Zipper-mediated oligomerization of the mixed lineage kinase SPRK/MLK-3 is not required for its activation by the GTPase cdc 42 but Is necessary for its activation of the JNK pathway. Monomeric SPRK L410P does not catalyze the activating phosphorylation of Thr258 of murine MITOGEN-ACTIVATED protein kinase kinase 4.
Src homology 3 domain-containing proline-rich kinase (SPRK)/mixed lineage kinase-3 is a serine/threonine kinase that has been identified as an upstream activator of the c-Jun NH(2)-terminal kinase (JNK) pathway. SPRK is capable of activating MKK4 by phosphorylation of serine and threonine residues, and mutant forms of MKK4 that lack the phosphorylation sites Ser(254) and Thr(258) block SPRK-induced JNK activation. A region of 63 amino acids following the kinase domain of SPRK is predicted to form a leucine zipper. The leucine zipper domain of SPRK has been shown to be necessary and sufficient for SPRK oligomerization, but its role in regulating activation of SPRK and downstream signaling remains unclear. In this study, we substituted a proposed stabilizing leucine residue in the zipper domain with a helix-disrupting proline to abrogate zipper-mediated SPRK oligomerization. We demonstrate that constitutively activated Cdc42 fully activates this monomeric SPRK mutant in terms of both autophosphorylation and histone phosphorylation activity and induces the same in vivo phosphorylation pattern as wild type SPRK. However, this catalytically active SPRK zipper mutant is unable to activate JNK. Our data show that the monomeric SPRK mutant fails to phosphorylate one of the two activating phosphorylation sites, Thr(258), of MKK4. These studies suggest that zipper-mediated SPRK oligomerization is not required for SPRK activation by Cdc42 but instead is critical for proper interaction and phosphorylation of a downstream target, MKK4.
Vacratsis
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Departments of Biochemistry and Physiology, Michigan State University, East Lansing, Michigan 48824, USA.
Gallo
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KA
D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D016350:Leucine Zippers; D048670:MAP Kinase Kinase 4; D020930:MAP Kinase Kinase Kinases / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D017354:Point Mutation; D055503:Protein Multimerization; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013912:Threonine; D014162:Transfection; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*; D018909:src Homology Domains; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11
D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020930:MAP Kinase Kinase Kinases; D046911:Macromolecular Substances; D020929:Mitogen-Activated Protein Kinase Kinases; D020928:Mitogen-Activated Protein Kinases; D010748:Phosphopeptides; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins; D013912:Threonine; D020764:cdc42 GTP-Binding Protein; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11; C482559:MAP2K4 protein, human; C482562:Map2k4 protein, mouse