Transformation suppressor activity of C3G is independent of its CDC25-homology domain

Abstract

The guanine nucleotide releasing protein C3G was initially identi®ed as a Crk SH3-binding protein and recently shown to exhibit exchange activity on Rap1 proteins. Overexpression in NIH3T3 cells of a full-length C3G cDNA isolated from human placenta markedly reduced the focus forming activity of cotransfected, malignantly activated, ras oncogenes (5 ± 7-fold). C3G also had a reverting eect on sis-mediated transformation, decreasing the number of c-sis-induced foci by a factor of 5 ± 10-fold. The observed inhibitory eect of C3G on focus-forming activity of Ras and Sis was always higher than that observed with Rap1A, a known target of C3G. The inhibition of focus formation observed in the presence of C3G was not due to toxic eects on cell viability, since transfected C3G cells exhibited the same survival and growth rates as untransfected NIH3T3 cells or cells transfected with plasmid vector alone. Surprisingly, as opposed to Rap1A, which has no eect on Raf-1 oncogene-mediated transformation, C3G also reduced dramatically (6 ± 8-fold) the number of v-raf-induced foci in transfected NIH3T3 cells. The inhibitory eect on Raf-induced transformation suggests that C3G has other functional targets in addition to Rap1. A C3G mutant (C3G DCat) lacking the catalytic domain (CDC25-H) but retaining the rest of the N-terminal sequences, including the Crkbinding domain, exhibited similar ability than full length C3G to inhibit focus formation. In contrast, a C3G mutant (C3G Cat), containing the catalytic domain only but lacking the rest of the N-terminal sequences, did not have any inhibitory eect on transformation mediated by the oncogenes tested. The C3G-derived gene products overexpressed in our transfected cell lines localized to the cytoplasm and did not change the basal MAPK or JNK activity of those cell lines nor their ability to activate the kinases in response to agonists. Our results suggest that the N-terminal region of C3G, and not its catalytic domain, may be responsible for the inhibitory eects observed.

The GTPase Rap1 Regulates Phorbol 12-Myristate 13-Acetate-stimulated but Not Ligand-induced β1Integrin-dependent Leukocyte Adhesion

“…Thus, when actin cytoskeleton-dependent re-structuring was disrupted by the high concentration of cytochalasin D, cell adhesion was inhibited. Our studies indicate that Rap1 activation is required for this second step, perhaps in part modulating receptor affinity as suggested by Guerrero et al (22).…”

“…However, in other studies treatment with C3 exoenzyme did not affect ␤ 2 integrin-dependent adhesion of neutrophils (13) or JY lymphocytic cells (14) or ␤ 1 integrin-dependent adhesion of peripheral blood T-cells (15) or U937 monocytic cells (16). Finally, several recent studies have implicated Rap1, a small GTPase with 53% amino acid sequence homology to K-Ras, in stimulated ␤ 1 (17)(18)(19) and ␤ 2 (10, 19 -21) integrin-dependent leukocyte adhesion, as well as ␤ 3 -dependent platelet adhesion (22).…”

A dominant negative RAS-specific guanine nucleotide exchange factor reverses neoplastic phenotype in K-ras transformed mouse fibroblasts

“…One class is mostly brain-speci®c and has been called CDC25 Mm , Ras Grf, hGRF55 by various groups (Martegani et al, 1992;Shou et al, 1992;Schweigho er et al, 1993;Wei et al, 1993Wei et al, , 1994Sturani et al, 1997). The other class is expressed ubiquitously and is called Sos Buday and Downward, 1993;Guerrero et al, 1996). Both GEFs are large, multi-domain proteins, whose catalytic domains are approximately 70% homologous.…”

Crk family adaptors–signalling complex formation and biological roles

“…GEF) domain at the C-terminus and a Ras-exchanger motif (REM; Guerrero et al, 1998), C3G probably has presumably other functionally important domains (Guerrero et al, 1998) which are not well characterized yet. For example, C3G binds to the large docking protein p130Cas which also interacts with AND-34, another already mentioned Rap1 activator (Kirsch et al, 1998) but it is not clear whether ternary C3G ± p130Cas ± AND-34 complexes can occur.…”