Worm Breeder's Gazette 13(1): 70 (October 1, 1993)

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.

A C. elegans homolog of MAP kinase, mpk-1 ,is involved in vulval development

Mark Lackner[1], Kerry Kornfeld[2], H. Robert Horvitz[2], Stuart K. Kim[1]

[1]Department of Developmental Biology, Stanford University Medical Center, Stanford, CA 94305
[2]Department of Biology, M.I.T., Cambridge, MA 02139.

Vulval induction in C. elegans is mediated by a receptor tyrosine kinase pathway that has been remarkably well conserved during n metazoan evolution. This conservation led us to predict that one or more members of the Mitogen Activated Protein (MAP) kinase family would function during vulval induction, as these protein serine/threonine kinases are activated by tyrosine kinase pathways in vertebrate cells and biochemical studies have shown that MAP kinase activation requires both ras and raf. Once activated, MAP kinases can directly phosphorylate transcription factors. Thus, MAP kinases may lie at a key step linking signaling to changes in gene expression.

To clone C. elegans MAP kinase homologs we used degenerate oligonucleotide primers corresponding to amino acid sequences conserved in all known MAP kinases. With these primers, we enzymatically amplified sequences from genomic DNA and cDNA using the polymerase chain reaction. Amplified fragments were cloned in a plasmid vector and sequenced. Sequence analysis of these cloned PCR fragments revealed that they were derived from two genes. We have named these MAP kinase-l( mpk-1 ) and mpk-2 ,because of their extensive sequence similarity to known MAP kinases. Sequence analysis of both genomic DNA and cDNA clones indicated that the predicted Mpk-1 protein is 72% identical to the human Erk 1 MAP kinase. Notably, mpk-1 encodes a TEY tri-peptide; in vitro, the threonine and tyrosine residues of this conserved motif have been shown to be the sole targets of MEK, a MAP kinase. Thus, a C. elegans MEK homolog may interact with mpk-1 during vulval induction.

In parallel to using a molecular approach to identify candidate genes involved in the let-23 tyrosine kinase pathway, we screened for mutations that affect vulval induction. Forty-three mutations representing 22 complementation groups were isolated as suppressers of the multivulva (Muv) phenotype caused by let-60 ( n1046 ),which encodes a Ras protein that is constitutively active. One complementation group, consisting of the single allele n2521 ,mapped between unc-79 and dpy-17 on chromosome III, the same interval that contains mpk-1 (Alan Coulson kindly localized mpk-1 to YACS Y47H5 and Y61H12 ).Transformation rescue experiments using n2521 ;1et-60(gf) animals showed that injection of DNA containing the mpk-1 (+)gene rescues the n2521 phenotype (i.e. n2521,nlO46;Exmpk-1(+) animals are Muv). Creating a small insertion in the mpk-1 open reading frame abolished rescuing ability. Further, sequence analysis of n2521 genomic DNA revealed that the n2521 allele contains a C-to-T substitution that alters amino acid 124 in the MPK-1 protein. This substitution changes a leucine that is conserved in all known MAP kinases to a phenylalanine. The findings that mpk-1 (+)DNA rescues the n2521 phenotype and that n2521 contains a mutation in the mpk-1 gene indicate that n2521 is an mpk-1 allele.

We have noted only one phenotype caused by mpk-1 ( n2521 ).In let-60 ( n1046 )mutants, all six cells in the vulval equivalence group P3 .p-P8.pundergo vulval differentiation and a population of animals is 93% Muv. In contrast, in mpk-1 ( n2521 ); Iet-60 (n1O46) mutants, generally only P5 .p-P7.padopt vulval fates and a population is 5% Muv. mpk-1 ( n2521 )mutants appear wild-type. n2521 is largely recessive, so n2521 is likely to cause a loss of mpk-1 function.

Studies in vertebrate cells have shown that MAP kinases are activated by ras-mediated signaling pathways. However, it has been difficult to demonstrate the functional significance of this activation in cultured cells. Our genetic results show that mpk-1 plays an important functional role in the ras-mediated cell signaling that occurs during vulval induction.