title: Resistance to phosphatase of thiophosphorylated epidermal growth factor receptor in A431 membranes.
abstract: 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.
mesh_terms: 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
chemical_list: D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D013462:Sulfur Radioisotopes; D008345:Manganese; D004815:Epidermal Growth Factor; D000255:Adenosine Triphosphate; D010749:Phosphoprotein Phosphatases; D008274:Magnesium