.gov/24088663","citation_count":7,"is_preprint":false},{"pmid":"36713382","id":"PMC_36713382","title":"PRKX down-regulates TAK1/IRF7 signaling in the antiviral innate immunity of black carp Mylopharyngodon piceus.","date":"2023","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/36713382","citation_count":6,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11571,"output_tokens":3239,"usd":0.041649,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10725,"output_tokens":3401,"usd":0.069325,"stage2_stop_reason":"end_turn"},"total_usd":0.110974,"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\": 1999,\n      \"finding\": \"PrKX is a catalytic subunit of cAMP-dependent protein kinase that phosphorylates the heptapeptide Kemptide, binds with high affinity to regulatory subunit type I (RIα, KD=10 nM) and heat-stable protein kinase inhibitor (KD=15 nM), but not to RIIα under physiological conditions (KD=2.3 µM). RIα inhibition of PrKX is reversed by nanomolar cAMP (Ka=40 nM). Microinjection data showed that RIα but not RIIα binds PrKX in vivo, preventing nuclear translocation in the absence of cAMP. RIIα is phosphorylated by PrKX in a cAMP-independent manner.\",\n      \"method\": \"Surface plasmon resonance, in vitro kinase assay (Kemptide phosphorylation), autophosphorylation assay, microinjection with fluorescent R subunits\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal in vitro methods (SPR, kinase assay, in vivo microinjection) in one rigorous study establishing substrate, binding partners, and regulation\",\n      \"pmids\": [\"10026146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"AAV type 2 Rep78 interacts with PRKX via a C-terminal zinc finger-like domain of Rep78 (Rep68, which lacks this domain, does not interact). This interaction inhibits PRKX autophosphorylation and kinase activity. PRKX was shown to activate CREB-dependent transcription when expressed in transfected cells, and this activation was suppressed by Rep78.\",\n      \"method\": \"Yeast two-hybrid screen, pMal-Rep pull-down assay, in vitro kinase assay, transfection/reporter assay\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid confirmed by pull-down and kinase assay; functional consequence demonstrated by reporter assay in single lab\",\n      \"pmids\": [\"9733829\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"AAV Rep78 and Rep52 (but not Rep68) form stable complexes with PrKX and inhibit its kinase activity; Rep52 inhibits PrKX trans- and autophosphorylation with a Ki of ~167 nM, as determined by surface plasmon resonance. Rep52 also inhibits PKA with Ki ~300 nM. Interaction was confirmed by coimmunoprecipitation in vitro and in vivo.\",\n      \"method\": \"Yeast two-hybrid, coimmunoprecipitation (in vitro and in vivo), surface plasmon resonance, in vitro kinase assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reciprocal Co-IP, SPR with kinetics, in vitro kinase assay; replicated independently across two labs (PMID 9733829 and 9742109)\",\n      \"pmids\": [\"9742109\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"PRKX activates migration of cultured renal epithelial cells in the presence of cAMP (blocked by H89); this effect was not observed with PKA. PRKX also activates branching morphogenesis of MDCK cells in collagen gels even without cAMP or HGF, an effect not seen with PKA or with a kinase-inactivated PRKX mutant, establishing kinase activity as essential.\",\n      \"method\": \"Transfection/overexpression in renal epithelial cells, migration assay, MDCK branching morphogenesis assay in collagen gels, kinase-dead mutant control\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — kinase-dead mutant controls, multiple cellular phenotype assays (migration and morphogenesis), PKA comparison in same study\",\n      \"pmids\": [\"12082174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"AAV2 Rep78 inhibition of PKA and PRKX is mapped to amino acids 526–561 (for PRKX) and 526–621 (for PKA). Steady-state kinetic analysis showed Rep78 increases the Km for the peptide substrate without affecting Vmax, indicating competitive (pseudosubstrate) inhibition. Homology was detected between the Rep78 inhibitory domain and the cellular pseudosubstrate inhibitor PKI.\",\n      \"method\": \"Deletion mapping, steady-state kinetic analysis of kinase inhibition, sequence homology analysis\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — kinetic mechanism established with deletion mapping in single lab; mechanistic interpretation supported by PKI homology\",\n      \"pmids\": [\"11773379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"AAV2 Rep78/Rep52 proteins contain a PKA/PrKX inhibitory domain homologous to the heat-stable PKA inhibitor PKI. Adenovirus replication was shown to be sensitive to PKA activity, and a mutant AAV2 lacking the Rep kinase-inhibitory domain failed to inhibit adenovirus replication, establishing that PKA/PrKX inhibition by Rep is required for AAV interference with adenovirus.\",\n      \"method\": \"Mutant virus construction, adenovirus replication assay, PKA activity modulation\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutant virus with defined domain deletion, functional replication assay; mechanistic link between PRKX/PKA inhibition and viral interference established in single lab\",\n      \"pmids\": [\"12660177\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"PRKX binds and phosphorylates the C-terminal tail of polycystin-1 (PKD1 gene product) at Ser-4166 in vitro. Expression of constitutively active PRKX in human ADPKD epithelial cells rescued adhesion and migration defects caused by PKD1 deficiency. Co-injection of constitutively active PRKX with inhibitory PKD1 construct into mouse embryonic kidney ureteric buds restored normal branching morphogenesis.\",\n      \"method\": \"In vitro kinase assay with polycystin-1 C-terminal peptide, transfection/rescue assay in ADPKD cell lines, embryonic kidney organ culture injection\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro phosphorylation with specific site identified, cellular rescue experiments, and organ culture rescue; single lab with multiple assay types\",\n      \"pmids\": [\"17980165\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The αH-αI loop residue Arg-283 in PrKX is crucial for its preferential interaction with RI over RII regulatory subunits. A R283L mutant of PrKX was able to form holoenzyme complexes with wild-type RII subunits. Conversely, the reciprocal L277R mutation in PKA Cα destabilized RII-containing holoenzymes and facilitated cAMP-mediated activation. This RI preference is evolutionarily conserved across PrKX-like kinases from human to Trypanosoma brucei.\",\n      \"method\": \"Bioluminescence resonance energy transfer (BRET) in living cells, surface plasmon resonance, site-directed mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis combined with two orthogonal biophysical methods (BRET and SPR) in living cells and in vitro; evolutionarily conserved finding tested across multiple species\",\n      \"pmids\": [\"20819953\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PRKX stimulates endothelial cell proliferation, migration, and vascular-like structure formation (three essential processes for angiogenesis), whereas PKA had an inhibitory effect on vascular-like structure formation. PRKX was found to interact (bind) with Pin-1, Magi-1, and Bag-3.\",\n      \"method\": \"Overexpression in endothelial cells, proliferation assay, migration assay, tube formation assay, co-immunoprecipitation/binding assay for Pin-1/Magi-1/Bag-3\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — functional assays with PKA comparison, binding partners identified; single lab, limited mechanistic depth on binding partners\",\n      \"pmids\": [\"21684272\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PRKX (together with PKACα) phosphorylates TAK1 at Ser-412, which is required for full TAK1 activation in response to proinflammatory stimuli (TNF-α, LPS, IL-1β). shRNA-based knockdown of PRKX reduced TAK1 Ser-412 phosphorylation and impaired downstream NF-κB signaling and proinflammatory cytokine induction. The corresponding site in zebrafish TAK1 (Ser-391) was shown by morpholino knockdown and rescue to play a conserved role in NF-κB activation.\",\n      \"method\": \"In vitro kinase assay, shRNA knockdown, phospho-specific immunoblot, zebrafish morpholino knockdown and rescue\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro kinase assay identifies specific phosphorylation site, shRNA knockdown with signaling readout, and in vivo zebrafish morpholino rescue with multiple orthogonal approaches\",\n      \"pmids\": [\"25028512\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In black carp, PRKX (bcPRKX) interacts with TAK1 (bcTAK1), suppresses bcTAK1/bcIRF7/IFN signaling, and promotes lysosome-dependent (not proteasome-dependent) degradation of bcTAK1. Chloroquine (lysosome inhibitor) prevented bcTAK1 degradation mediated by bcPRKX, whereas the proteasome inhibitor MG-132 did not.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence, luciferase reporter assay, shRNA knockdown, inhibitor treatment (chloroquine vs MG-132), plaque assay\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and functional assays with specific inhibitors to distinguish lysosome vs proteasome pathway; fish ortholog, single lab\",\n      \"pmids\": [\"36713382\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PRKX is a cAMP-dependent serine/threonine protein kinase (phylogenetically distinct from classical PKA) that is selectively inhibited by RI-type regulatory subunits through an αH-αI loop (Arg-283)-dependent pseudosubstrate mechanism, is activated by nanomolar cAMP, phosphorylates substrates including Kemptide, RIIα, polycystin-1 (Ser-4166), and TAK1 (Ser-412), and drives renal epithelial cell migration/morphogenesis and endothelial angiogenesis; its kinase activity is competitively inhibited by AAV Rep proteins (Rep52/Rep78) via a PKI-like pseudosubstrate domain, and it can promote lysosomal degradation of TAK1 to modulate innate immune signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PRKX is a cAMP-dependent serine/threonine protein kinase that is structurally and regulatorily distinct from classical PKA and drives epithelial morphogenesis, cell migration, and angiogenesis [#0, #3, #8]. It phosphorylates the model substrate Kemptide and is held inactive by high-affinity binding to type I regulatory subunit RIα and to the heat-stable protein kinase inhibitor PKI, while binding RIIα only weakly; this RIα-mediated inhibition is relieved by nanomolar cAMP, which also permits nuclear translocation [#0]. Its selectivity for RI over RII regulatory subunits is dictated by the αH-αI loop residue Arg-283, since an R283L mutant gains the ability to form holoenzymes with RII, a determinant conserved across PrKX-like kinases [#7]. Through its kinase activity PRKX promotes renal epithelial cell migration and branching morphogenesis of MDCK cells, effects that require an intact catalytic function and are not reproduced by PKA [#3]. PRKX binds and phosphorylates the polycystin-1 C-terminal tail at Ser-4166, and constitutively active PRKX rescues adhesion, migration, and branching defects caused by PKD1 deficiency, linking the kinase to renal tubular morphogenesis [#6]. In innate immunity, PRKX phosphorylates TAK1 at Ser-412 to enable full TAK1 activation and downstream NF-κB signaling in response to proinflammatory stimuli [#9], and in a teleost ortholog it instead promotes lysosome-dependent degradation of TAK1 to suppress IFN signaling [#10]. PRKX kinase activity is competitively inhibited by AAV Rep proteins (Rep78/Rep52) through a PKI-like pseudosubstrate domain that raises the substrate Km without altering Vmax, an inhibition required for AAV interference with adenovirus replication [#2, #4, #5].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established that the viral AAV Rep proteins physically engage and inhibit PRKX, defining the first regulatory partners of the kinase and a route by which a virus could subvert cAMP signaling.\",\n      \"evidence\": \"Yeast two-hybrid, pull-down/Co-IP, in vitro kinase assay, and reporter assays with Rep78/Rep52 versus Rep68\",\n      \"pmids\": [\"9733829\", \"9742109\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the precise inhibitory domain or kinetic mechanism\", \"CREB-dependent transcriptional output not tied to specific endogenous targets\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Defined PRKX as a bona fide cAMP-dependent kinase with distinct regulatory-subunit preference, answering how it is held inactive and activated.\",\n      \"evidence\": \"Surface plasmon resonance, in vitro Kemptide kinase assay, and microinjection of fluorescent R subunits\",\n      \"pmids\": [\"10026146\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological substrates beyond Kemptide not identified\", \"Mechanistic basis for RI versus RII selectivity not yet defined\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Showed PRKX kinase activity drives renal epithelial migration and branching morphogenesis distinct from PKA, establishing a cellular role.\",\n      \"evidence\": \"Overexpression in renal epithelial and MDCK cells with migration and collagen-gel branching assays and a kinase-dead control\",\n      \"pmids\": [\"12082174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous substrates mediating the morphogenetic effect not identified\", \"Differences from PKA at the molecular level unexplained\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined Rep78 inhibition as a competitive pseudosubstrate mechanism homologous to PKI, explaining how the virus blocks the kinase.