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'', phos phorylates both catalytic subunit and inhibitor-2 (I2) and both proces ses are inhibited by a cdc2-inhibitory peptide. We compared the phosph orylation of I2 by cdc2-cyclin B and by the PP1-activator Glycogen Syn thase Kinase 3 (GSK3). Each kinase introduced less than 0.1. mol phosp hate/mol into I2 bound to PPI and the same two tryptic phosphopeptides were obtained from I2, which contained phospho-T only. The same resul ts were obtained also with isolated I2 phosphorylated by GSK3. Since G SK3 phosphorylates only T-72, cdc2-cyclin B is also likely to phosphor ylate 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 phosph o-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 isolat ed I2 only. (C) 1995 Academic Press, Inc.