ILKAP

ILKAP is a PP2C-family serine/threonine phosphatase that functions as a negative regulator of integrin-linked kinase (ILK) signaling and a broader hub controlling pro-survival kinase pathways and Wnt-driven gene expression (PMID:11331582, PMID:14990992). It was first identified as a catalysis-independent binding partner of ILK1 that, when catalytically active, selectively inhibits ECM- and growth-factor-stimulated ILK kinase activity without affecting Raf-1 (PMID:11331582). Downstream, active ILKAP selectively blocks ILK-dependent GSK3β Ser9 phosphorylation while sparing AKT Ser473, and endogenous loss-of-function by siRNA mirrors this selectivity, defining the ILK–GSK3β axis as its principal target (PMID:11331582, PMID:14990992). Through this axis ILKAP suppresses Tcf/Lef-dependent transcription and cyclin D1 expression, arresting cells in G1 and limiting anchorage-independent growth (PMID:11331582, PMID:14990992). ILKAP is predominantly nuclear, imported via an N-terminal NLS (residues 71–86, with Lys-78/Arg-79 critical) that binds importin α1/α3/α5 (PMID:23329845), and it also controls CRM1-dependent nuclear export of ILK (PMID:18635968). In the nucleus it binds and dephosphorylates additional substrates including RSK2 and HIF-1α to promote apoptosis (PMID:23329845, PMID:29742494), and it binds phosphopeptide substrates of PP2Cδ such as p38, ATM, Chk1, Chk2, and RSK2 (PMID:22348942). ILKAP loss broadly activates pro-survival kinases (ILK, AKT, RSK, DNA-PK), conferring resistance to genotoxic and chemotherapeutic stress (PMID:26460618, PMID:27065457), and its protein level is lowered by MAEL-driven lysosomal degradation (PMID:29371914). In hepatocellular carcinoma ILKAP can instead stabilize β-catenin by dephosphorylating GSK3β and CK1 and bridging TCF4–β-catenin to enhance Wnt targets (PMID:38379270), and it supports glycolytic reprogramming via PGAM1 (PMID:41454076).

1. 2001 High

   Established that ILKAP physically associates with ILK1 and acts as a phosphatase that selectively suppresses ILK signaling, defining its core function as a negative regulator of ILK rather than a general phosphatase.

   Evidence Yeast two-hybrid, co-precipitation, and conditional expression with immune-complex kinase assays plus an H154D catalytic-dead control in HEK293 cells

   PMID:11331582

   Open questions at the time

   • Direct dephosphorylation of ILK or an intermediate not biochemically resolved
   • Binding shown to be catalysis-independent but functional inhibition catalysis-dependent — coupling mechanism unexplained
2. 2001 High

   Defined downstream selectivity by showing ILKAP blocks ILK→GSK3β Ser9 phosphorylation and Tcf/Lef transactivation but spares AKT, placing it specifically on the Wnt/GSK3β branch.

   Evidence Conditional expression with phosphorylation assays, TOPFlash reporter, and active-site/paralog (PP2Cα) controls

   PMID:11331582

   Open questions at the time

   • Whether GSK3β is a direct ILKAP substrate or an indirect consequence of ILK inhibition not distinguished
3. 2004 High

   Confirmed via endogenous loss-of-function that ILKAP physiologically targets the ILK–GSK3β axis and links its activity to cell-cycle and growth control.

   Evidence siRNA knockdown with phosphorylation assays, cyclin D1 readout, cell-cycle and soft-agar assays, and ILK rescue in LNCaP cells

   PMID:14990992

   Open questions at the time

   • Cyclin D1 regulation inferred through ILK–GSK3β; alternative inputs not excluded
4. 2008 Medium

   Extended ILKAP function to spatial control of ILK by showing it promotes CRM1-dependent nuclear export of ILK, coupling its regulation to DNA synthesis.

   Evidence Live-cell imaging, nuclear fractionation, leptomycin B treatment, and DNA synthesis assays in keratinocytes

   PMID:18635968

   Open questions at the time

   • Mechanism by which ILKAP enhances CRM1 export unresolved
   • Single cell-type context
5. 2012 Medium

   Characterized ILKAP substrate-binding preferences, showing phospho-dependent recognition of PP2Cδ substrates and implicating it in DNA-damage and stress kinase signaling.