\",\n      \"evidence\": \"Deletion mapping and steady-state kinetic analysis of inhibition with sequence homology analysis\",\n      \"pmids\": [\"11773379\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab kinetic characterization\", \"Did not establish in vivo consequences of inhibition\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Linked Rep-mediated PRKX/PKA inhibition to AAV interference with adenovirus replication, giving the inhibition a biological purpose.\",\n      \"evidence\": \"Mutant AAV2 lacking the Rep kinase-inhibitory domain and adenovirus replication assays\",\n      \"pmids\": [\"12660177\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative contribution of PRKX versus PKA inhibition not separated\", \"Downstream effectors of the interference not mapped\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identified polycystin-1 Ser-4166 as a PRKX substrate and placed PRKX downstream/parallel to PKD1 in renal tubular morphogenesis.\",\n      \"evidence\": \"In vitro kinase assay on the polycystin-1 C-terminal peptide, ADPKD cell rescue, and embryonic kidney organ-culture injection\",\n      \"pmids\": [\"17980165\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo phosphorylation of full-length polycystin-1 not demonstrated\", \"Functional output of Ser-4166 phosphorylation not defined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Pinpointed Arg-283 in the αH-αI loop as the determinant of RI-over-RII selectivity, explaining the divergent regulation of PRKX from PKA.\",\n      \"evidence\": \"BRET in living cells, SPR, and reciprocal site-directed mutagenesis (R283L PrKX, L277R PKA Cα) tested across species\",\n      \"pmids\": [\"20819953\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological significance of RI selectivity in cells not established\", \"Structural model of the holoenzyme interface not resolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Extended PRKX function to angiogenesis and named candidate protein partners, broadening its biological scope beyond renal cells.\",\n      \"evidence\": \"Endothelial proliferation, migration, and tube-formation assays with PKA comparison and binding assays for Pin-1, Magi-1, Bag-3\",\n      \"pmids\": [\"21684272\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding partners not validated by reciprocal or structural studies\", \"No substrate connecting PRKX to the angiogenic phenotype identified\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified TAK1 Ser-412 as a PRKX substrate required for NF-κB activation, embedding PRKX in proinflammatory signaling.\",\n      \"evidence\": \"In vitro kinase assay, shRNA knockdown with phospho-specific immunoblot, and zebrafish morpholino knockdown/rescue\",\n      \"pmids\": [\"25028512\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stimulus-specific regulation of PRKX upstream of TAK1 not defined\", \"Shared activity with PKACα not fully deconvoluted\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed an opposing, lysosome-dependent mechanism in which a PRKX ortholog degrades TAK1 to suppress IFN signaling, indicating context-dependent regulation of TAK1.\",\n      \"evidence\": \"Co-IP, immunofluorescence, luciferase reporter, shRNA knockdown, and chloroquine versus MG-132 inhibitor treatment in black carp\",\n      \"pmids\": [\"36713382\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Demonstrated in fish ortholog only\", \"Mechanism reconciling activation versus degradation of TAK1 not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PRKX selects among its substrates in different tissues and how its activation versus degradation of TAK1 is controlled remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of substrate recognition\", \"Upstream signals controlling PRKX activation in vivo unknown\", \"Reconciliation of pro-inflammatory and IFN-suppressive roles unestablished\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 6, 9]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 3, 6, 9]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [9, 10]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [3, 6, 8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"PRKAR1A\", \"PKIA\", \"REP78\", \"REP52\", \"PKD1\", \"TAK1\", \"PIN1\", \"MAGI1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":7,"faith_pct":85.71428571428571}}