Affinage

PKN3

Serine/threonine-protein kinase N3 · UniProt Q6P5Z2

Length
889 aa
Mass
99.4 kDa
Annotated
2026-06-10
15 papers in source corpus 11 papers cited in narrative 11 extracted findings
Cross-family judge faithfulness: 6/6 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

PKN3 is an AGC-family serine/threonine protein kinase that functions as a Rho GTPase effector coupling Rho signaling to actin cytoskeletal dynamics, invasive growth, and tissue remodeling (PMID:10441506, PMID:22217540). It interacts physically with Rho-family GTPases and preferentially associates with RhoC, an interaction that stimulates its catalytic activity and drives malignant growth and invasiveness (PMID:22217540). PKN3 expression and catalytic activity are both regulated downstream of activated PI3K, and PKN3 is required for invasive prostate tumor cell growth in 3D culture and orthotopic models (PMID:15282551). Through a proline-rich linker bearing class II SH3-binding motifs, PKN3 binds and phosphorylates the RhoGAP proteins Graf and Graf2 (PMID:10441506, PMID:11432776) and the adaptor p130Cas, with which it colocalizes in pro-invasive structures to promote growth and invasion (PMID:30422386). It also phosphorylates ARHGAP18, enhancing its GAP activity toward active RhoA and thereby establishing a negative-feedback loop within Rho signaling (PMID:33092266). Beyond tumor cells, PKN3 governs endothelial actin stress fiber formation and adherens junction integrity and is needed for glycoprotein maturation, angiogenesis, and melanoma metastasis (PMID:22609186, PMID:26742562), and acts in a Wnt5a-Ror2-Daam2-Rho axis where it binds and activates c-Src to support osteoclast bone-resorbing activity (PMID:31400545).

Mechanistic history

Synthesis pass · year-by-year structured walk · 11 steps
  1. 1999 Medium

    Establishing PKN3 as a distinct PKN isoform with intrinsic kinase activity and a defined subcellular address answered whether it was a functional kinase separate from other PKN family members.

    Evidence Recombinant expression in COS7 cells with autophosphorylation/peptide kinase assays and immunolocalization in NIH3T3 cells

    PMID:10441506

    Open questions at the time
    • No physiological substrate identified
    • Upstream activator of catalytic activity not defined
    • Function of the proline-rich motifs not yet tested
  2. 2001 High

    Identifying Graf and Graf2 as binding partners and substrates connected PKN3's proline-rich linker to Rho-GAP signaling, the first mechanistic link to Rho-mediated pathways.

    Evidence Yeast two-hybrid, direct SH3-domain pulldown, Co-IP in COS-7 cells, and in vitro kinase assay

    PMID:11432776

    Open questions at the time
    • Functional consequence of Graf/Graf2 phosphorylation on RhoGAP activity not measured
    • Cellular phenotype of the interaction not established
  3. 2004 High

    Placing PKN3 downstream of PI3K and demonstrating its requirement for invasive growth defined its disease-relevant role in tumor progression.

    Evidence Expression profiling, inducible shRNA knockdown in 3D culture and orthotopic mouse tumor model, catalytic activity measurement

    PMID:15282551

    Open questions at the time
    • Direct PI3K-to-PKN3 signaling intermediates not mapped
    • Substrates mediating the invasive phenotype not identified
  4. 2011 Medium

    Demonstrating preferential RhoC association and Rho-dependent catalytic activation identified the GTPase that activates PKN3 and links it to invasiveness.

    Evidence Reciprocal Co-IP, catalytic activity assay, breast cancer overexpression, and orthotopic knockdown models

    PMID:22217540

    Open questions at the time
    • Structural basis of RhoC selectivity over other Rho GTPases not defined
    • Single lab
  5. 2012 Medium

    Showing that PKN3 loss disrupts endothelial actin and adherens junctions extended its role beyond tumor cells to vascular cell biology.

    Evidence RNAi knockdown in HUVEC with actin/AJ immunofluorescence, TNF-α stimulation, ICAM-1 and Pyk2 readouts

    PMID:22609186

    Open questions at the time
    • Direct substrates in endothelial cells not identified
    • Mechanism linking ICAM-1 signaling to actin dynamics unresolved
  6. 2016 High

    Genetic knockout revealed PKN3's requirement for angiogenesis and metastasis and traced these to a glycoprotein maturation defect, providing a unifying cellular mechanism.

