Lipocortins 1 and 2 as substrates for the insulin receptor kinase in rat liver.
Lipocortins 1 and 2 are major substrates for the epidermal growth factor receptor and the pp60v-src tyrosine kinases in transformed cells. In the present study, we have characterized the phosphorylation of lipocortins 1 and 2 by the insulin receptor tyrosine kinase in vitro and in vivo. In vitro, the solubilized insulin receptor, partially purified from rat liver, catalyzed phosphorylation of human recombinant lipocortin 1 and purified bovine lipocortin 2. Phosphorylation of lipocortin 1 was increased 15-fold upon stimulation with 10(-7) M insulin. The apparent Km of the reaction was 3.3 microM and was not affected by insulin stimulation. Insulin stimulated phosphate incorporation into lipocortin 2 by 20-fold (apparent Km greater than 20 microM). Both lipocortins were phosphorylated exclusively on tyrosine residues as judged by phosphoamino acid analysis. Based upon peptide mapping, lipocortin 1 was phosphorylated on Tyr-21, a site phosphorylated by other tyrosine kinases. Polyclonal anti-phosphotyrosine antibodies recognized the tyrosine-phosphorylated lipocortin 2, but not lipocortin 1 in its phosphorylated form. In hepatocytes from normal and dexamethasone-treated rats, lipocortin 1 content was less than 50 ng/10(6) cells. Insulin-induced phosphorylation of lipocortin 1 was detected in intact hepatocytes from corticosteroid-treated animals but not in cells from normal rats. No phosphorylation of lipocortin 2 was found, although its content was approximately 100 ng/10(6) cells from normal animals and increased to approximately 1 microgram/10(6) cells following treatment of rats with dexamethasone for 4 days. Thus, although lipocortins 1 and 2 are in vitro substrates of the insulin receptor kinase, only lipocortin 1 is phosphorylated in an insulin-dependent manner in intact hepatocytes, and this is only observed after dexamethasone treatment of the rats.
Karasik
A
A
Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02215.
Pepinsky
R B
RB
Shoelson
S E
SE
Kahn
C R
CR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017302:Annexins; D002118:Calcium / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000378:metabolism*; D007163:Immunosorbent Techniques; D007328:Insulin / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008345:Manganese / Q000494:pharmacology; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D017302:Annexins; D006023:Glycoproteins; D007328:Insulin; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D008345:Manganese; D003907:Dexamethasone; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin; D002118:Calcium