Abstract
In Saccharomyces cerevisiae, adenylate cyclase activity is controlled by Ras1p and Ras2p. Activation of the Ras proteins is in turn controlled by the GTPase-activating proteins (GAPs), Ira1p and Ira2p, and the guanine nucleotide exchange factor (GNEF), Cdc25p. We have characterized Cdc25p enzymologically in order to gain information about the mechanism of Cdc25p-mediated guanine nucleotide exchange and to appreciate how the activity of a GNEF is integrated as a part of a basic molecular switch module consisting of Ras, GNEF, and GAP. Using Ras2p and a catalytic fragment of Cdc25p, both expressed in and purified from Escherichia coli, we determined that Cdc25p has a Km for Ras2p-GDP of 160 nM and a maximal rate of 0.20 s-1. The Km of Cdc25p for Ras2p complexed to GTP is 3-fold greater than that for Ras2p complexed to GDP. The Km of free GDP is about 2-fold higher than the Km of free GTP. This suggests that Cdc25p activates Ras2p primarily by equilibrating Ras2p with the pool of free guanine nucleotides in the cell rather than by driving Ras2p inexorably into the activated state. This renders Ras activation potentially subject to energy charge fluctuations in the cell. The free guanine nucleotide affects kcat, indicating that the rate-limiting step is nucleotide association. Finally, we demonstrated that dominant negative alleles of Ras2p are potent competitive inhibitors of Cdc25p. These data, in conjunction with the kinetic data, are consistent with the hypothesis that Cdc25p catalyzes guanine nucleotide exchange by stabilizing a nucleotide-free intermediate of Ras.
Highlights
In Saccharomyces cerevisiae, adenylate cyclase activ- The ratesof both exchangeof GTP for GDP and hydrolysis of ity is controlled by Raslp andRas2p
Ras proteins is in turn controlledby the GTPase-activat- of GTP hydrolysis is controlled by GTPase-activating proteins ing proteins (GAPS), Iralp and Iragp, and the guanine (GAPs).l In Saccharomyces cereuisiae, these membrane-bound nucleotide exchange factor (GNEF),Cdc25p
The first GNEFs to be characterized were identified in yeast and included Cdc25p, which acts in the glucose-responsive RaskAMP signal transduction pathway of yeast (9-111, and Scd25p, the gene for which wasisolated as a suppressor of a cdc25 mutant and whose role in mediating signal transduction is unclear [12]
Summary
Vol 269, No 24, Issue of J u n e 17, pp. 16541-16548, 1994 Printed in U.S.A. Cdc25p, the Guanine Nucleotide ExchangeFactor for the Ras Proteins of Saccharomyces cereuisiae, Promotes Exchangeby Stabilizing Ras in a Nucleotide-freeState*. Ras proteins aremonomeric guanine nucleotide binding pro- nucleotide than when bound to either GTP or GDP [19] These teinswith highly conserved sequences andstructuresthat observations have led to the hypothesis that Cdc25p effects serve in mosteukaryotic cells as switches in various signal catalysis by stabilizing a nucleotide-free transition intermeditransduction pathways[1].The ability of Ras proteinst o act as ate of Ras. The dominant negative mutant, with its reduced switches derives fromtheir ability t o exist intwo distinct func- affinity for nucleotide, would persist in a nucleotide-free state tional states [2, 3]. Enzymology of Cdc25p nant negative alleles suggest different rate-limiting steps dur- activity was monitoredby mixing 20pl of the final Ras2p.GTP sample ing GNEF-catalyzed nucleotide exchange. The rate of intrinsic GTPase activity of wild type Ras2p has been shown bteo 6.4 mmol of GTP/mol of Ras2p at 30 "C, which indicates that the conversion of the GTPloaded Ras2p represents lessthan 6% of the Ras2p.GTP sample during lecular switch module consisting of Ras, GNEF, and GAP. the incubationperiod with Cdc25p [27]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
