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.
Puntoni
F
F
Department of Biomedicine, University of Pisa, Italy.
Villa-Moruzzi
E
E
D000818:Animals; D016203:CDC2 Protein Kinase / Q000302:isolation & purification / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D016213:Cyclins / Q000302:isolation & purification / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D038341:Glycogen Synthase Kinases; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D010766:Phosphorylation; D017354:Point Mutation; D054645:Protein Phosphatase 1; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D016213:Cyclins; D010748:Phosphopeptides; D011994:Recombinant Proteins; D038341:Glycogen Synthase Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D016203:CDC2 Protein Kinase; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1