    Evidence PKN3 knockout mice, aortic ring and corneal pocket assays, tail-vein melanoma model, siRNA knockdown, cell-surface glycoprotein analysis

    PMID:26742562

    Open questions at the time
    • How PKN3 kinase activity controls glycosylation machinery not defined
    • Direct Golgi substrates unknown
  7. 2018 High

    Identifying p130Cas as a direct binding partner and substrate established PKN3 as the first Ser/Thr kinase for p130Cas and linked it to pro-invasive structures independent of Src transformation.

    Evidence Co-IP, SH3-polyproline binding assay, in vitro phosphorylation, colocalization, and MEF loss-of-function

    PMID:30422386

    Open questions at the time
    • Phosphosites on p130Cas and their downstream signaling consequences not mapped
    • In vivo relevance not tested
  8. 2019 High

    Placing PKN3 in a Wnt5a-Ror2-Daam2-Rho axis and showing it activates c-Src defined its non-canonical Wnt-dependent role in osteoclast bone resorption.

    Evidence Pkn3 knockout mice, Co-IP with c-Src and Pyk2, c-Src kinase activity assay, osteoclast differentiation and bone histomorphometry

    PMID:31400545

    Open questions at the time
    • Whether PKN3 directly phosphorylates c-Src not established
    • Molecular basis of Wnt5a-Ror2-dependent binding unresolved
  9. 2020 High

    Identifying ARHGAP18 as a substrate whose phosphorylation enhances RhoGAP activity revealed a negative-feedback architecture in PKN3-Rho signaling.

    Evidence Analog-sensitive PKN3 phosphoproteomics, in vitro kinase assay, Co-IP, and RhoA GAP activity assay

    PMID:33092266

    Open questions at the time
    • In vivo contribution of this feedback loop to PKN3 phenotypes not tested
    • Phosphosite-resolved mechanism of GAP enhancement not defined
  10. 2022 Medium

    Discovery of a potent 4-anilinoquinoline inhibitor provided a chemical tool to interrogate PKN3 biology.

    Evidence In vitro kinase inhibition (IC50 = 14 nM) and cell-based activity assay

    PMID:35403825

    Open questions at the time
    • Selectivity across the kinome not characterized
    • Limited cellular potency
  11. 2026 Medium

    Exploiting a unique threonine gatekeeper residue enabled a highly selective bisubstrate inhibitor, addressing the need for specific PKN3 chemical probes.

    Evidence Structure-guided design, fluorescence binding assay (KD = 0.2 nM), selectivity profiling against 397 kinases

    PMID:41752364

    Open questions at the time
    • Cellular and in vivo efficacy of ARC-2603 not reported
    • Single study

Open questions

Synthesis pass · forward-looking unresolved questions
  • How PKN3's distinct substrate set (Graf, p130Cas, ARHGAP18, c-Src axis) is integrated into a single coherent control of invasion, glycoprotein maturation, and Rho feedback in specific tissue contexts remains unresolved.
  • No structural model of PKN3 regulation by Rho GTPases
  • Phosphosite-resolved substrate map incomplete
  • Mechanism connecting kinase activity to Golgi glycoprotein maturation unknown

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0016740 transferase activity 4 GO:0140096 catalytic activity, acting on a protein 4 GO:0098772 molecular function regulator activity 2
Localization
GO:0005634 nucleus 1 GO:0005794 Golgi apparatus 1
Pathway
R-HSA-162582 Signal Transduction 4 R-HSA-1643685 Disease 3
Partners
ARHGAP10ARHGAP18ARHGAP26BCAR1PTK2BRHOCSRC

Evidence

Reading pass · 11 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1999 PKN3 (PKNbeta) is a novel isoform of PKN with distinct structural features: proline-rich sequences (class II SH3-binding motifs PXXPXR) in the region N-terminal to the catalytic domain. Recombinant PKN3 expressed in COS7 cells displays autophosphorylation and peptide kinase activity, but is significantly less responsive to arachidonic acid than PKNalpha. Immunochemical analysis localized PKN3 to the nucleus and perinuclear Golgi apparatus, largely absent from the cytoplasm in NIH3T3 cells. Recombinant expression in COS7 cells, autophosphorylation assay, peptide kinase assay, arachidonic acid stimulation, immunofluorescence/immunochemical localization Biochemical and biophysical research communications Medium 10441506
2001 PKN3 (PKNbeta) physically interacts with the SH3 domains of Graf and Graf2 (RhoGAP proteins) via its proline-rich linker region. The SH3 domains of Graf and Graf2 bind directly to PKN3 in vitro (pulldown with purified proteins) and co-immunoprecipitate with PKN3 in COS-7 cells. The catalytically active form of PKN3 phosphorylates Graf and Graf2 in vitro, implicating PKN3 in Rho-mediated signaling via these GAP proteins. Yeast two-hybrid screening, direct pulldown with purified E. coli-expressed SH3 domains, co-immunoprecipitation in COS-7 cells, in vitro kinase assay Journal of biochemistry High 11432776
2004 PKN3 is regulated downstream of activated PI3K at both the expression level and the catalytic activity level. PKN3 is required for invasive prostate cell growth, as demonstrated by shRNA-mediated knockdown in 3D culture assays and in an orthotopic mouse tumor model. Gene expression profiling combined with 3D culture, inducible shRNA knockdown in vitro and in vivo orthotopic mouse tumor model, catalytic activity measurement The EMBO journal High 15282551
2011 PKN3 physically interacts with Rho-family GTPases and preferentially associates with RhoC compared to other PKN family members. PKN3 catalytic activity is increased in the presence of Rho GTPases. RhoC preferentially associates with PKN3 over its closely related PKN family members, and this interaction promotes malignant growth and invasiveness. Co-immunoprecipitation, overexpression of exogenous PKN3 in breast cancer cells, orthotopic mouse tumor knockdown models, in vitro invasiveness assays Molecular oncology Medium 22217540
2012 PKN3 knockdown in human endothelial cells (HUVEC) impairs actin stress fiber formation, disrupts adherens junction integrity, reduces cell motility, and attenuates TNF-α-induced ICAM-1 surface expression. Loss of PKN3 function also affects Pyk2 phosphorylation, suggesting PKN3 links ICAM-1 signaling with actin/AJ dynamics. RNAi-mediated knockdown in HUVEC, immunofluorescence for actin and adherens junctions, TNF-α stimulation, ICAM-1 surface expression assay, Pyk2 phosphorylation measurement European journal of cell biology Medium 22609186
2016 PKN3 knockout mice are viable but exhibit impaired angiogenesis (reduced micro-vessel sprouting in ex vivo aortic ring and in vivo corneal pocket assays) and impaired lung metastasis of melanoma cells. PKN3 knockdown by siRNA induces a glycosylation defect of cell-surface glycoproteins including ICAM-1, integrin β1, and integrin α5 in HUVECs, suggesting defective glycoprotein maturation underlies these phenotypes. PKN3 knockout mouse generation, ex vivo aortic ring assay, in vivo corneal pocket assay, tail-vein melanoma metastasis model, siRNA knockdown, cell-surface glycoprotein analysis Scientific reports High 26742562
2018 PKN3 directly interacts with the adaptor protein p130Cas; this interaction is mediated by the p130Cas SH3 domain binding to the centrally located PKN3 polyproline sequence. PKN3 is the first identified Ser/Thr kinase to bind and phosphorylate p130Cas. PKN3 and p130Cas colocalize in pro-invasive cell structures, and their interaction promotes mouse embryonic fibroblast growth and invasiveness independently of Src transformation. Co-immunoprecipitation, direct binding assays (SH3 domain–polyproline interaction), in vitro kinase/phosphorylation assay, colocalization by immunofluorescence, loss-of-function in MEFs Molecular oncology High 30422386
2019 PKN3 (Pkn3) acts as a Rho effector downstream of Wnt5a-Ror2-Daam2-Rho signaling in osteoclasts. PKN3 expression increases during osteoclast differentiation. PKN3 binds to c-Src and Pyk2 in a Wnt5a-Ror2 signaling-dependent manner, enhances c-Src kinase activity, and is essential for bone-resorbing activity of osteoclasts. Pkn3-deficient mice have greater trabecular bone mass due to decreased osteoclast bone-resorbing activity. Pkn3 knockout mice, co-immunoprecipitation (Pkn3 with c-Src and Pyk2), c-Src kinase activity assay, osteoclast differentiation assays, bone histomorphometry Journal of oral biosciences High 31400545
2020 PKN3 phosphorylates ARHGAP18 (a RhoGAP family protein) in vitro. The interaction between PKN3 and ARHGAP18 is mediated via the N-terminal part of ARHGAP18 and is increased upon ARHGAP18 phosphorylation by PKN3. Phosphorylation of ARHGAP18 by PKN3 enhances its GAP domain activity and contributes to negative regulation of active RhoA, constituting a negative feedback mechanism in Rho signaling. Phosphoproteomic screen using analog-sensitive PKN3, in vitro kinase assay, co-immunoprecipitation, RhoA GAP activity assay International journal of molecular sciences High 33092266
2022 A 4-anilinoquinoline compound (7-iodo-N-(3,4,5-trimethoxyphenyl)quinolin-4-amine) was identified as a potent PKN3 inhibitor with IC50 = 14 nM in biochemical assay and micromolar cell activity, providing a chemical tool to study PKN3 biology. In vitro kinase inhibition assay (IC50 determination), cell-based activity assay ChemMedChem Medium 35403825
2026 PKN3 possesses a threonine gatekeeper residue that is unique within the AGC kinase group, enabling design of highly selective inhibitors. A bisubstrate-analog inhibitor ARC-2603 binds PKN3 with KD = 0.2 nM and shows 5500-fold selectivity over PKAcα, confirmed across a panel of 397 protein kinases. Rational inhibitor design exploiting gatekeeper residue, fluorescence-based binding assay (KD measurement), selectivity profiling against 397 kinases Molecules (Basel, Switzerland) Medium 41752364

