The Mekl-Erk1 node : A place of convergence

At present, five mitogen-activated protein (MAP) kinase families have been identified in vertebrates. At least three of these MAP kinases have been shown to operate within highly conserved intracellular signalling modules composed of sequentially activating protein kinases. Cell surface receptors in response to extracellular stimuli invoke MAP kinase cascades to regulate gene expression within the nucleus. The best characterized MAP kinases are the extracellular signal-regulated kinases (Erks), the jun N-terminal kinases (Jnks) and the high osmolarity glycerol kinases (Hogs). Mitogenic signals that stimulate the G protein Ras activate the Raf->Mek->Erk->Rsk module. In turn, the Nik->Mekkl->Mkk4->Jnk and the Mkk3->Hog->MAPKAPK modules respond to environmental stress signals. Recent progress in MAP kinase signalling has revealed that MAPKs respond to a variety of environmental cues that promote cell growth, differentiation and death. The experimental work described in this doctoral thesis was initiated six years ago as an independent study into the regulation of MAP kinase. At the time, purified MAP kinase was shown to be a prolyl-directed seryl/threonyl kinase that was regulated by phosphorylation on tyrosyl and threonyl residues. In addition, a partial cDNA sequence for a MAP kinase termed Erk1 was shown to be homologous to Fus3 (for fusion), a kinase that regulated the mating pathway in Saccharomyces cerevisiae. Results from genetic epistasis experiments revealed that Fus3 was positioned downstream in a cascade that involved several protein kinases. Consequently, we hypothesized that MAP kinase probably functioned as a key intermediary in the transmission of information from the plasma membrane to the nucleus. To understand the function MAP kinase plays in regulating the flow of information from plasma membrane to the transcriptional machinery in the nucleus, a cDNA clone of Erk1 was isolated from a human liver library. The mRNA message encoded a 379-amino acid protein which was 12 residues longer (MAAAAAQGGGGG) than Erk1 described previously from rat and, therefore, represented the full-length sequence. By using recombinant Erk1 protein, a MAP kinase activator activity was purified from 1-MeAde-treated sea star oocytes. Immunoblotting with antipeptide antibodies directed against mouse Mek1 (MAP/Erk kinase) and yeast Ste7 (Sterile 7) revealed that the sea star activator protein was a Mek1 homologue. Unexpectedly, the purified activator was able to increase Erk1 MBP phosphotransferase activity without directly phosphorylating the enzyme. This contrasts results obtained by other researcher groups that showed direct phosphorylation by the protein kinase activator Mek1 leads to Erk1 activation. Therefore, the sea star Mek-like protein may activate human Erk1 by a different mechanism. The data presented here indicates that sea star Mek may activate human Erkl via allosteric modulation. MAP kinase family subgroups Erk, Jnk and Hog are activated by phosphorylation on threonyl and tyrosyl residues that are located within a tripeptide Thr- Xaa-Tyr (where the intervening residue Xaa codes for Glu in Erk1, Gly in Hog and Pro in Jnk) regulatory motif. Phosphorylation of these sites in MAP kinases is performed by one of two specific pairs of MAP kinase kinases. Erk1 and Erk2 are the only known substrates for Mek1 and Mek2. A similar narrow specificity has been observed for Jnks and Hogs by their immediate activators Mkk4/Mkk7 and Mkk3/Mkk6, respectively. A systematic analysis of the TEY tripeptide sequence in Erk1 was undertaken to understand the importance each residue plays in the substrate specificity of mammalian Mek1. Constitutively active mouse Mek1 was used to phosphorylate a battery of threonyl and tyrosyl phosphorylation site mutants. Mutation of either Thr-202 or Tyr- 204 reduced the efficiency at which Mek1 was able to phosphorylate the Erk1 mutant proteins. In fact, serine substitution for Thr-202 was the only allele that retained MBP kinase activity. The intervening residue located between the regulatory threonyl and tyrosyl phosphorylation sites of MAP kinase may also serve as an important specificity determinant to prevent inappropriate cross-phosphorylation by MAP kinase kinases from parallel modules. The replacement of Erk1 TEY motif with the Jnk (TPY) and Hog (TGY) regulatory sites also reduced Mek1 specificity and markedly decreased Erk MBP phosphotransferase activity. Taken together, these results indicate that the TEY motif in Erk1 is an important consensus sequence for Mek1 recognition.Medicine, Faculty ofExperimental Medicine, Division ofMedicine, Department ofGraduat