Identification of Tyr-185 as the site of tyrosine autophosphorylation of recombinant mitogen-activated protein kinase p42mapk.
Tyrosine phosphorylation of 42-kDa mitogen-activated protein kinase (p42mapk) occurs during expression of the recombinant protein in Escherichia coli, as well as during in vitro phosphorylation of the protein purified from this source. Structural analyses were performed to identify the site(s) of tyrosine phosphorylation of recombinant p42mapk, both during expression of the protein in E. coli and during in vitro incubations with ATP/Mg2+/Mn2+. Mass spectrometry and phosphopeptide mapping showed that tyrosine phosphorylation of recombinant p42mapk occurs on Tyr-185, the site of regulatory tyrosine phosphorylation that occurs in mitogen-stimulated mammalian cells.
Rossomando
A J
AJ
Department of Microbiology, University of Virginia, Charlottesville, VA 22908.
Wu
J
J
Michel
H
H
Shabanowitz
J
J
Hunt
D F
DF
Weber
M J
MJ
Sturgill
T W
TW
D000595:Amino Acid Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D013058:Mass Spectrometry; D019950:Mitogen-Activated Protein Kinase 1; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D019000:Phosphotyrosine; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D011506:Proteins; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1