   Evidence Solid-phase phosphopeptide affinity pull-downs (p38, ATM, Chk1, Chk2, RSK2) from cell lysates

   PMID:22348942

   Open questions at the time

   • Binding affinity does not establish catalytic dephosphorylation of these substrates in cells
   • Flanking-context determinants only partially mapped
6. 2013 High

   Localized ILKAP to the nucleus via a defined importin-binding NLS and identified nuclear RSK2 as a substrate whose inhibition drives apoptosis.

   Evidence NLS deletion/point mutagenesis, immunofluorescence, fractionation, importin co-IP, plus RSK2 kinase and apoptosis assays

   PMID:23329845

   Open questions at the time

   • Whether nuclear vs cytoplasmic pools have distinct substrate repertoires not dissected
   • RSK2 dephosphorylation site not mapped
7. 2015 Medium

   Implicated ILKAP in DNA-damage survival signaling by showing it is required for radiation-induced DNA-PK activation in a p53-dependent manner.

   Evidence siRNA knockdown with γH2AX/53BP1 foci, phospho-DNA-PK blots, and clonogenic survival in GBM cells

   PMID:26460618

   Open questions at the time

   • Direct vs indirect effect on DNA-PK phosphorylation unresolved
   • p53-dependence mechanism not defined
8. 2016 Medium

   Positioned ILKAP as a multi-kinase regulatory hub whose loss confers chemoresistance by simultaneously activating RSK, ILK, and AKT.

   Evidence siRNA knockdown, phospho-Western blots, viability/apoptosis assays, and combined pharmacological kinase inhibition in ovarian cancer cells

   PMID:27065457

   Open questions at the time

   • Which kinase dephosphorylations are direct not established
   • Single tumor context
9. 2017 Medium

   Identified MAEL-driven lysosomal degradation as a mechanism controlling ILKAP protein level and its substrate phosphorylation in tumors.

   Evidence Co-expression/silencing, lysosome inhibitors, substrate phospho-blots, xenografts, and adenoviral ILKAP rescue

   PMID:29371914

   Open questions at the time

   • Molecular route of lysosomal targeting (e.g. direct MAEL interaction) not defined
10. 2018 Medium

    Added HIF-1α as a nuclear ILKAP substrate, linking ILKAP to hypoxic apoptosis via HIF-1α–p53 interaction.

    Evidence Co-IP, HRE luciferase reporter, overexpression/shRNA, and viability assays under hypoxia

    PMID:29742494

    Open questions at the time

    • HIF-1α dephosphorylation site not identified
    • Single-lab functional model
11. 2024 Medium

    Revealed a context-dependent pro-Wnt role in hepatocellular carcinoma where ILKAP stabilizes β-catenin and bridges TCF4–β-catenin, contrasting with its earlier Wnt-suppressive activity.

    Evidence Co-IP, phospho/ubiquitination blots, immunofluorescence, luciferase reporter, and zebrafish xenograft metastasis assays

    PMID:38379270

    Open questions at the time

    • Reconciliation with the earlier GSK3β/Tcf-suppressive model not addressed
    • Direct dephosphorylation of CK1 vs GSK3β priority unresolved
12. 2025 Medium

    Connected ILKAP to metabolic reprogramming by placing PGAM1-dependent glycolysis downstream of ILKAP for proliferation and invasion.

    Evidence siRNA knockdown, RNA-seq, ECAR measurement, and PGAM1 rescue in xenografts

    PMID:41454076

    Open questions at the time

    • Mechanism linking ILKAP phosphatase activity to PGAM1 expression unknown
    • Direct vs transcriptional control unclear

• How ILKAP's context determines whether it suppresses or promotes Wnt/β-catenin signaling, and the structural basis coupling its catalysis-independent ILK binding to catalysis-dependent inhibition, remain unresolved.
• No structural model of the ILKAP–ILK complex
• Direct catalytic substrates vs indirect effects not systematically separated
• Determinants of opposite Wnt outcomes across tumor types unknown