IDENTIFICATION OF 2 ESSENTIAL PHOSPHORYLATED THREONINE RESIDUES IN THE CATALYTIC DOMAIN OF MEKK1 - INDIRECT ACTIVATION BY PAK3 AND PROTEIN-KINASE-C

The 78-kDa protein kinase Mekk1 plays an important role in the stress response pathway that involves the activation of downstream kinases Se k1 and stress-activated protein kinase/c-Jun NH2-terminal kinase. Cons erved serine and threonine residues located between the kinase subdoma ins VII and VIII of many protein kinases are phosphorylated for maxima l kinase activation. Two threonine residues within this region in Mekk 1 at positions 560 and 572, but not the serine at 557, were shown to b e essential for catalytic activity in this study. When these threonine residues were replaced with alanine, there was a significant loss in phosphotransferase activity toward the primary substrate, Sek1, and a large decrease in autophosphorylation activity. Site-directed mutagene sis demonstrated that these threonine residues cannot be replaced with either serine or glutamic acid for preservation of phosphotransferase activity. Further examination of the Mekk1 mutants isolated from P-32 -labeled transfected COS cells showed that Thr-560 and Thr-572 were in deed phosphorylated after two-dimensional tryptic-chymotryptic phospho peptide analysis. Additional determinants in the NH2-terminal domain o f Mekk1 also play a role in the regulation of Mekk1 activity. Although Pak3 and PKC can activate Mekk1 in vivo, this interaction is indirect and independent, since there was no direct phosphorylation of Mekk1 b y Pak3 or PKC or of Pak3 by PKC, respectively.