Source papers

Stage 0 corpus · 15 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2004 PKN3 is required for malignant prostate cell growth downstream of activated PI 3-kinase. The EMBO journal 87 15282551
2001 PKNbeta interacts with the SH3 domains of Graf and a novel Graf related protein, Graf2, which are GTPase activating proteins for Rho family. Journal of biochemistry 43 11432776
2011 The interaction of PKN3 with RhoC promotes malignant growth. Molecular oncology 42 22217540
2016 PKN3 is the major regulator of angiogenesis and tumor metastasis in mice. Scientific reports 37 26742562
1999 Identification and characterization of PKNbeta, a novel isoform of protein kinase PKN: expression and arachidonic acid dependency are different from those of PKNalpha. Biochemical and biophysical research communications 32 10441506
2015 Novel Nrf2/ARE activator, trans-Coniferylaldehyde, induces a HO-1-mediated defense mechanism through a dual p38α/MAPKAPK-2 and PK-N3 signaling pathway. Chemical research in toxicology 27 26275128
2012 Depletion of protein kinase N3 (PKN3) impairs actin and adherens junctions dynamics and attenuates endothelial cell activation. European journal of cell biology 20 22609186
2022 Efficient delivery of PKN3 shRNA for the treatment of breast cancer via lipid nanoparticles. Bioorganic & medicinal chemistry 19 35752145
2019 Regulation of osteoclast function via Rho-Pkn3-c-Src pathways. Journal of oral biosciences 15 31400545
2018 The interaction of p130Cas with PKN3 promotes malignant growth. Molecular oncology 15 30422386
2020 A Screen for PKN3 Substrates Reveals an Activating Phosphorylation of ARHGAP18. International journal of molecular sciences 6 33092266
2022 Identification of 4-Anilinoquin(az)oline as a Cell-Active Protein Kinase Novel 3 (PKN3) Inhibitor Chemotype. ChemMedChem 4 35403825
2025 PKN3 as a key regulator in cancer - From signaling pathways to targeted therapies. Bioorganic chemistry 3 40633483
2000 Pharbitis class-1 knotted-like homeobox gene, PKn3, shares similar characteristics to those of class-2 knotted-like genes. Plant cell reports 2 30754929
2026 Selective Inhibitor of Protein Kinase PKN3 Generated by Conjugation of a Structurally Optimized Bumped N-(2-Aminoethyl)-8-anilinoisoquinoline-5-sulfonamide (H-9) with d-Arginine-Rich Chain. Molecules (Basel, Switzerland) 0 41752364

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