REGULATION OF THE ACTIVITY OF MEK-KINASE-1 (MEKK1) BY AUTOPHOSPHORYLATION WITHIN THE KINASE ACTIVATION DOMAIN

MEK kinase 1 (MEKK1) shares sequence identity with the yeast kinases S tell and Byr2, and is capable of phosphorylation and activation of bot h mitogen-activated protein/extracellular signal-related protein kinas e (MAP/ERK) kinase (MEK) and stress-activated protein kinase (SAPK)/ER K kinase (SEK) in vitro. In vivo, however, MEKK1 predominantly activat es the SEK/SAPK kinase cascade. Mechanisms of activation of MEKK1 are unclear. We have identified a major site of autophosphorylation (Thr-5 75) within the 'activation loop' of MEKK1 between the kinase subdomain s VII and VIII. Phosphatase treatment of a constitutively active MEKK1 decreased kinase activity by 59%. Dephosphorylated T575 was rapidly r e-(auto)phosphorylated by MEKK1. Mutation of T575 to alanine decreased MEKK1 transphosphorylation activity with a SEK substrate to approx. 3 0% of wild-type. Mutation of a second threonine residue (Thr-587) to a lanine eliminated the phosphorylation of MEK or SEK substrate but not autophosphorylation. MEKK1 autophosphorylation is an intramolecular re action because active MEKK1 cannot transphosphorylate a kinase-inactiv e MEKK1. Inactive MEKK1 was not phosphorylated on Thr-575 within cells , suggesting that the phosphorylation of Thr-575 in vivo results from autophosphorylation rather than phosphorylation by an upstream kinase. Autoactivation of MEKK1 via autophosphorylation of Thr-575 might be a n immediate response to initial kinase activation through non-phosphor ylation mechanisms.