Worm Breeder's Gazette 14(4): 44 (October 1, 1996)

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.

gap-1 encodes a GTPase activating protein that inhibits vulval induction.

Alex Hajnal, Charles Whitfield, Stuart K. Kim

Department of Developmental Biology, Stanford University School of Medicine, Stanford CA, 94305

During vulval development, the anchor cell signal normally induces P6.p
to express the primary cell fate by activating a conserved receptor
tyrosine kinase/Ras/Map Kinase signaling pathway. Here we describe the
genetic analysis and the molecular cloning of gap-1 (previously known as
suv-1; Kim,S.K. and Horvitz,H.R.,WBG 11(3),68), a component of this
signaling pathway.
The mutant phenotype of gap-1 suggests that it inhibits vulval induction
by the let-60 ras signaling pathway. Null mutations in gap-1 (see below)
suppress the vulvaless phenotype caused by mutations that reduce but do
not eliminate signaling activity at a step upstream of lin-45 raf. For
example, gap-1(ga133) suppresses the vulvaless phenotype caused by
partial reduction-of-function alleles of lin-3 which encodes the anchor
cell signal, let-23 which encodes a receptor tyrosine kinase, sem-5
which encodes a GRB-2 homolog and let-60 which encodes Ras (Table). In
addition, gap-1(ga133) suppresses the vulvaless phenotype of null
alleles of lin-2, lin-7 and lin-10, three genes required for receptor
activation. However, gap-1(ga133) does not suppress the vulvaless
phenotype of a weak allele of lin-45, which encodes Raf and acts
downstream of let-60 ras. Finally, gap-1(ga133) does not exhibit a
vulval phenotype in a single mutant. These results suggest that gap-1
mutations do not allow vulval induction independently of the anchor cell
signal, but rather appear to increase the sensitivity of the Pn.p cells
towards the anchor cell signal.
We were able to define a 9kb DNA fragment of the cosmid T24C12 that
contains gap-1, first by using RFLP mapping, then by finding DNA
rearrangements associated with gap-1 alleles and finally by
transformation rescue experiments. The only predicted open reading frame
on this DNA fragment encodes a protein of 629 amino acids that is
similar to the Drosophila GTPase activating protein GAP1 (27% identical
and 53% similar) and to human GAP1(m) (25% identical and 42% similar).
Sequence analysis of strong gap-1 alleles has identified four potential
null alleles. The first (ga133) is a 334 bp deletion causing a
frameshift after amino acid 163 and the others (n1691, ga14 and ga12)
are stop mutations at positions 151, 174 and 182, respectively. All four
alleles are predicted to express non-functional proteins because they
each lack the putative catalytic domain. GAP proteins inhibit the
signaling activity of Ras by stimulating its intrinsic GTPase activity,
which results in a decrease in the levels of Ras in the active,
GTP-bound form. Thus, GAP-1 may directly inhibit the signaling activity
of LET-60 RAS during vulval induction.
Interestingly, double mutants containing gap-1(ga133) in combination
with mutations that reduce let-23 receptor activity such as let-23(sy1),
lin-2(n397), lin-7(e1449) or lin-10(n1390) cause a hyperinduced
phenotype in up to 80% of the animals (a gonad-dependent multivulva
phenotype). This phenotype is not observed in double mutants containing
gap-1(ga133) and mutations in genes that act at other steps in the
signaling pathway (Table). These observations are paradoxical since
single mutants in either lin-2, lin-7, lin-10 or let-23 have a vulvaless
phenotype due to reduced receptor activity but double mutants containing
gap-1(ga133) and the same mutations have a hyperinduced phenotype
indicating increased signaling activity. One explanation for this
reversal of phenotypes, previously proposed by Aroian et al. (Genetics
(1991) 128,251), is that let-23 may have two functions; one function is
to activate the let-60 ras signaling pathway, and the other function
might be to inhibit vulval induction through a pathway that is yet to be
determined. In single mutants, the vulvaless phenotype is the result of
reduced activation by let-23 whereas in double mutants with
gap-1(ga133), the hyperinduced phenotype might be due to reduced
inhibition by let-23.

genotype                     % Egl     % WT      % Hin     n
gap-1(ga133)                 0         100       0         280
lin-3(e1417)                 88+/-5    12+/-5    0         189
lin-3(e1417);gap-1(ga133)    42+/-6    8+/-6     0         238
let-23(sy1)                  80+/-4    0+/-4     0         427
let-23(sy1);gap-1(ga133)     3+/-2     4+/-5     73+/-6    238
lin-2(n397)                  93+/-4    7+/-4     0         185
gap-1(ga133)lin-2(n397)      4+/-3     2+/-6     74+/-6    197
lin-7(e1449)                 85+/-4    3+/-4     1+/-1     357
lin-7(e1449);gap-1(ga133)    2+/-2     7+/-5     81+/-5    228
lin-10(n1390)                94+/-3    6+/-3     0         203
lin-10(n1390);gap-1(ga133)   6+/-3     31+/-7    63+/-7    177
sem-5(n2019)                 91+/-3    9+/-3     0         292
gap-1(ga133)sem-5(n2019)     41+/-6    59+/-6    0         306
let-60(n1876)*               100       0         0        >200**
let-60(n1876);gap-1(ga133)*  14+/-14   86+/-14   0         20
lin-45(n2018ts) @14C         76+/-6    24+/-6    0         186
lin-45(n2018ts);gap-1(ga133) 72+/-5    28+/-5    0         341
* the progeny of n1876/+ animals was scored
** from Beitel et al.,(1990) Nature 348:503