GSKIP Is Homologous to the Axin GSK3β Interaction Domain and Functions as a Negative Regulator of GSK3β

Abstract

Although prominent FRAT/GBP exhibits a limited degree of homology to Axin, the binding sites on GSK3 for FRAT/GBP and Axin may overlap to prevent the effect of FRAT/GBP in stabilizing beta-catenin in the Wnt pathway. Using a yeast two-hybrid screen, we identified a novel protein, GSK3beta interaction protein (GSKIP), which binds to GSK3beta. We have defined a 25-amino acid region in the C-terminus of GSKIP that is highly similar to the GSK3beta interaction domain (GID) of Axin. Using an in vitro kinase assay, our results indicate that GSKIP is a good GSK3beta substrate, and both the full-length protein and a C-terminal fragment of GSKIP can block phosphorylation of primed and nonprimed substrates in different fashions. Similar to Axin GID(381-405) and FRATtide, synthesized GSKIPtide is also shown to compete with and/or block the phosphorylation of Axin and beta-catenin by GSK3beta. Furthermore, our data indicate that overexpression of GSKIP induces beta-catenin accumulation in the cytoplasm and nucleus as visualized by immunofluorescence. A functional assay also demonstrates that GSKIP-transfected cells have a significant effect on the transactivity of Tcf-4. Collectively, we define GSKIP as a naturally occurring protein that is homologous with the GSK3beta interaction domain of Axin and is able to negatively regulate GSK3beta of the Wnt signaling pathway.

The A-Kinase Anchoring Protein (AKAP) Glycogen Synthase Kinase 3β Interaction Protein (GSKIP) Regulates β-Catenin through Its Interactions with Both Protein Kinase A (PKA) and GSK3β

“…2G). generated a PKA-binding-deficient (GSKIP-N42I) variant of GSKIP, and as a control, the previously described GSK3␤-binding-deficient GSKIP variant, GSKIP-L130P (27,37). We initially analyzed the interaction of the GSKIP variants with PKA by cAMP-agarose precipitation.…”

“…We then examined whether GSKIP competes with Axin1/2 for GSK3␤ binding by co-immunoprecipitation and Western blotting, as GSKIP and Axin1/2 share a common motif for binding GSK3␤ (Fig. 7B) (27). In cells expressing Axin1 or Axin2, the amount of GSKIP bound to GSK3␤ was significantly reduced (Fig.…”

“…GSKIP and AKAP220 differ in their cellular location: GSKIP is cytoplasmic (9,27), whereas AKAP220 is predominantly found at the plasma membrane and on peroxisomes (41,(51)(52)(53). GSKIP does not interact with ␤-catenin, whereas AKAP220 forms a complex with ␤-catenin and cadherin at the plasma membrane, which competes for ␤-catenin in Wnt signaling (51,54).…”

“…GSKIP seems able to induce ␤-catenin accumulation in the cytoplasm and the nucleus and to activate transcription through direct interaction with GSK3␤ when overexpressed in HeLa and neuroblastoma SH-SY5Y cells (27,28). In SH-SY5Y cells, the GSKIP overexpression blocked neurite outgrowth during retinoic acid-mediated differentiation.…”

GSKIP (GSK3-beta interaction protein)

“…These AKAPs proteins interact with the regulatory domain of PKA and facilitate their phosphorylation. GSKIP directly interacts with GSK3B through its C-terminal conserved GSK3B-binding domain (GID; amino acid 115-139) and negatively regulates GSK3B in the Wnt/beta-catenin signaling pathway (Chou et al, 2006). The overexpression of GSKIP may mimic activation of the Wnt pathway involved in hematopoietic stem cell homeostasis and normal megakaryopoiesis (Li et al, 2008) and in the development of leukemia stem cells in AML (Wang.…”