Temperature-dependent tyrosine phosphorylation of microtubule-associated protein kinase in epidermal growth factor-stimulated human fibroblasts.
Treatment of normal human fibroblasts with epidermal growth factor (EGF) results in the rapid (0.5 min) and simultaneous tyrosine phosphorylation of the EGF receptor (EGFr) and several other proteins. An exception to this tyrosine phosphorylation wave was a protein (42 kDa) that became phosphorylated on tyrosine only after a short lag time (5 min). We identified this p42 kDa substrate as the microtubule-associated protein (MAP) kinase using a monoclonal antibody to a peptide corresponding to the C-terminus of the predicted protein (Science 249, 64-67, 1990). EGF treatment of human fibroblasts at 37 degrees C for 5 min resulted in the tyrosine phosphorylation of 60-70% of MAP kinase as determined by the percent that was immunoprecipitated with antiphosphotyrosine antibodies. Like other tyrosine kinase growth factor receptors, the EGFr is activated and phosphorylated at 4 degrees C but is not internalized. Whereas most other substrates were readily tyrosine phosphorylated at 4 degrees C, MAP kinase was not. When cells were first stimulated with EGF at 4 degrees C and then warmed to 37 degrees C without EGF, tyrosine phosphorylation of MAP kinase was again observed. Treatment of cells with the protein kinase C activator phorbol myristate acetate (PMA) also resulted in the tyrosine phosphorylation of MAP kinase, and again only at 37 degrees C. Tryptic phosphopeptide maps demonstrated that EGF and PMA both induced the phosphorylation of the same peptide on tyrosine and threonine. This temperature and PMA sensitivity distinguishes MAP kinase from most other tyrosine kinase substrates in activated human fibroblasts.
Campos-González
R
R
Lucille P. Markey Cancer Center, University of Kentucky, Lexington 40536-0084.
Glenney
J R
JR
D000595:Amino Acid Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002460:Cell Line / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010703:Phorbol Esters / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification*; D013696:Temperature; D014443:Tyrosine / Q000378:metabolism*
D010703:Phorbol Esters; D014443:Tyrosine; D004815:Epidermal Growth Factor; D011494:Protein Kinases; D066246:ErbB Receptors; D017871:Calcium-Calmodulin-Dependent Protein Kinases