Constitutive mutant and putative regulatory serine phosphorylation site of mammalian MAP kinase kinase (MEK1).
In response to various external stimuli, MAP kinases are activated by phosphorylation on tyrosine and threonine by MAP kinase kinase (MAPKK), a dual specificity kinase. This kinase is in turn activated via Raf-1 and MAPKK kinase (MAPKKK). To determine regulatory phosphorylation sites of MAPKK, we isolated a Chinese hamster cDNA, that we epitope-tagged and expressed in fibroblasts. This hamster MAPKK (MEK1 isoform) can reactivate recombinant p44mapk when immunoprecipitated from growth factor-stimulated cells or when incubated with an active form of MAPKKK. Mutations at either of two residues that are conserved among kinases, D208N or S222A, abolished MAPKK activity. However, only S222A/MAPKK showed a reduction in phosphorylation in response to active MAPKKK and exerted a dominant negative effect on the serum-stimulated endogenous MAPKK. Finally, replacing Ser222 with Asp, a negatively charged residue, restored MAPKK activity independently of the upstream kinase. These results strongly suggest that Ser222 represents one key MAPKKK-dependent phosphorylation site switching on and off the activity of MAPKK, an event crucial for growth control.
Centre de Biochimie-CNRS, Université de Nice, France.
G
G
Pagès
A
A
Brunet
G
G
L'Allemain
J
J
Pouysségur
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006133:Growth Substances / Q000494:pharmacology; D006801:Humans; D048369:MAP Kinase Kinase 1; D020930:MAP Kinase Kinase Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism*
D018076:DNA, Complementary; D006133:Growth Substances; D011994:Recombinant Proteins; D012694:Serine; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases