Tyrosine and threonine phosphorylation of an immunoaffinity-purified 44-kDa MAP kinase.
We have approached the functioning of a MAP kinase, which is thought to be a "switch kinase" in the phosphorylation cascade initiated from various receptor tyrosine kinases including the insulin receptor. To do so, antipeptide antibodies were raised against the C-terminal portion of ERK1 (extracellular signal-regulated kinase 1), a protein kinase belonging to the family of MAP kinases. With these antipeptide antibodies, we observed the following: (i) a 44-kDa protein can be specifically recognized both under native and denaturing conditions; (ii) a 44-kDa phosphoprotein can be revealed in 32P-labeled cells; its phosphorylation is stimulated by insulin, sodium orthovanadate, and okadaic acid; (iii) a MBP kinase activity can be precipitated, which phosphorylates MBP on threonine residues, and which is stimulated by insulin, sodium orthovanadate, okadaic acid, and fetal calf serum; (iv) this MBP kinase activity appears to be correlated with the in vivo induced phosphorylation of the 44-kDa protein. We next studied the in vitro phosphorylation of this 44-kDa/ERK1-immunoreactive protein. A time- and manganese-dependent phosphorylation was stimulated by the in vitro addition of sodium orthovanadate. Phosphoamino acid analysis of the in vitro phosphorylated 44-kDa protein revealed both threonine and tyrosine phosphorylation. Importantly, this in vitro phosphorylation of MAP kinase results in activation of phosphorylation of added MBP substrate. As a whole, our data indicate that the 44-kDa phosphoprotein identified by our antipeptide antibodies very likely corresponds to a MAP kinase.(ABSTRACT TRUNCATED AT 250 WORDS)
Scimeca
J C
JC
INSERM U145, Faculté de Médecine, Nice, France.
Ballotti
R
R
Nguyen
T T
TT
Filloux
C
C
Van Obberghen
E
E
D000818:Animals; D015153:Blotting, Western; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D005347:Fibroblasts / Q000201:enzymology; D051379:Mice; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008934:Mitogens / Q000494:pharmacology; D008970:Molecular Weight; D004676:Myelin Basic Protein / Q000378:metabolism; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D019000:Phosphotyrosine; D011494:Protein Kinases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D008934:Mitogens; D004676:Myelin Basic Protein; D010769:Phosphothreonine; D019000:Phosphotyrosine; D014443:Tyrosine; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases