A New RAS Mutation That Suppresses the CDC25 Gene Requirement ...

MOLECULAR AND CELLULAR BIOLOGY, July 1988, p. 2980-2983 0270-7306/88/072980-04$02.00/0 Copyright © 1988, American Society for Microbiology

Vol. 8, No. 7

A New RAS Mutation That Suppresses the CDC25 Gene Requirement for Growth of Saccharomyces cerevisiae JACQUES H. CAMONIS* AND MICHEL JACQUET Groupe Information Genetique et Developpement, Universite de Paris-Sud, 91405 Orsay Cedex, France Received 8 February 1988/Accepted 6 April 1988

In the yeast Saccharomyces cerevisiae, the activation of adenylate cyclase requires the products of the RAS genes and of CDC25. We isolated several dominant extragenic suppressors of the yeast cdc25 mutation. They did not suppress a thermosensitive allele of the adenylate cyclase gene (CDC35). One of these suppressors was a mutated RAS2 gene in which the transition C/G -* T/A at position 455 resulted in replacement of threonine 152 by isoleucine in the protein. The same mutation in a v-Ha-ras gene reduces the affinity of p21 for guanine nucleotides (L. A. Feig, B. Pan, T. M. Roberts, and G. M. Cooper, Proc. Natl. Acad. Sci. USA 83:4607-4611, 1986). These results support a model in which the CDC25 gene product is the GDP-GTP exchange factor regulating the activity of the RAS gene product.

RAS genes were first described as the transforming genes of the Kirsten and Harvey oncogenic retroviruses. They belong to a family of ubiquitous eucaryotic genes involved in growth control and in cell differentiation. RAS gene products are GTP-binding proteins with an intrinsic GTPase activity (for a review, see reference 2). By analogy to transducin and Gs and Gi proteins, cellular RAS proteins are believed to be transducers; the corresponding receptor and effector have not yet been definitely identified. In Saccharomyces cerevisiae, the RASISC and the RAS2SC genes are closely related to the ras genes of higher eucaryotic organisms (12, 22) and their products are positive regulators of adenylate cyclase (5). Experimental evidence suggests that the adenylate cyclase signaling pathway is responsible for the switch between the resting state and progress through the cell division cycle (3) in response to nitrogen availability (4). The production of cyclic AMP also requires the CDC25 gene product (6, 7, 25). This requirement for growth is bypassed by modification of RAS proteins (RAS2Va-l9 RASJLeu-6S) similar to the modifications which lead to mammalian ras oncogenic activation (6, 19, 25). The CDC25 gene product is suggested to be the upstream element in the RAS-adenylate cyclase cascade; it may constitute the "detector" of some still unknown molecule(s) which would reflect nitrogen availability (5-7, 11, 25). Isolation of cdc25 suppressors. To extend our knowledge of the elements involved in the CDC25-RAS-CDC35-encoded cascade, we looked for extragenic suppressors of cdc25 mutations which do not suppress cdc35 mutations. Cells containing a thermosensitive cdc25 mutation (strain DlBR205-1200: MATot cdc25-2 adel,2,6,7 arg4 thr4 ural gall) were plated on rich medium at the restrictive temperature (36°C). Several clones capable of growth at 36°C were genetically analyzed, and three of them were shown to harbor different dominant extragenic suppressors, which we named SU25-1 SU25-3 SU25-8. The cdc25-suppressing activity of each mutant segregated as a single gene. All three suppressed the cdc25-2 and cdc25-5 mutations and thus are not allele specific. None of them suppressed the thermosensitive cdc35-10 mutation of the adenylate cyclase gene (data not shown). Each suppressor was studied for possible allelism with genes known to be involved in the CDC25-RAS*

CDC35 pathway. Analysis of 118 tetrads does not permit detection of any recombination between the SU25-3 gene, located by its capacity to suppress a thermosensitive cdc25 mutation, and the RAS2 locus, located by the LEU2 gene inserted therein (Table 1) (8). Thus, the SU25-3 suppressor is closely linked to the RAS2Sc gene (genetic distance, A here) is indicated by an arrow. (B) Restriction map of the region common to all the cloned cdc25suppressing DNA fragments. The difference in the wild-type RAS2Sc gene is the additional EcoRV site indicated by an arrow. The length of one kilobase pair (kbp) is indicated.

36 "C.

On

fift Lb L.

A

B

A

A

FIG. 2. Suppression of the thermosensitive cdc25 phenotype. OL971-11B cells (cdc25 ura3) were transformed with the following DNAs. (A) Rows: 1, Chimeric RAS2 gene composed of the 5' ClaI-PstI fragment of the wild-type RAS2 gene and the 3' PstIHindIII fragment of the mutated RAS2* gene in the integrative plasmid pEMBLyi32: 2, chimeric RAS2 gene composed of the 5' ClaI-PstI fragment of the mutated gene and the 3' PstI-HindIII fragment of the wild-type gene in pEMBLyi32: 3. SRA3 gene cloned in YCp5O (positive control). (B) Rows: 1. ClaI-HindIII fragment encompassing the mutated RAS2* gene in pEMBLyi32 (pRAS*): 2, ClaI-HindIII fragment encompassing the wild-type RAS2 gene in pEMBLYi32 (pRAS); 3. plasmid YCp5O (negative control). The ability of each plasmid to complement the cdc25 mutation was tested in several different clones, and two of them are presented. The SRA3 gene, also named TPKI, encodes the catalytic subunit of the cyclic AMP-dependent protein kinase (9, 28).

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limiting and probably regulated step is believed to be the exchange of the bound GDP for GTP. In response to regulatory signals, an exchange protein could modify the conformation of the RAS proteins and thus increase the rate of dissociation of bound GDP. This would accelerate cytoplasmic nucleotide exchange, which would lead to a higher proportion of active GTP-bound RAS proteins, since the concentration of cytoplasmic GTP is in the millimolar range (13, 14), which is significantly higher than the cytoplasmic GDP concentration (23). The effect of mutations reducing the affinity for guanine nucleotides might be to eliminate the need for the exchange factor, since RAS proteins, frozen in a lower-affinity state, would be in a continuous equilibrium with the more abundant GTP and thus active enough to stimulate their effector. Indeed, some mutations (modifying p21 at positions 116, 117, and 119) which increase the Kit of p21 for GDP and GTP were shown to be oncogenic (24, 27. 30). The modification of the yeast RAS2 protein we identified could define an activating mutation by desensitization with respect to a regulatory exchange factor. Analogous mutations of the cellular p21, with a similar effect on the K,,. would allow higher eucaryotic cells to bypass the signals which serve to regulate p21 binding of GDP or GTP. for instance, in tumors. The occurrence of a spontaneous RAS2Ie-l 52 mutation which suppresses the (dc25 mutation strengthens the idea of a close relationship between the CDC25 and the RAS gene products. The fact that a RAS mutation (RAS211c-152) which probably allows spontaneous GDP-GTP exchange suppresses the CDC25 requirement, as do mutations which reduce the GTPase activity (RAS2vt''l9 RASll-'-'). suggests that the CDC25 gene product is involved in recycling the active RAS proteins. If the CDC25 protein is the exchange factor, it will define a new class of exchange factors different from transmembrane receptors. Although the mammalian mas protein also bypasses the CDC25 requirement in yeast cells, the question of the existence of functionally related exchange factors in mammalian cells can be raised

(19). We thank R. Guilbaud for her excellent technical assistance and Muriel Decraene for manuscript preparation. Anne Roels help was invaluable during the genetic analysis. We thank 0. Fasano and A. Parmeggiani for the gift of the cloned RAS2.C gene. K. Tatchell and J. F. Cannon for yeast strains JC302-26 and JC302-26B/1091. F. Fabre for strain D1BR205-1200. G. Cesareni for the pEMBLyi32 plasmid, Linda Sperling for critical reading of the manuscript, and E. Boy-Marcotte and H. Garreau for fruitful discussions. This research was supported by grants from Institut National de la Sante et de la Recherche Medicale A.R.C., and Ligue Nationale Franqaise contre le Cancer.

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