Tyrosine phosphorylation of two cytosolic proteins of 50 kDa and 35 kDa in rat liver by insulin-receptor kinase in vitro.
Insulin-receptor tyrosine kinase can phosphorylate a variety of artificial substrates in vitro. Its physiological substrate(s), however, remains unknown. In the present study, we show that immobilized insulin receptors phosphorylate tyrosine residues of two cytosolic proteins of 50 kDa and 35 kDa in rat liver. Phosphorylation of these two proteins required Mn2+- or Mg2+-ATP as the phosphate donor. Phosphorylation was time- and temperature-dependent. Furthermore, the rate of phosphorylation of the two proteins was related to the autophosphorylated state of the insulin receptor. The pI of the phosphorylated 50 kDa and 35 kDa proteins was 5.4 and 5.6 respectively. These proteins were present in low abundance. They were not related to each other, nor to the insulin receptor, as demonstrated by in-gel proteolytic digestion and by immunoprecipitation using antibodies produced against them. They were specific substrates for the insulin receptor kinase, since they were not phosphorylated by epidermal-growth-factor-receptor kinase. These observations suggest that the 50 kDa and 35 kDa cytosolic proteins may be endogenous substrates for the insulin-receptor kinase.
Kwok
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YC
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Yip
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CC
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D011232:Chemical Precipitation; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007122:Immunoelectrophoresis; D066298:In Vitro Techniques; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D010750:Phosphoproteins / Q000276:immunology; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin
D000596:Amino Acids; D010750:Phosphoproteins; D011506:Proteins; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin