New insights into MKK7 regulation in mammalian cells

Identification of a regulated site in MKK7 activation loop Abstract: Eukaryotic cells respond to external stimuli by activating mitogen-activated protein kinase (MAPK) pathways. MAPK signaling pathways include the ERK, p38 and JNK cascades. All MAPK family members are activated by the dual phosphorylation of a tripeptide motif located in their activation loop. JNK is phosphorylated and activated by two upstream MAPKKs, termed MKK4 and MKK7. These kinases are themselves activated through phosphorylation of serine/threonine residues in their activation loop by upstream MAPKKKs. MKK7 is thought to be activated primarily by cytokines while MKK4 is primarily activated by environmental stress. For my M.Sc. project, I have validated and characterized several phospho-MKK7 specific antibodies directed against MKK7 Ser271, Thr275 and Ser277, which are phospho-acceptor residues located in the MKK7 activation loop. By producing MKK7 mutant constructs in which each of the phospho-acceptor sites were substituted with Ala or Asp, I have demonstrated that we succeeded in generating phosphospecific antibodies against each of the phospho-acceptor sites. Of the three sites, only Thr275 is regulated by external stimuli whereas Ser271 and Ser277 appear to be constitutively phosphorylated. Both TNFoc and sorbitol treatment induce phosphorylation of the Thr275 residue in MKK7 activation loop; surprisingly, our results show that osmotic stress activates MKK7 more strongly than TNFa. By using RNA interference and immunoprecipitation, we have demonstrated that in endogenously expressed MKK7, Ser271 and Ser277 are constitutively phosphorylated while Thr275 is stimuli-regulated. Together, these studies provide fundamental new insights into MKK7 regulation in mammalian cells