THE MEK KINASE-ACTIVITY OF THE CATALYTIC DOMAIN OF RAF-1 IS REGULATEDINDEPENDENTLY 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 liberat ed Raf-1 catalytic domain in fibroblast cell lines. We demonstrate tha t 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. Increa sing intracellular cAMP inhibits Raf-1 activation stimulated by phorbo l myristate acetate, but not the activation of Raf-BXB. Serine 621, bu t not serine 499, is essential for Raf-BXB MEK kinase activity. Becaus e Raf-BXB does not bind Ras, the data establishes a Ras-independent si gnal in directly regulating the activity of the Raf-1 catalytic domain .