C3G mediated suppression of malignant transformation involves activation of PP2A phosphatases at the subcortical actin cytoskeleton.

In previous work, we demonstrated that C3G suppresses Ras oncogenic transformation by a mechanism involving inhibition of ERK phosphorylation. Here we present evidences indicating that this suppression mechanism is mediated, at least in part, by serine/threonine phosphatases of the PP2A family. Thus: (i) ectopic expression of C3G or C3GDeltaCat (mutant lacking the GEF activity) increases specific ERK-associated PP2A phosphatase activities; (ii) C3G and PP2A interact, as demonstrated by immunofluorescence and co-immunoprecipitation experiments; (iii) association between PP2A and MEK or ERK increases in C3G overexpressing cells; (iv) phosphorylated-inactive PP2A level decreases in C3G expressing clones and, most importantly, (v) okadaic acid reverts the inhibitory effect of C3G on ERK phosphorylation.

Characterization of p87C3G, a novel, truncated C3G isoform that is overexpressed in chronic myeloid leukemia and interacts with Bcr-Abl.

A novel C3G isoform, designated p87C3G, lacking the most amino terminal region of the cognate protein has been found to be overexpressed in two CML cell lines, K562 and Boff 210, both expressing Bcr-Abl p210. p87C3G expression is also highly augmented in patients diagnosed with chronic myeloid leukemia (CML) Ph+, in comparison with healthy individuals, and returns to basal levels after treatment with STI571. p87C3G co-immunoprecipitates with both CrkL and Bcr-Abl in CML cell lines and co-immunoprecipitation between p87C3G and Bcr-Abl was also detected in primary cells from CML patients.

C3G-mediated suppression of oncogene-induced focus formation in fibroblasts involves inhibition of ERK activation, cyclin A expression and alterations of anchorage-independent growth.

We showed previously that exogenous overexpression of C3G, a guanine nucleotide releasing factor (GEF) for Rap1 and R-Ras proteins, blocks the focus-forming activity of cotransfected, activated, sis, ras and v-raf oncogenes in NIH 3T3 cells. In this report, we show that C3G also interferes with dbl and R-Ras focus-forming activity and demonstrate that the transformation suppressor ability of C3G maps to its Crk-binding region (SH3-b domain). Using full-length C3G and C3GDeltaCat mutant, lacking catalytic domain, we showed here that overexpression of cotransfected C3G or C3GDeltaCat inhibited oncogenic Hraslys12-mediated phosphorylation of ERK, without altering Ras and Raf-1 kinase activation.