The 78-kDa protein kinase Mekk1 plays an important role in the stress response pathway that involves the activation of downstream kinases Sek1 and stress-activated protein kinase/c-Jun NH2-terminal kinase. Conserved serine and threonine residues located between the kinase subdomains VII and VIII of many protein kinases are phosphorylated for maximal kinase activation. Two threonine residues within this region in Mekk1 at positions 560 and 572, but not the serine at 557, were shown to be 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 mutagenesis 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 32P-labeled transfected COS cells showed that Thr-560 and Thr-572 were indeed phosphorylated after two-dimensional tryptic-chymotryptic phosphopeptide analysis. Additional determinants in the NH2-terminal domain of 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 by Pak3 or PKC or of Pak3 by PKC, respectively.