Mitogen-induced tyrosine-phosphorylated 41- and 43-kDa proteins are family members of extracellular signal-regulated kinases/microtubule-associated protein 2 kinases.
Two antipeptide antibodies, one against the peptide corresponding to residues 307-327 (alpha Y91) and one against the peptide corresponding to the C-terminal portion (alpha C92) of the deduced amino acid sequence of the extracellular signal-regulated kinase 1 (ERK1), precipitated two 41-kDa and/or two 43-kDa phospho-proteins from mitogen-stimulated Swiss 3T3 cells. Electrophoretic mobilities on two-dimensional gels of the immunoprecipitated 41- and 43-kDa phosphoproteins were similar to those of the 41- and 43-kDa cytosol proteins, whose increased tyrosine phosphorylation we and others had originally identified in various mitogen-stimulated cells (Cooper, J. A., Sefton, B. M., and Hunter, T. (1984) Mol. Cell. Biol. 4, 30-37; Kohno, M. (1985) J. Biol. Chem. 260, 1771-1779); phosphopeptide map analysis revealed that they were respectively identical molecules. All those phosphoproteins contained phosphotyrosine, and the more acidic forms contained additional phosphothreonine. Immunoprecipitated 41- and 43-kDa phosphoproteins had serine/threonine kinase activity toward myelin basic protein (MBP) and microtuble-associated protein 2 (MAP2). With the combination of two-dimensional gel electrophoresis and the kinase assay in MBP-containing polyacrylamide gels of the alpha Y91 immunoprecipitates, with or without phosphatase 2A treatment, we showed that only their acidic forms were active. These results clearly indicate that 41- and 43-kDa proteins, the increased tyrosine phosphorylation of which is rapidly and commonly induced by mitogen stimulation of fibroblasts, are family members of ERKs/MAP2 kinases and that phosphorylation both on tyrosine and threonine residues is necessary for their activation.
Chatani
Y
Y
Department of Biology, Gifu Pharmaceutical University, Japan.
Tanaka
E
E
Tobe
K
K
Hattori
A
A
Sato
M
M
Tamemoto
H
H
Nishizawa
N
N
Nomoto
H
H
Takeya
T
T
Kadowaki
T
T
D016475:3T3 Cells; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D051379:Mice; D008970:Molecular Weight; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D014443:Tyrosine
D000596:Amino Acids; D010750:Phosphoproteins; D010982:Platelet-Derived Growth Factor; D016222:Fibroblast Growth Factor 2; D014443:Tyrosine; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases