Program of the 2001 International Worm Meeting - Sternberg Lab ...

304
1Barr MM and Sternberg PW, Nature 401:
386-389 1999
305. CATION DIFFUSION
FACILITATOR PROTEINS AND
ZINC IONS REGULATE
RAS-MEDIATED SIGNALING
Janelle Jakubowski, Kerry Kornfeld
Department of Molecular Biology and
Pharmacology, Washington University School
of Medicine, St. Louis, MO 63110
305
A conserved receptor tyrosine kinase-Ras-MAP
kinase signal transduction cascade is required
for specification of the C. elegans vulva. In a
screen for suppressors of the multivulva (Muv)
phenotype caused by a constitutively active
let-60 ras gene, we identified the strong
loss-of-function mutation n2527. The n2527
mutation causes a weak vulvaless phenotype;
epistasis experiments suggest that it functions
downstream of let-60 and upstream of lin-1 if
the genes act in a linear pathway. To
positionally clone the gene affected by n2527,
we created a local, high-density,
single-nucleotide polymorphism map and
positioned n2527 within a 9.6-kb interval. The
molecular lesion was identified by sequencing,
and the candidate gene was confirmed by
transformation rescue. The n2527 mutation
creates a premature STOP codon in a previously
uncharacterized gene that we named cdf-1, since
it encodes a predicted protein with significant
similarity to members of the cation diffusion
facilitator (CDF) family. CDF-1 is similar to
mammalian ZnT-1 which reduces the
intracellular concentration of Zn 2+ ions.
CDF proteins have not been previously shown
to be involved in signal transduction. We
investigated if regulation of Ras signaling is an
evolutionarily conserved property of CDF
proteins. When expressed under the control of
the cdf-1 promoter, rat ZnT-1 rescues the
cdf-1(n2527) suppression of let-60(gf) ras Muv
phenotype. Thus a vertebrate CDF protein can
function in worms to promote Ras signaling. We
also tested if CDF-1 could function in vertebrate
signal transduction using Xenopus laevis
oocytes. In a sensitized background,
overexpression of CDF-1 induces MAP kinase
phosphorylation and Xenopus oocyte meiotic
maturation, suggesting that worm CDF-1 can
stimulate Ras signaling in a vertebrate.