The MEK kinase activity of the catalytic domain of RAF-1 is regulated independently of Ras binding in T cells.
Deletion of the amino-terminal domain of Raf-1, which contains the Ras-binding region, results in the constitutive activation of the liberated Raf-1 catalytic domain in fibroblast cell lines. We demonstrate that the MEK kinase activity of the isolated Raf-1 catalytic domain, Raf-BXB, is not constitutively active, but is regulated in Jurkat T cells. Raf-BXB is activated by engaging the antigen receptor-CD3 complex, or treating cells with phorbol myristate acetate or okadaic acid. Increasing intracellular cAMP inhibits Raf-1 activation stimulated by phorbol myristate acetate, but not the activation of Raf-BXB. Serine 621, but not serine 499, is essential for Raf-BXB MEK kinase activity. Because Raf-BXB does not bind Ras, the data establishes a Ras-independent signal in directly regulating the activity of the Raf-1 catalytic domain.
Whitehurst
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CE
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-8884.
Owaki
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H
Bruder
J T
JT
Rapp
U R
UR
Geppert
T D
TD
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002460:Cell Line; D004789:Enzyme Activation; D000939:Epitopes / Q000032:analysis; D004988:Ethers, Cyclic / Q000494:pharmacology; D006388:Hemagglutinins / Q000276:immunology; D006801:Humans; D048728:MAP Kinase Kinase Kinase 1; D008387:Marine Toxins; D051379:Mice; D008969:Molecular Sequence Data; D019319:Okadaic Acid; D009842:Oligopeptides / Q000276:immunology; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D018631:ras Proteins / Q000378:metabolism*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000939:Epitopes; D004988:Ethers, Cyclic; D006388:Hemagglutinins; D008387:Marine Toxins; D009842:Oligopeptides; D010080:Oxazoles; D011518:Proto-Oncogene Proteins; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D019319:Okadaic Acid; C059041:calyculin A; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human; C482578:Map3k1 protein, mouse; D010749:Phosphoprotein Phosphatases; D018631:ras Proteins; D013755:Tetradecanoylphorbol Acetate