Fig. 4. GSK3ß activity increases during PC12 cell differentiation. (A) The GSK3ß activity in extracts from untreated PC12 cells and from cells exposed to NGF for five days was determined using a recombinant fragment of MAP1B (1B750) as a substrate. Phosphorylation was assayed by immunoblotting with mAb SMI-31 (1B750-P); recombinant protein was detected with anti-GST serum (1B750). Activity was virtually undetectable in unstimulated cells (DIV 0), but was readily detected five days after NGF addition (DIV 5). Inhibition of this activity by LiCl confirms that GSK3 is the kinase phosphorylating MAP1B in these cell extracts (DIV 5, + Li+). This increase in activity is not related to an increase in GSK3ß expression, which shows only a modest increase over the same period (GSK3ß). (B) GSK3ß activity towards recombinant MAP1B (1B750) in extracts from PC12 cells exposed to NGF from zero to five days was assayed as above. The level of phosphorylated 1B750 was divided by the level of GSK3ß, each determined by immunoblotting and normalised to five DIV, to give a measure of the activity of GSK3ß relative to its abundance during differentiation. Results are means±s.e.m. from four independent experiments. The increase in kinase activity induced by NGF reflects closely the level of endogenous MAP1B-P (Fig. 1).
