MicroinJection of either Ki-ms,.Vl.2 p21 or the GDP-bound form of Ki-ras p21 plus smg GDP dissociation stimulator (GDS), a stimulatory GDP/GTP exchange protein for Ki-ras p21, smg/rapllKrev-l p21, and rho p21, into quiescent Swiss 3T3 cells induced DNA synthesis irrespective of the presence or absence of insulin. The guanosine 5'-(3-0-thio)triphosphate (GTPyS)-bound form of smg p21B or the GDP-bound form of smg p21B plus smg GDS also induced DNA The smg p21 family, consisting of two members, A and B, belongs to the ras p21-related small GTP-binding protein (G protein) superfamily (for reviews, see references 3 and 41). smg p21A is identical to raplA p21 and Krev-1 p21, and smg p21B is identical to raplB p21 (22,24,28,35,36). Among many small G proteins, smg p21 has the same amino acid sequence as does the effector region of ras p21 (3,22,24,28,35,36,41). This structural property suggests that smg p21 can share the effector(s) with ras p21 and exert actions similar or antagonistic to those of ras p21. Consistently, Krev-1 p21 has been shown to suppress the transforming activity of v-Ki-ras p21 in NIH 3T3 cells (24). smg p21B and raplA p21 inhibit the ras p21 GTPase-activating protein (GAP) activity in a manner competitive with ras p21 in a cell-free system (10, 13). ras p21 GAP has been shown to interact with the effector domain of ras p21 and to stimulate its GTPase activity (for a review, see reference 29). Moreover, our recent studies have revealed that overexpression of smg p21 in NIH 3T3 cells inhibits the ras p21-, plateletderived growth factor (PDGF)-, and 12-O-tetradecanoylphorbol-13-acetate-induced activation of the c-fos promoter/ enhancer element but does not inhibit the c-raf-l-induced activation of this element (39). There are several lines of evidence that ras p21 is a downstream molecule of the PDGF receptor and protein kinase C and that the c-raf-1 protein kinase mediates at least a part of the actions of ras p21 (6,7,16,18,25,32,40). These results indicate that smg p21 may antagonize ras p21 actions presumably by competing for the proteins interacting with the effector domain of ras p21. Although ras p21 GAP interacts with the effector domain of ras p21, there is increasing evidence that this protein is a negative regulatory protein which converts ras p21 from the GTP-bound active form to the GDP-bound inactive form (29, * Corresponding author. 33,43,45). At present, the effector protein of ras p21 in mammalian cells is unknown.The conversion of smg p21 from the GDP-bound inactive form to the GTP-bound active form is stimulated by a GDP/GTP exchange protein, named smg GDP dissociation stimulator (GDS) (17,44). This smg GDS is active not only on smg p21 but also on Ki-ras p21 and rho p21 (31). On the other hand, smg p21 is directly phosphorylated by cyclic AMP-dependent protein kinase (protein kinase A) and cyclic GMP-dependent protein kinase (protein kinase G) at the same serine residue (Ser-179), which is located between the polybasic region and the geranylgeranylated cysteine residue in the C...

smg p25A, also known as the rab3A protein, is a small GTP-binding protein that has been implicated in intracellular vesicle transport and the secretion of neurotransmitters. It has been shown to bind reversibly to membranes, though its cDNA-predicted sequence contains no obvious membrane-binding domains. However, smg p25A does contain a cDNA-predicted C-terminal Cys-Ala-Cys sequence at positions 218 through 220, which suggests that it may be posttranslationally modified. In the present study we used two different approaches to investigate this possibility. First, we incubated pheochromocytoma cells with [3H]mevalonolactone, examined the proteins that became labeled by two-dimensional gel electrophoresis, and demonstrated that two of these proteins exactly corresponded to smg p25A. Second, we purified smg p25A from bovine brain membranes and analyzed both the full-length protein and a proteolytically derived C-terminal peptide by a combination of high performance liquid chromatography and mass spectrometry. This approach revealed that the protein's C-terminal region is methyl-esterified and contains two geranylgeranyl groups linked via thioether bonds to Cys-218 and Cys-220. Since smg p25A is one of several small GTP-binding proteins that share a C-terminal Cys-Xaa-Cys consensus sequence (where Xaa is an unspecified amino acid), our results suggest that these proteins may be similarly geranylgeranylated and methyl-esterified.

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smg p21A and -B are small GTP-binding proteins that share putative effector and consensus C-terminal sequences with ras p21 proteins. In the present report, we showed that human platelet smg p21B became labeled when intact platelets were incubated with exogenous [3H]mevalonolactone and when a purified preparation of smg p21B was incubated with bovine brain membranes and S-adenosyl-L- [methyl-3H]methionine. In addition, we demonstrated by gas chromatography/mass spectrometry that treatment of smg p21B with Raney nickel released a geranylgeranyl moiety in a molar ratio of about 1:1. In contrast, treatment of smg p21B with NH2OH or KOH yielded no evidence for the presence of a palmitoyl thioester. Extensive digestion of sing p21B with Achromobacter protease I yielded two C-terminal tripeptides that contained serine and cysteine in a molar ratio of 2:1. Both peptides were modified by a thioether-linked geranylgeranyl group. One of the peptides comigrated with a 3H-labeled proteolytic product of methylated smg p21B on reverse-phase HPLC and this peptide appeared at the same retention time as that of the other peptide after being treated with KOH. Since the cDNA-predicted C-terminal sequence of smg p21B contains a unique Ser-Ser-Cys peptide within its C-terminal domain, -Lys-Lys-Ser-Ser-Cys-Gln-Leu-Leu'8", these results indicate that smg p2IB is posttranslationally modified by geranylgeranylation of Cys-181 and suggest that further modifications cause proteolytic removal of the three predicted C-terminal amino acids followed by partial methylation of the cysteinyl carboxyl group. smg p21A and -B, also referred to as the raplA or Krev-1 and raplB proteins, respectively, belong to a superfamily of ras p21/ras p21-like small GTP-binding proteins (G proteins) (refs.