Genetic analysis of the Kirsten-ras-revertant 1 gene: potentiation of its tumor suppressor activity by specific point mutations.

Abstract

Kirsten-ras-revertant 1 (Krev-1) cDNA encodes a ras-related protein and exhibits an activity of inducing flat revertants at certain frequencies (2-5% of total transfectants) when introduced into a v-K-ras-transformed mouse NIH 3T3 cell line, DT. Toward understanding the mechanism of action of Krev-l protein, we constructed a series of point mutants of Krev-1 cDNA and tested their biological activities in DT cells and HT1080 human fibrosarcoma cells harboring the activated N-ras gene. Substitutions of the amino acid residues in the putative guanine nucleotide-binding regions (Asp'7 and Asn'I'), in the putative effector-binding domain (residue 38), at the putative acylation site (Cys"'5), and at the unique Thr" all decreased the transformation suppressor activity. On the other hand, substitutions such as Gly'2 to Val'2 and Gln63 to Glu63were found to significantly increase the transformation suppressor/tumor suppressor activity of Krev-1. These findings are consistent with the idea that Krev-1 protein is regulated like many other G proteins by the guanine triphosphate/ guanine diphosphate-exchange mechanism probably in response to certain negative growth-regulatory signals.Kirsten-ras-revertant 1 (Krev-1) cDNA was recovered from one of the flat revertants isolated from populations of a v-Kras-transformed NIH 3T3 cell derivative, DT, following transfection with a human fibroblast cDNA expression library (1). When transfected into DT cells, the plasmid expressing Krev-1 cDNA induces flat revertants at certain frequencies (2-5% of total transfectants), and relatively high levels of expression seem to be required to induce morphological reversion (2). Krev-1 (also known as rap 1A and smg p21) encodes a guanine-nucleotide binding protein with a molecular weight of 21,000 whose amino acid sequence shares strong similarity (around 50% amino acid identity) with the products of the classical ras protooncogenes-namely, H-ras, K-ras, and N-ras (2-4). Similarities are especially high in the essential regions known in H-ras protein as guanine nucleotide-binding regions, the effector-binding region, and the C-terminal acylation site (see Fig. 1 for locations), which have been determined by in vitro mutagenesis (5-10) and later by x-ray crystallography (11, 12). The classical ras oncoproteins are also known to be activated by specific point mutations, such as amino acid substitutions at residues 12, 59, 61, and 63, to become highly transforming (reviewed in refs. 13 and 14). These mutations are believed to somehow disturb the guanine triphosphate (GTP)-hydrolyzing (GTPase) activity associated with ras proteins, thereby arresting these proteins in their active, GTP-bound form. In Krev-1 protein, some of the critical target amino acids for mutational activation of ras are identical (Gly12 and Thr59), and others are distinct from the corresponding amino acids in normal ras proteins (Thr61/ Gln63 in Krev-1 vs. Gln61/Glu63 in ras) (2).In this study we took advantage of the strong structural similarity between Krev-1 protei...

Biochemical characterization of C3G: an exchange factor that discriminates between Rap1 and Rap2 and is not inhibited by Rap1A(S17N)

“…In those studies in which S17 mutants of Rap1A has been examined, this mutant was found to be inactive (S17D in ref. Kitayama et al, 1990; S17N in refs. Maly et al, 1994 andGabig et al, 1995), as predicted from the observed decrease in nucleotide a nity, in analogy to the Ras situation.…”

Increasing complexity of Ras signaling

“…This antagonism has been attributed to the ability of Rap1A to form nonproductive complexes with key Ras e ectors important for Ras transforming activity. This may occur because Rap1A may bind, but fail to activate some Ras e ectors such as Raf-1 (Kitayama et al, 1989(Kitayama et al, , 1990. Alternatively, since Rap1A is located in the endoplasmic reticulum and Golgi, it may sequester Ras e ectors in a subcellular location that prevents their complete activation to or from functional signaling complexes (Beranger et al, 1991;Sato et al, 1994;Cook et al, 1993).…”

The Ras-related Protein Rheb Is Farnesylated and Antagonizes Ras Signaling and Transformation

“…Rheb Antagonizes Ras Signaling and TransformationSince Rheb lacked the growth promoting activity seen with constitutively activated mutants of Ras, TC21, or R-Ras, we next determined if Rheb was more similar to KRev-1/Rap1A and, instead, could antagonize Ras function (32,33). For these analyses, we used the constitutively activated KRev-1(63E) mutant as a positive control for these co-transfection focus formation inhibition assays (33).…”

“…Exceptions include TC21/R-Ras2 (10, 11), R-Ras (12,13), RhoA (14 -18), RhoB (19), and Rac1 (17,20), where constitutively activated versions of these Ras-related proteins can cause tumorigenic transformation of NIH 3T3 cells. The transforming activities of TC21 and R-Ras reflect the fact that these two Ras-related proteins share complete identity with the core Ras effector domain (Ras residues [32][33][34][35][36][37][38][39][40] and, consequently, may activate downstream signaling pathways in common with those that mediate Ras transforming potential (21). However, although the Raf-1 serine/threonine kinase is clearly a critical effector important for Ras signaling and transformation (22)(23)(24)(25)(26)(27), neither TC21 nor R-Ras cause activation of Raf-1 (28,29).…”

“…Although KRev-1/Rap1A also shares complete identity with the core Ras effector domain, it lacks any transforming potential and, instead, antagonizes the ability of Ras to transform cells (32)(33)(34). Since KRev-1 can interact with the Raf-1 serine/ threonine kinase (35), as well as other candidate Ras effectors (e.g.…”