The kinase activation loop is the key to mixed lineage kinase-3 activationvia both autophosphorylation and hematopoetic progenitor kinase 1 phosphorylation

We have demonstrated previously that Cdc42 induced MLK-3 homodimerization l eads to both autophosphorylation and activation of MLK-3 and postulated tha t auto phosphorylation is an intermediate step of MLK-3 activation followin g its dimerization, In this report we sought to refine further the mechanis m of MLK-3 activation and study the role of the putative kinase activation loop in MLK-3 activation. First we mutated the three potential phosphorylat ion sites in MLK-3 putative activation loop to alanine in an effort to abro gate MLK-3 autophosphorylation, Mutant T277A displayed almost no autophosph orylation activity and was nearly nonfunctional; mutant S281A, that display ed a low level of autophosphorylation, only slightly activated its downstre am targets, whereas the T278A mutant, that exhibited autophosphorylation co mparable to that of the wild type, was almost fully functional. Thus, these residues within the activation loop are critical for MLK-3 autophosphoryla tion and activation. In addition, when the Thr(277) and Ser(281) residues w ere mutated to negatively charged glutamic acid to mimic phosphorylated ser ine/threonine residues, the resulting mutants were fully functional, implyi ng that these two residues may serve as the autophosphorylation sites. Inte restingly, HPK1 also phosphorylated MLK-3 activation loop in vitro, and Ser 281 was found to be the major phosphorylation site, indicating that HPK1 al so activates MLK-3 via phosphorylation of the kinase activation loop.