| 2006 |
GSKIP was identified as a GSK3β-binding protein via yeast two-hybrid screen; a 25-amino acid C-terminal region of GSKIP is highly similar to the GSK3β interaction domain (GID) of Axin. In vitro kinase assays showed GSKIP is a GSK3β substrate and that both full-length GSKIP and its C-terminal fragment block phosphorylation of primed and non-primed GSK3β substrates. A synthetic GSKIPtide competes with and blocks phosphorylation of Axin and β-catenin by GSK3β. Overexpression of GSKIP induces β-catenin accumulation in cytoplasm and nucleus and activates Tcf-4 transcriptional activity, defining GSKIP as a negative regulator of GSK3β in the Wnt signaling pathway. |
Yeast two-hybrid screen, in vitro kinase assay, peptide competition assay, immunofluorescence, Tcf-4 reporter assay |
Biochemistry |
High |
16981698
|
| 2009 |
GSKIP binding to GSK3β shares overlapping sites (scaffold-binding region I, SBR-I residues 260–300) with AxinGID and FRATtide, as mapped by single-point mutations in GSK3β. GSK3β V267G mutation reduces binding to GSKIP and AxinGID but not FRATtide, while Y288F mutation abolishes FRATtide binding without affecting GSKIP or AxinGID. A novel C-terminal helix region of GSK3β (SBR-II, residues 339–383) is required for FRATtide binding but not GSKIP or AxinGID binding. |
GSK3β single-point mutagenesis, co-immunoprecipitation/binding assays, molecular simulation |
Molecular and cellular biochemistry |
Medium |
20043192
|
| 2009 |
In SH-SY5Y neuroblastoma cells, GSKIP overexpression prevents neurite outgrowth, inhibits GSK3β-mediated phosphorylation of tau at Ser396, increases nuclear β-catenin and cyclin D1 levels, and downregulates N-cadherin expression, reducing recruitment of β-catenin to the membrane. siRNA depletion of β-catenin blocks neurite outgrowth, establishing GSKIP as a regulator of the GSK3β/β-catenin and N-cadherin/β-catenin pools during neuronal differentiation. |
Overexpression, siRNA knockdown, immunofluorescence, Western blotting in SH-SY5Y cells with retinoic acid differentiation |
Journal of cellular biochemistry |
Medium |
19830702
|
| 2011 |
Molecular dynamics simulation of GSK3β complexed with a peptide derived from GSKIP (GSKIPtide) showed that GSKIPtide binds a hydrophobic pocket formed by an α-helix and an extended loop near the GSK3β C-terminus; this binding mode is closer to AxinGID than to FRATtide. V267G mutation in GSK3β reduces GSKIPtide binding affinity by ~70%, and Y288F abolishes FRATtide binding but does not affect GSKIPtide, consistent with experimental mutagenesis data. |
Molecular dynamics simulation validated against experimental mutagenesis data |
Biopolymers |
Low |
21328310
|
| 2015 |
GSKIP forms a working complex PKA/GSKIP/GSK3β/Drp1 that mediates Drp1 Ser637 phosphorylation in the cAMP/PKA/Drp1 axis. GSKIP wild-type overexpression increases Drp1 S637 phosphorylation 7–8-fold versus PKA-binding-defective (V41/L45) and GSK3β-binding-defective (L130) GSKIP mutants under H2O2/forskolin challenge. Silencing either GSKIP or GSK3β (but not GSK3α) dramatically reduces Drp1 S637 phosphorylation. Kinase-dead GSK3β-K85R (retains GSKIP binding) sustains Drp1 phosphorylation, whereas K85M (loses GSKIP binding) does not, indicating GSK3β acts as an anchoring protein rather than a kinase in this complex. Phosphomimetic Drp1 S637D (but not S637A) rescues the elongated mitochondrial morphology lost in GSKIP mutant-overexpressing cells, placing Drp1 downstream of PKA/GSKIP/GSK3β signaling. |
Site-directed mutagenesis, overexpression, siRNA knockdown, phosphorylation assays, mitochondrial morphology imaging in HEK293 cells |
Biochimica et biophysica acta |
High |
25920809
|
| 2015 |
GSKIP deficiency in a conditional knockout mouse causes lethality at birth with cleft palate and delayed ossification. At the molecular level, GSKIP loss decreases GSK3β phosphorylation at Ser-9 (starting at E10.5), leading to enhanced GSK3β activity, establishing GSKIP as an in vivo regulator of GSK3β activity required for palatal shelf fusion. |
Conditional knockout mouse model, immunohistochemistry, Western blotting for GSK3β Ser-9 phosphorylation |
The Journal of biological chemistry |
High |
26582204
|
| 2015 |
Germline duplication of GSKIP (and ATG2B) enhances hematopoietic progenitor differentiation, including megakaryocyte differentiation, by increasing progenitor sensitivity to thrombopoietin (TPO), and cooperates with acquired JAK2, MPL, and CALR mutations during myeloproliferative neoplasm development, as demonstrated in iPSC and primary cell models. |
Induced pluripotent stem cell (iPSC) models, primary hematopoietic cell assays, genetic epistasis with JAK2/MPL/CALR mutations |
Nature genetics |
Medium |
26280900
|
| 2016 |
GSKIP functions as an AKAP that simultaneously binds PKA and GSK3β, and both interactions are required for regulation of β-catenin. GSKIP facilitates PKA-mediated stabilizing phosphorylation of β-catenin at Ser-675 and facilitates GSK3β-mediated destabilizing phosphorylation at Ser-33/Ser-37/Thr-41. GSKIP acts as a scavenger that recruits PKA and GSK3β away from the β-catenin destruction complex without forming a complex with β-catenin itself. AKAP220, which also binds PKA and GSK3β via a conserved GID, did not affect Wnt signaling, indicating specificity of the GSKIP mechanism. |
Mutant overexpression (PKA-binding and GSK3β-binding defective GSKIP), co-immunoprecipitation, phosphorylation assays, Wnt reporter assays, comparison with AKAP220 |
The Journal of biological chemistry |
High |
27484798
|
| 2018 |
The PKA-RII binding domain (V41/L45 residues) of GSKIP is required for forming the PKA/GSKIP/GSK3β/Drp1 working complex and for Drp1 Ser637 phosphorylation. Yeast two-hybrid and co-immunoprecipitation show the V41/L45P mutant causes a gain-of-function in GSKIP dimerization that further influences GSK3β binding, while L130 (GSK3β-binding site) mediates GSKIP dimerization. Molecular modeling indicates mammalian GSKIP can form a dimer through the L130 residue rather than V41/L45. |
Yeast two-hybrid, co-immunoprecipitation, site-directed mutagenesis, molecular modeling |
Biochimica et biophysica acta. Molecular cell research |
Medium |
29694914
|
| 2019 |
GSKIP anchoring enhances PKA-mediated phosphorylation of Tau at Ser409; overexpression of GSKIP WT produces greater Tau Ser409 phosphorylation than PKA-binding-defective (V41/L45) or GSK3β-binding-defective (L130) mutants. In vitro kinase assays show that the combination of GSK3β with PKA (but not CaMKII) provides a conformational context for Tau Ser409 phosphorylation. In APPWT/D678H iPSC-derived cells, PKA-mediated Tau phosphorylation is enhanced relative to controls, implicating the cAMP/PKA/GSKIP/GSK3β axis in Alzheimer-relevant Tau hyperphosphorylation. |
In vitro kinase assay, overexpression of GSKIP mutants, CRISPR/Cas9 isogenic iPSC mutants, Western blotting |
Journal of clinical medicine |
Medium |
31640277
|
| 2020 |
GSKIP overexpression in cardiomyocytes subjected to hypoxia/reoxygenation (H/R) injury upregulates nuclear Nrf2 and increases Nrf2/ARE transcriptional activity associated with increased GSK3β Ser-9 phosphorylation (GSK3β inhibition). Pharmacological GSK3β inhibition rescues the phenotype caused by GSKIP depletion, placing GSKIP upstream of GSK3β in regulating Nrf2/ARE antioxidant signaling. Nrf2 inhibition reverses the cardioprotective effect of GSKIP overexpression. |
Overexpression, siRNA knockdown, pharmacological GSK3β inhibition, Nrf2/ARE reporter assay, Western blotting in cardiomyocytes |
Biochemical and biophysical research communications |
Medium |
32828530
|
| 2021 |
Double knockout of Atg2b and Gskip (but not either gene alone) in mice causes severely decreased hematopoiesis, reduction in long-term HSC pool size due to increased cell death, and lethality in utero with anemia. Loss of both genes increases expression of oxidative phosphorylation genes without affecting autophagy, revealing a synergistic role for GSKIP and ATG2B in HSC maintenance through a non-autophagy mechanism. |
Double and single knockout mouse models, flow cytometry of HSC populations, bone marrow/fetal liver analysis, transcriptomic gene expression |
Molecular and cellular biology |
Medium |
34748402
|
| 2023 |
CRISPR/Cas9 knockout of GSKIP in SH-SY5Y cells produces a cell aggregation phenotype and reduced cell growth via suppression of GSK3β/β-catenin pathways and cell cycle progression, linked to EMT/MET signaling rather than differentiation. Phosphorylated β-catenin at S675 and S552 (but not S33/S37/T41) translocates to the nucleus in GSKIP-KO cells. Reintroduction of GSKIP into KO cells restores cell migration and tumorigenesis, and neurite outgrowth upon RA treatment is still observed in GSKIP-KO clones. |
CRISPR/Cas9 knockout, rescue by GSKIP re-expression, gene set enrichment analysis, Western blotting for β-catenin phosphorylation, migration assays |
Journal of cell communication and signaling |
Medium |
37133713
|