Epidermal growth factor, but not insulin, stimulates tyrosine phosphorylation of an endogenous protein of Mr 95,000 in triton extracts of human placental syncytiotrophoblast membranes.
1. Triton extracts of syncytiotrophoblast membranes were incubated with [gamma-32P]ATP, MgCl2 and MnCl2. Addition of epidermal growth factor (EGF) resulted in increased phosphorylation not only of the EGF receptor and a Mr-35,000 protein as previously described, but also a protein of Mr 95,000 on both tyrosine and serine residues. In addition, a small increase in the phosphorylation of a protein of Mr 105,000 was observed. Spermine had a similar effect on the phosphorylation of the Mr-95,000 protein, without affecting the phosphorylation of the other proteins. In the absence of MnCl2, the effect of spermine on the phosphorylation of Mr-95,000 protein was still evident, whereas that of EGF was greatly diminished. 2. The Mr-95,000 protein bound poorly to wheat-germ-lectin-Sepharose and was not precipitated by antisera specific for insulin and EGF receptors. The protein continued to exhibit serine and tyrosine phosphorylation on addition of [gamma-32P]ATP, MgCl2 and MnCl2 to a glycoprotein-depleted fraction prepared by chromatography on wheat-germ-lectin-Sepharose. The extent of phosphorylation was no longer increased by spermine or EGF, but was inhibited by heparin. 3. It is suggested that the Mr-95,000 protein not only is a possible direct substrate for the EGF-receptor (but not the insulin receptor) tyrosine kinase but is a substrate for other endogenous kinases, including a protein tyrosine kinase which is probably not a glycoprotein, and a protein serine kinase with properties similar to those of casein kinase II.
Tavaré
J M
JM
Department of Biochemistry, University of Bristol Medical School, U.K.
Diggle
T A
TA
Denton
R M
RM
D000596:Amino Acids / Q000032:analysis; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D013096:Spermine / Q000494:pharmacology; D013268:Stimulation, Chemical; D014327:Trophoblasts / Q000187:drug effects / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D000596:Amino Acids; D007328:Insulin; D011506:Proteins; D013096:Spermine; D014443:Tyrosine; D004815:Epidermal Growth Factor; D006493:Heparin