Phosphorylation of the inhibitor-2 of protein phosphatase-1 by cdc2-cyclin B and GSK3.

cdc2-cyclin B activates protein phosphatase-1 (PP1) "in vitro", phosphorylates both catalytic subunit and inhibitor-2 (I2) and both processes are inhibited by a cdc2-inhibitory peptide. We compared the phosphorylation of I2 by cdc2-cyclin B and by the PP1-activator Glycogen Synthase Kinase 3 (GSK3). Each kinase introduced less than 0.1 mol phosphate/mol into I2 bound to PP1 and the same two tryptic phosphopeptides were obtained from I2, which contained phospho-T only. The same results were obtained also with isolated I2 phosphorylated by GSK3. Since GSK3 phosphorylates only T-72, cdc2-cyclin B is also likely to phosphorylate this site. This was confirmed by using I2 that had been mutated at this site. On the other hand cdc2-cyclin B introduced up to 0.8 mol/mol phosphate into isolated I2 and four phosphopeptides were obtained. The two new peptides contained phospho-T and one of them also phospho-S. These data indicate the presence of at least one T and one S that are phosphorylated only by cdc2-cyclin B and are accessible on isolated I2 only.