Abstract
Glycogen-specific kinase (GSK3β) is an integral regulator of the Wnt signalling pathway as well as many other diverse signalling pathways and processes. Dys-regulation of GSK3β is implicated in many different pathologies, including neurodegenerative disorders as well as many different tumour types. In the context of tumour development, GSK3β has been shown to play both oncogenic and tumour suppressor roles, depending upon tissue, signalling environment or disease progression. Although multiple substrates of the GSK3β kinase have been identified, the wider protein networks within which GSK3β participates are not well known, and the consequences of these interactions not well understood. In this study, LC-MS/MS expression analysis was performed using knockout GSK3β colorectal cancer cells and isogenic controls in colorectal cancer cell lines carrying dominant stabilizing mutations of β-catenin. Consistent with the role of GSK3β, we found that β-catenin levels and canonical Wnt activity are unaffected by knockout of GSK3β and therefore used this knockout cell model to identify other processes in which GSK3β is implicated. Quantitative proteomic analysis revealed perturbation of proteins involved in cell-cell adhesion, and we characterized the phenotype and altered proteomic profiles associated with this. We also characterized the perturbation of metabolic pathways resulting from GSK3β knockout and identified defects in glycogen metabolism. In summary, using a precision colorectal cancer cell-line knockout model with constitutively activated β-catenin we identified several of the diverse pathways and processes associated with GSK3β function.
Highlights
Glycogen synthase kinase-3 beta (GSK3β) is a serine/threonine kinase in the glycogen synthase kinase subfamily with diverse roles and interaction partners in a wide range of signalling pathways and cellular functions [1]
We identified a set of 440 proteins that were uniquely detected in HCT116-GSK3β-KO or HCT116-GSK3β-WT cells or significantly (p
Adenomatous Polyposis Coli (APC) contains several phosphorylation consensus sequences for both GSK3β and CSK1, and GSK3β has been shown to mediate the interaction between APC and Axin in the destruction complex [20]
Summary
Glycogen synthase kinase-3 beta (GSK3β) is a serine/threonine kinase in the glycogen synthase kinase subfamily with diverse roles and interaction partners in a wide range of signalling pathways and cellular functions [1]. Following pre-phosphorylation by casein kinase 1 (CK1), GSK3β phosphorylates β-catenin at serine residues 33, 37, and 41 which targets β-catenin for proteosomal degradation [2]. When Wnt signalling is activated by the family of Wnt ligands binding to receptors, such as Frizzled, GSK3β and CK1 phosphorylate low-density lipoprotein receptorrelated protein 6 (LRP6) at conserved PPSPXS motifs, which initiates the recruitment of the scaffold protein Axin. It is this interaction with the Axin/LRP6 complex that surrounds GSK3β with phosphorylated residues and directly inhibits its ability to phosphorylate β-catenin [3, 4]. Following inhibition of GSK3β, β-catenin accumulates in the cytosol and undergoes nuclear import where it activates target transcription factors and gene transcription [4]
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