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.