Insulin and epidermal growth factor stimulate phosphorylation of a 170-kDa protein in intact hepatocytes immunologically related to lipocortin 1.
Lipocortin 1 is a steroid-induced, calcium-regulated membrane binding protein (Mr = 39,000) which is a substrate for the epidermal growth factor receptor kinase in intact cells. Using a polyclonal antibody to human recombinant lipocortin 1, we have identified a 170-kDa phosphoprotein in freshly isolated rat hepatocytes which shares antigenic determinants with lipocortin 1. The protein was recognized by four different anti-lipocortin 1 antisera, and antibody binding was inhibited by a 100-fold molar excess of human recombinant lipocortin 1 over antibody. Based on Coomassie Blue staining, the 170-kDa lipocortin-related protein is abundant (approximately 100 ng/10(6) cells) in rat liver, while lipocortin 1 itself is found in very low amounts. Epidermal growth factor and insulin stimulated phosphorylation of this 170-kDa protein in intact rat hepatocytes. The increase in phosphorylation was more pronounced in hepatocytes from dexamethasone-treated animals. The phosphorylation occurred exclusively on serine residues and was maximal 30-60 min after hormone addition. The 170-kDa protein was localized in the cytoplasm in the absence of calcium, while increasing calcium concentration led to partial association with the membrane compartment in rat liver. This 170-kDa protein represents a new member of the class of proteins whose serine phosphorylation is regulated by insulin and EGF and may belong to the family of lipocortin-related molecules.
Karasik
A
A
Research Division, Joslin Diabetes Center, Boston, Massachusetts 02215.
Pepinsky
R B
RB
Kahn
C R
CR
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D017302:Annexins; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D006023:Glycoproteins; D007328:Insulin / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D010740:Phospholipases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011506:Proteins / Q000276:immunology / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D000596:Amino Acids; D017302:Annexins; D006023:Glycoproteins; D007328:Insulin; D011506:Proteins; D004815:Epidermal Growth Factor; D010740:Phospholipases