Resistance to phosphatase of thiophosphorylated epidermal growth factor receptor in A431 membranes.
Epidermal growth factor (EGF) increases the phosphorylation of its receptor and other membrane proteins, and these proteins can be rapidly dephosphorylated by membrane-bound protein phosphatase [Carpenter, G., King L., Jr., & Cohen, S. (1979) J. Biol. Chem. 254, 4884]. We report that [35S]-adenosine 5'-[gamma-thio]triphosphate is as effective as [gamma-32P]ATP as substrate for the EGF receptor-associated protein kinase in A431 membranes. Both the kinetics and the extent of the EGF-dependent thiophosphorylation at 0 degrees C are similar to those obtained with [gamma-32P]ATP, provided that ATP hydrolysis by the membrane preparation is inhibited by addition of adenosine 5'-[beta, gamma-imino]-triphosphate. The thiophosphorylation reaction requires Mn2+ but differs from the phosphorylation reaction in the inability of Mg2+ to serve as cofactor. Both EGF-dependent phosphorylated and thiophosphorylated membrane proteins yield the same two major bands of Mr 145,000-160,000 in autoradiograms of NaDodSO4/polyacrylamide gel electrophorograms. The rate of dephosphorylation of membrane proteins that have been thiophosphorylated in the presence of EGF is dramatically slower (factors of 1/20 to 1/40) than that of the phosphorylated proteins at both 0 degrees C and 32 degrees C. This increased metabolic stability of the thiophosphorylated proteins will be useful for investigation of the role of phosphorylation in the biological effects of EGF.
D000255:Adenosine Triphosphate; D000818:Animals; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D007457:Iodine Radioisotopes; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D013379:Substrate Specificity; D013462:Sulfur Radioisotopes
D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D013462:Sulfur Radioisotopes; D008345:Manganese; D004815:Epidermal Growth Factor; D000255:Adenosine Triphosphate; D010749:Phosphoprotein Phosphatases; D008274:Magnesium