Mutations of Ha-ras p21 that define important regions for the molecular mechanism of the SDC25 C-domain, a guanine nucleotide dissociation stimulator.

Abstract

The SDC25 C‐domain is a very active guanine nucleotide dissociation stimulator (GDS) isolated from Saccharomyces cerevisiae which acts equally well on Ha‐ras p21 and yeast RAS2. These properties make the SDC25 C‐domain a suitable tool to study the basic mechanism of a GDS. The action of the SDC25 C‐domain was analysed by mutation of structurally important regions of p21. Substitutions that influence the coordination of Mg2+.GDP or the interaction of the guanine ring were found to stimulate the intrinsic dissociation of GDP and suppress the action of the SDC25 C‐domain. No relevant effects were observed with mutations in the phosphate binding loop L1 or by deleting the last 23 C‐terminal residues of p21. Substitutions in the switch region 1 (loop L2) and 2 (loop L4) of p21 strongly impaired the action of this GDS; however, we show that this effect is not related to a decreased affinity of the SDC25 C‐domain for the mutated p21. No functional competition could be found between this GDS and the catalytic domain of the human GTPase activating protein (GAP). This indicates that GDS and GAP bind to different sites of the p21.nucleotide complex, even though the same mutations in loops L2 and L4 regions affect the activity of both effectors. Since these two regions appear not to be involved directly in the interaction with GDS, we conclude that the negative effect induced by their mutation is related to their function as switches of selective conformations during the GDP to GTP exchange reaction catalysed by GDS.

An Evolutionarily Conserved Domain in a Subfamily of Rabs Is Crucial for the Interaction with the Guanyl Nucleotide Exchange Factor Mss4

“…The last 23 aa of Ras have been shown to be dispensable for binding, in agreement with the results reported here for the Rab3a-Mss4 interaction (24,25). However, a mutational analysis of Ras has shown that the switch II domain (which consists of L4/␣2), but not the switch I domain, is important for binding to its GEFs (26 -28).…”

SDC25, a dispensable Ras guanine nucleotide exchange factor of Saccharomyces cerevisiae differs from CDC25 by its regulation.

“…Indeed, these two GEFs are inactive on other Ras-related proteins . Chimeric proteins between the two carboxy domains of Sdc25p and Cdc25p are active GEF on Ras proteins, whereas chimeric proteins between Sdc25p and Bud5p, the specific GEF for Budl/Rsrlp, are inactive proteins (Camus et al, 1994 (Mistou et al, 1992;Haney and Broach, 1994;Jacquet et al, 1995). The Sos GEFs have a low specific activity in vitro on unprocessed Ras proteins.…”

Inhibition of Ras oncogenic activity by Ras protooncogenes

“…[13][14][15][16][17][18][19][20][21] Other, older reports have analyzed the interaction of the protooncogenic effector domain mutants with various guanine exchange factors and GTPase activating proteins. [22][23][24][25] In addition, mutating the highly conserved cysteine at position 186 of the CAAX box domain to serine results in a nonfarnesylated protein that fails to associate with membranes and becomes cytosolic. 26,27 Localization to the membranes and transforming activity of oncogenic Ras/S186 are rescued when Ras is targeted to the membranes by a myristoylation signal in its N-terminal domain.…”