Tyrosine phosphorylation of pp185 by insulin receptor kinase in a cell-free system.
Insulin treatment of rat H-35 hepatoma cells causes rapid tyrosine phosphorylation of a high molecular weight protein termed pp185 besides autophosphorylation of the beta-subunit of the insulin receptor (IR) in an intact cell system. To elucidate the molecular basis for tyrosine phosphorylation of pp185, cell-free phosphorylation of pp185 was performed using phosphotyrosine-containing proteins (PYPs) purified from detergent-solubilized cell lysates by immunoprecipitation with anti-phosphotyrosine antibody. After insulin treatment of cells, marked increases of tyrosine phosphorylation of pp185 and IR were observed compared to noninsulin-treated cells. Site-specific antibodies that specifically inactivate IR kinase inhibited tyrosine phosphorylation of pp185 as well as the beta-subunit of IR. PYPs purified from detergent-free cell extracts contained pp185 but little IR; tyrosine phosphorylation of pp185 did not occur. Addition of IR kinase purified from human placenta to these PYPs restored insulin-dependent tyrosine phosphorylation of pp185. These results suggest that tyrosine phosphorylation of pp185 is catalyzed directly by IR kinase in this cell-free system.
Tashiro-Hashimoto
Y
Y
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Tobe
K
K
Koshio
O
O
Izumi
T
T
Takaku
F
F
Akanuma
Y
Y
Kasuga
M
M
D000818:Animals; D000937:Antigen-Antibody Reactions; D002474:Cell-Free System; D015180:Electrophoresis, Gel, Two-Dimensional; D007328:Insulin / Q000494:pharmacology*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011972:Receptor, Insulin; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D007328:Insulin; D010750:Phosphoproteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin