Insulin-stimulated tyrosine phosphorylation of a 43 kDa protein in rat liver membranes.
The insulin receptor (IR) tyrosine kinase is essential for the regulation of different cellular functions by insulin. This may occur by a direct phosphorylation of membrane and/or cytoplasmic proteins by the IR tyrosine kinase. Hence it is important to identify putative physiological substrates for the IR tyrosine kinase. In this study we found that the glycoprotein fraction from rat liver membranes contain a 43 kDa protein (pp43) which, like the beta-subunit of IR, is phosphorylated in an insulin-dependent manner. A 25-fold enhancement of 32P incorporation into pp43 by insulin was found under optimal conditions. Half-maximal phosphorylation of pp43 and the beta-subunit of IR were attained at 66 nM and 60 nM insulin, respectively. Mn2+ (Ka = 1.0 mM) was much better than Mg2+ (Ka = 6.3 mM) in supporting pp43 phosphorylation. Insulin-stimulated phosphorylation of pp43 (t1/2 = 3.6 min) proceeded at a much slower rate compared to that of the beta-subunit of IR (t1/2 = 1.2 min). Phosphoamino acid analysis of pp43 revealed that both tyrosine and serine are phosphorylated in the ratio 4:1. Tyrosine, but not serine, phosphorylation was increased 12-fold by insulin. Phosphorylation of pp43 occurred on 4 major tryptic peptides. Comparison to the tryptic phosphopeptides from IR beta-subunit suggest that pp43 was not derived from IR beta-subunit by proteolysis. Our results suggest that pp43 may be an endogenous substrate for the IR tyrosine kinase.
Klee
U
U
Department of Biology, University of Waterloo, Ontario, Canada.
Singh
T J
TJ
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D007328:Insulin / Q000494:pharmacology*; D008565:Membrane Proteins / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism*; D008970:Molecular Weight; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D015183:Restriction Mapping; D012694:Serine / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism*
D007328:Insulin; D008565:Membrane Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D012694:Serine; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin