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 phosphorylated on tyrosine residues. Inhibitor 2 was 32P-labeled by the insulin receptor kinase in vitro, in the presence of polylysine. Phosphorylation of inhibitor 2 was accompanied by decreased electrophoretic mobility. 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 digestion, one predominant phosphopeptide was recovered at the anode. The ability of inhibitor 2 to inhibit type-1 phosphatase activity was diminished with increasing phosphorylation up to a stoichiometry of 1 mole phosphate incorporated/mole of inhibitor 2, where inhibitory activity was completely lost. These data demonstrate that inhibitor 2 can be phosphorylated on tyrosine residues by the insulin receptor kinase, resulting in a molecule with decreased ability to inhibit type-1 phosphatase activity.
Williams
J P
JP
Department of Pathology, University of Alabama at Birmingham 35294, USA.
Jo
H
H
Hunnicutt
R E
RE
Brautigan
D L
DL
McDonald
J M
JM
D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors; D006801:Humans; D009124:Muscle Proteins / Q000737:chemistry*; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry*; D011972:Receptor, Insulin / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*; D000097847:Protein Phosphatase Inhibitory Proteins
D004791:Enzyme Inhibitors; D009124:Muscle Proteins; D011506:Proteins; D011972:Receptor, Insulin; D014443:Tyrosine; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins