Fig. 6. Post-translational modifications of GSK3ß during PC12 cell differentiation. Whole-cell extracts from PC12 cell cultures treated with NGF for the indicated times were immunoblotted with phosphospecific antibodies. (A) The levels of tyrosine-phosphorylated GSK3ß show a transient reduction following NGF treatment (Ptyr); changes in tyrosine phosphorylation correlate with the appearance of faster migrating bands reactive with a GSK3ß specific antibody (GSK3ß). Reduced phosphorylation is detectable by 15 minutes and has returned to unstimulated levels by three hours (Ptyr, lower band: note, the antibody used detects a conserved epitope present in both GSK3
and ß, hence the visualisation of a doublet on this blot). The levels of serine-phosphorylated GSK3ß (Pser) show a rapid increase within five minutes following NGF addition. Subsequently, phosphorylation gradually declines up to three hours, but does not return to baseline levels. (B) Longer-term changes in GSK3ß post-translational modifications were examined by immunoblotting. Overall levels of GSK3ß show a shallow rise following NGF addition (GSK3ß). This expression pattern is similar to that shown by tyrosine-phosphorylated GSK3ß. Indeed, the relative level of tyrosine-phosphorylated GSK3ß changes little when compared to total kinase over the five days of NGF treatment (Ptyr and D). NGF induces the expression of a band that migrates significantly more slowly on SDS gels than the GSK3ß present prior to NGF addition (GSK3ß and F). These two bands can be resolved more clearly on a 10% acrylamide gel (C). GSK3ß phosphorylation on serine-9 was readily detected in cells prior to NGF addition; note that these cultures had not been incubated in low serum medium prior to NGF addition. GSK3ß phosphorylation on serine-9 was maintained for two to three days after which it decreased to approximately half of the initial levels after five DIV (Pser and E). (D-F) quantitative immunoblot analysis of the relative levels of tyrosine- (D) or serine- (E) phosphorylated GSK3ß and the proportion of GSK3ß showing reduced electrophoretic mobility (F) from PC12 cells treated with NGF from 0 to 5 days. Results are means±s.e.m. from three independent experiments.
