ILKAP

ILKAP is a PP2C-family serine/threonine phosphatase that functions as a negative regulator of multiple pro-survival and proliferative kinase pathways by selectively dephosphorylating GSK3β, RSK2, HIF-1α, and CK1. It was identified as a binding partner of integrin-linked kinase (ILK1) and selectively inhibits ILK1-mediated GSK3β Ser9 phosphorylation without affecting PKB/AKT Ser473, thereby suppressing Wnt/Tcf signaling and cyclin D1 expression, reducing S-phase entry and anchorage-independent growth (PMID:11331582, PMID:14990992). ILKAP shuttles between nucleus and cytoplasm via an importin α–dependent NLS and CRM1-dependent export; nuclear ILKAP dephosphorylates RSK2 to induce apoptosis and promotes CRM1-dependent nuclear export of ILK to limit ILK-driven proliferation (PMID:23329845, PMID:18635968). ILKAP protein stability is controlled by MAEL-dependent lysosomal degradation, and ILKAP additionally modulates β-catenin stability through dephosphorylation of GSK3β/CK1, regulates HIF-1α–p53–dependent apoptosis, and sustains PGAM1-driven glycolysis in hepatocellular carcinoma (PMID:29371914, PMID:38379270, PMID:29742494, PMID:41454076).

1. 2001 High

   Established ILKAP as a PP2C-family phosphatase that physically associates with ILK1 and selectively dephosphorylates GSK3β Ser9 (but not AKT Ser473) to suppress Wnt/Tcf signaling, answering how ILK kinase activity is negatively regulated.

   Evidence Yeast two-hybrid, co-precipitation, catalytic mutant H154D, ILK immune complex kinase assay, Tcf/Lef luciferase reporter in HEK293 cells

   PMID:11331582

   Open questions at the time

   • Direct phosphatase activity on GSK3β peptide not demonstrated in vitro
   • Endogenous interaction stoichiometry unknown
   • Mechanism of selectivity for GSK3β over AKT not resolved
2. 2004 High

   Reciprocal gain- and loss-of-function confirmed endogenous ILKAP restrains ILK–GSK3β signaling to control cell cycle progression and anchorage-independent growth, establishing ILKAP as a tumor-suppressive phosphatase.

   Evidence siRNA knockdown and stable overexpression in LNCaP cells, flow cytometry, soft-agar colony assay, cyclin D1 Western blot

   PMID:14990992

   Open questions at the time

   • Whether ILKAP directly dephosphorylates GSK3β or acts indirectly through ILK remains unresolved
   • Mechanism linking cyclin D1 loss to G1 arrest not dissected beyond GSK3β
3. 2008 Medium

   Revealed that ILKAP promotes CRM1-dependent nuclear export of ILK, restricting nuclear ILK accumulation and ILK-driven DNA synthesis, thereby establishing ILKAP as a regulator of ILK subcellular distribution.

   Evidence Live-cell imaging, nuclear fractionation, leptomycin B treatment, BrdU incorporation in keratinocytes

   PMID:18635968

   Open questions at the time

   • Whether ILKAP directly escorts ILK or modifies a separate export signal is unknown
   • Not tested in non-keratinocyte cell types
4. 2012 Medium

   Phosphopeptide affinity studies identified p38, ATM, Chk1, Chk2, and RSK2 as candidate ILKAP substrates, expanding the substrate repertoire beyond GSK3β.

   Evidence Solid-phase phosphopeptide pull-down with competitive binding assays

   PMID:22348942

   Open questions at the time

   • No in vitro dephosphorylation assays performed to confirm catalytic activity on these substrates
   • Binding ≠ dephosphorylation; functional validation needed for each candidate
5. 2013 High

   Mapped an NLS (residues 71–86, critical K78/R79) mediating importin α–dependent nuclear import and showed nuclear ILKAP dephosphorylates RSK2 to induce apoptosis and reduce cyclin D1, answering how ILKAP reaches the nucleus and identifying its nuclear substrate.

