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.

Mek-2, a Caenorhabditis elegans MAP Kinase Kinase, Functions in Ras-Mediated Vulval Induction and Other Developmental Events.

Yan Wu1, Min Han1, Kun-Liang Guan2

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
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