Characterisation of the phosphorylation of beta-catenin at the GSK-3 priming site Ser45.
Activation of the canonical Wnt signalling pathway results in stabilisation and nuclear translocation of beta-catenin. In the absence of a Wnt signal, beta-catenin is phosphorylated at four conserved serine and threonine residues at the N-terminus of the protein, which results in beta-catenin ubiquitination and proteasome-dependent degradation. The phosphorylation of three of these residues, Thr41, Ser37, and Ser33, is mediated by glycogen synthase kinase-3 (GSK-3) in a sequential manner, beginning from the C-terminal Thr41. It has recently been shown that the GSK-3 dependent phosphorylation of beta-catenin requires prior priming through phosphorylation of Ser45. However, it is not known whether phosphorylation of Ser45 is carried out by GSK-3 itself or by an alternative kinase. In this study, the phosphorylation of beta-catenin at Ser45 was characterised using a phospho-specific antibody. GSK-3beta was found to be unable to phosphorylate beta-catenin at Ser45 in vitro and in intact cells. However, inhibition of GSK-3 in intact cells reduced Ser45 phosphorylation, suggesting that GSK-3 kinase activity is required for the phosphorylation event. In vitro, CK1, but not CK2, phosphorylates Ser45. Ser45 phosphorylation in intact cells is not mediated by CK1varepsilon, a known positive regulator of Wnt signalling, as overexpression of this kinase leads to decreased phosphorylation levels. In conclusion, phosphorylation of beta-catenin at the GSK-3 priming site Ser45 is not mediated by GSK-3 itself, but by an alternative kinase, indicating that beta-catenin is not an unprimed substrate for GSK-3 in vivo. Priming of GSK-3 dependent phosphorylation of beta-catenin by a different kinase could have important implications for the regulation of Wnt signalling.
Hagen
Thilo
T
The Wolfson Institute for Biomedical Research, University College London, London WC1E 6BT, UK. t.hagen@ucl.ac.uk
Vidal-Puig
Antonio
A
D019943:Amino Acid Substitution; D000906:Antibodies / Q000378:metabolism; D000918:Antibody Specificity; D001665:Binding Sites / Q000502:physiology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D015398:Signal Transduction / Q000502:physiology; D013379:Substrate Specificity / Q000502:physiology; D015534:Trans-Activators; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism; D051176:beta Catenin
D000906:Antibodies; C495270:CTNNB1 protein, human; D003598:Cytoskeletal Proteins; D015534:Trans-Activators; D025801:Ubiquitin; D051176:beta Catenin; D038341:Glycogen Synthase Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3