   Evidence NLS deletion mutant, importin co-IP, RSK2 kinase assay, apoptosis assay, immunofluorescence in HeLa cells

   PMID:23329845

   Open questions at the time

   • Direct dephosphorylation of RSK2 not shown with purified components
   • Relative contribution of nuclear vs. cytoplasmic ILKAP pools to apoptosis not quantified
6. 2015 Medium

   ILKAP depletion sensitized glioblastoma cells to radiation and impaired DNA-PK phosphorylation, linking ILKAP to the DNA damage response for the first time.

   Evidence siRNA knockdown, γH2AX/53BP1 foci, DNA-PK phosphorylation Western blot, clonogenic survival in glioblastoma cells

   PMID:26460618

   Open questions at the time

   • Whether ILKAP directly dephosphorylates DNA-PK or acts indirectly is unresolved
   • Only tested in p53-wildtype glioblastoma context
7. 2016 Medium

   Showed ILKAP functions as a dual phosphatase hub controlling both RSK and ILK/AKT in ovarian cancer, with combined inhibition required to phenocopy ILKAP activity in cisplatin-induced death.

   Evidence siRNA knockdown, pharmacological inhibitors of RSK and ILK, apoptosis and viability assays in ovarian cancer cells

   PMID:27065457

   Open questions at the time

   • Contradicts earlier finding that ILKAP does not affect AKT; cell-type dependence not clarified
   • ILKAP direct activity on AKT versus ILK not biochemically separated
8. 2017 Medium

   Identified MAEL as an upstream negative regulator that targets ILKAP for lysosomal degradation, increasing phosphorylation of p38, CHK1, and RSK2—answering how ILKAP protein levels are controlled.

   Evidence Co-expression/silencing, lysosome inhibitor rescue, adenoviral ILKAP overexpression, xenograft in gastric cancer model

   PMID:29371914

   Open questions at the time

   • Mechanism by which MAEL directs ILKAP to lysosomes (adapter, ubiquitin signal) is unknown
   • Not confirmed in non-gastric cancer contexts
9. 2018 Medium

   Identified HIF-1α as a direct ILKAP-interacting substrate whose dephosphorylation is required for HIF-1α–p53 complex formation and hypoxia-induced apoptosis.

   Evidence Co-immunoprecipitation, HRE-luciferase reporter, shRNA knockdown, overexpression, viability assays

   PMID:29742494

   Open questions at the time

   • Specific phosphosite(s) on HIF-1α targeted by ILKAP not mapped
   • In vitro phosphatase assay with purified ILKAP and HIF-1α not performed
10. 2024 Medium

    Demonstrated ILKAP dephosphorylates GSK3β and CK1 to stabilize β-catenin by reducing its ubiquitination, and bridges TCF4–β-catenin interaction to promote Wnt target gene expression and HCC metastasis—revealing an unexpected pro-oncogenic role.

    Evidence Co-immunoprecipitation, ubiquitination assay, Wnt reporter, siRNA knockdown, zebrafish xenograft

    PMID:38379270

    Open questions at the time

    • Contradicts earlier tumor-suppressive role via GSK3β dephosphorylation; context-dependent regulation not reconciled
    • TCF4–β-catenin bridging mechanism (scaffold vs. dephosphorylation) not dissected
11. 2025 Medium

    Linked ILKAP to glycolytic reprogramming by showing ILKAP sustains PGAM1 expression and extracellular acidification in HCC, with PGAM1 rescue reversing ILKAP-knockdown proliferation defects.

    Evidence RNA sequencing, siRNA knockdown, PGAM1 rescue, Seahorse ECAR assay, xenograft in HCC cells

    PMID:41454076

    Open questions at the time

    • Whether ILKAP regulates PGAM1 transcriptionally or post-translationally is unknown
    • No direct phosphatase–substrate relationship established between ILKAP and PGAM1

• A unifying model reconciling ILKAP's apparently contradictory tumor-suppressive (GSK3β dephosphorylation suppressing Wnt) and tumor-promoting (β-catenin stabilization, PGAM1-driven glycolysis) activities across tissue contexts is lacking.
• No structural data for ILKAP exist to explain substrate selectivity
• In vitro reconstitution of direct phosphatase activity on most claimed substrates has not been performed
• Tissue- and context-specific regulation of ILKAP activity/expression is poorly defined