Autophosphorylation in vitro of recombinant 42-kilodalton mitogen-activated protein kinase on tyrosine.
Mitogen-activated protein kinase (MAP kinase) is a serine/threonine protein kinase that becomes enzymatically activated and phosphorylated on tyrosine and threonine following treatment of quiescent cells with a variety of stimulatory agonists. Phosphorylation on both tyrosine and threonine is necessary to maintain full activity, and these two regulatory phosphorylations occur close to each other, separated by a single glutamate. To study the mechanisms by which MAP kinase becomes phosphorylated and activated, we have cloned a full-length cDNA encoding MAP kinase and have expressed the enzyme in Escherichia coli as a soluble nonfusion protein. We find that the enzyme displays a basal, intramolecular autophosphorylation on tyrosine-185 that is accompanied by activation of the enzyme's kinase activity towards an exogenous substrate. The tyrosine-phosphorylated protein displays a small fraction of the activity seen with the fully activated, doubly phosphorylated enzyme isolated from mammalian cells but is activated 10- to 20-fold relative to the unphosphorylated enzyme. These findings raise the possibility that regulation of MAP kinase activity in response to agonist stimulation could occur in part through the enhancement of autophosphorylation on tyrosine.
Wu
J
J
Department of Microbiology, University of Virginia, Charlottesville 22908.
Rossomando
A J
AJ
Her
J H
JH
Del Vecchio
R
R
Weber
M J
MJ
Sturgill
T W
TW
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004252:DNA Mutational Analysis; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013696:Temperature
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D010769:Phosphothreonine; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli