Furthermore, the GSK3β-binding domain is reported to act synergistically with cAMP/PKA signaling to inhibit GSK3β activity through a cytoplasmic destruction complex with β-catenin, leading to the negative regulation of Wnt signaling (Dema et al. GSKIP is a cytosolic scaffolding protein that retains protein kinase A (PKA) RII-binding sites at the N-terminal residue V41/L45 and a GSK3β-binding domain at the C-terminal residue L130 (Chou et al. 2010) originally cloned and identified as a glycogen synthase kinase-3β (GSK3β) interacting protein (Chou et al.
Glycogen synthase kinase interaction protein (GSKIP) is the smallest A-kinase anchor protein (AKAP) (Hundsrucker et al. Collectively, these results suggested that GSKIP may function as an oncogene to form an aggregation phenotype for cell survival in harsh environments through EMT/MET rather than differentiation in the GSKIP-KO of SH-SY5Y cells. Notably, phosphor-β-catenin (S675) and β-catenin (S552) but not phosphor-β-catenin (S33/S37/T41) translocated into the nucleus for further gene activation. Conversely, reintroduction of GSKIP into GSKIP-KO clones restored cell migration and tumorigenesis. Gene set enrichment analysis indicated that GSKIP-KO was related to epithelial mesenchymal transition/mesenchymal epithelial transition (EMT/MET) and Wnt/β-catenin/cadherin signaling pathways, suppressing cell migration and tumorigenesis through the inhibition of Wnt/β-catenin mediated EMT/MET. The GSKIP-KO clones exhibited an aggregation phenotype through suppression of GSK3β/β-catenin pathways and cell cycle progression rather than cell differentiation. However, neuron outgrowth was still observed in GSKIP-KO clones treated with RA. Several GSKIP-KO clones resulted in an aggregation phenotype and reduced cell growth without retinoic acid (RA) treatment. To further investigate how GSKIP functions in neurons, CRISPR/Cas9 technology was utilized to knock out GSKIP (GSKIP-KO) in SH-SY5Y. GSK3β interacting protein (GSKIP) is a small A-kinase anchor protein previously reported to mediate the N-cadherin/β-catenin pool for differentiation in SH-SY5Y cells through overexpression of GSKIP to present the neuron outgrowth phenotype.
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