TYROSINE PHOSPHORYLATION OF PHOSPHATASE INHIBITOR-2

Inhibitor 2 is a heat-stable protein that complexes with the catalytic subunit of type-1 protein phosphatase. The reversible phosphorylation of Thr 72 of the inhibitor in this complex has been shown to regulate phosphatase activity. Here we show that inhibitor 2 can also be phosp horylated on tyrosine residues. Inhibitor 2 was P-32-labeled by the in sulin receptor kinase in vitro, in the presence of polylysine. Phospho rylation of inhibitor 2 was accompanied by decreased electrophoretic m obility. Dephosphorylation of inhibitor 2 by tyrosine phosphatase 1B, restored normal electrophoretic mobility. Phosphotyrosine in inhibitor 2 was detected by immunoblotting with antiphosphotyrosine antibodies and phosphoamino acid analysis. In addition, following tryptic digesti on, one predominant phosphopeptide was recovered at the anode. The abi lity of inhibitor 2 to inhibit type-1 phosphatase activity was diminis hed with increasing phosphorylation up to a stoichiometry of 1 mole ph osphate incorporated/mole of inhibitor 2, where inhibitory activity wa s completely lost. These data demonstrate that inhibitor 2 can be phos phorylated on tyrosine residues by the insulin receptor kinase, result ing in a molecule with decreased ability to inhibit type-1 phosphatase activity. (C) 1995 Wiley-Liss, Inc.