Worm Breeder's Gazette 13(5): 24 (February 1, 1995)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
|1||Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, CO 80309.|
|2||Department of Biological Chemistry and the Institute of Gerontology, University of Michigan Medical School, Ann Arbor, MI 48109|
Activated Ras initiates a cascade of sequential phosphorylation events, including the protein kinases Raf, MEK and MAP kinase. The Let-60 Ras mediated signal transduction pathway controls vulval induction in Caenorhabditis elegans. Both Lin-45 Raf and Sur-1 MAP kinase have been determined to be essential factors during vulval induction, however, the C. elegans mek gene has not been identified. In this report, we have cloned a C. elegans mek gene, mek-2, and demonstrated that the Mek-2 protein possesses the biochemical properties of MAP kinase kinases: the C. elegans Mek-2 protein can phosphorylate and activate a human MAP kinase (ERK1) and Mek-2 itself can be phosphorylated and activated by immunoprecipitated mammalian Raf. The predicted amino acid sequence of Mek-2 shares 55% identity to human MEK1. The mek-2 gene plays a key role in the let-60 ras-mediated vulval induction pathway, as loss-of-function mutations in the gene (ku114 and h294) significantly reduce the signal transmitted through Ras. mek-2(ku114) completely suppressed the Multivulva (Muv) phenotype of a hyperactive let 60 ras mutation, and animals homozygous for mek-2(ku114) also displayed a partial larval lethal phenotype. Animals homozygous for mek-2(h294) exhibited a highly penetrant sterile and Vulvaless phenotype. Molecular analysis of the mek-2 mutations demonstrated that ku114 and h294 contained substitutions of Pro237 by a serine and Glu238 by a lysine, respectively. Both Pro237 and Glu238 are absolutely conserved in the kinase family. Changes of either Pro237 or Glu238 are likely to result in reduction of kinase activity, consistent with the notion that ku114 and h294 are loss-of-function mutations. It has been demonstrated in mammalian cells that activation of MEK requires phosphorylation of two conserved serine residues. Mutations of these two serine residues to acidic amino acids result in a constitutively active form of MEK while mutations to alanines result in a dominant-negative MEK. We have constructed the corresponding mutations in C. elegans mek-2 to create a constitutively active or dominant-negative mek-2. Microinjection of the former resulted in Muv and other mutant phenotypes, while microinjection of the latter not only suppressed the Muv phenotype of an activated let-60 ras mutation but also caused an egg-laying defective phenotype in otherwise wild type animals. Our results demonstrate that mek-2 acts between lin-45 raf and sur-l/mpk-l in a signal transduction pathway used in the control of vulval differentiation and other developmental events.