We report the purification of a Ras-GTPase-activating protein (GAP)-binding protein, G3BP, a ubiquitously expressed cytosolic 68-kDa protein that coimmunoprecipitates with GAP. G3BP physically associates with the SH3 domain of GAP, which previously had been shown to be essential for Ras signaling. The G3BP cDNA revealed that G3BP is a novel 466-amino-acid protein that shares several features with heterogeneous nuclear RNA-binding proteins, including ribonucleoprotein (RNP) motifs RNP1 and RNP2, an RG-rich domain, and acidic sequences. Recombinant G3BP binds effectively to the GAP SH3 domain G3BP coimmunoprecipitates with GAP only when cells are in a proliferating state, suggesting a recruitment of a GAP-G3BP complex when Ras is in its activated conformation.

The p53 tumour suppressor is frequently inactivated in human tumours. One form of inactivation results from overexpression of MDM2, that normally forms a negative auto-regulatory loop with p53 and inhibits its activity through complex formation. We have investigated whether disrupting the MDM2-p53 complex in cells that overexpress MDM2 is sucient to trigger p53 mediated cell death. We ®nd that expression of a peptide homologue of p53 that binds to MDM2 leads to increased p53 levels and transcriptional activity. The consequences are increased expression of the downstream eectors MDM2 and p21, inhibition of colony formation, cell cycle arrest and cell death. There is also a decrease in E2F activity, that might have been due to the known physical and functional interactions of MDM2 with E2F1/DP1. However, this decrease is p53 dependent, as are also colony formation, cell cycle arrest and cell death. These results show that a peptide homologue of p53 is sucient to induce p53 dependent cell death in cells overexpressing MDM2, and support the notion that disruption of the p53-MDM2 complex is a target for the development of therapeutic agents.

No abstract

Proteins of the Ras superfamily, Ras, Rac, Rho, and Cdc42, control the remodelling of the cortical actin cytoskeleton following growth factor stimulation. A major regulator of Ras, Ras-GAP, contains several structural motifs, including an SH3 domain and two SH2 domains, and there is evidence that they harbor a signalling function. We have previously described a monoclonal antibody to the SH3 domain of Ras-GAP which blocks Ras signalling in Xenopus oocytes. We now show that microinjection of this antibody into Swiss 3T3 cells prevents the formation of actin stress fibers stimulated by growth factors or activated Ras, but not membrane ruffling. This inhibition is bypassed by coinjection of activated Rho, suggesting that the Ras-GAP SH3 domain is necessary for endogenous Rho activation. In agreement, the antibody blocks lysophosphatidic acid-induced neurite retraction in differentiated PC12 cells. Furthermore, we demonstrate that microinjection of full-length Ras-GAP triggers stress fiber polymerization in fibroblasts in an SH3-dependent manner, strongly suggesting an effector function besides its role as a Ras downregulator. These results support the idea that Ras-GAP connects the Ras and Rho pathways and, therefore, regulates the actin cytoskeleton through a mechanism which probably does not involve p190 Rho-GAP.

Oncogenes and tumour suppressor genes control the balance between apoptotic death and anti-apoptotic survival signals determining whether a cell proliferates or dies. Through which eectors might oncoproteins generate sensitivity to apoptosis remains to be determined. Ras GTPase activating protein (Ras-GAP) is a key element in the Ras signalling pathway, being both a negative regulator and possibly an eector of Ras. Ras-GAP acts as a regulator of transcription, and possibly connects Ras to stress-activated protein kinases. A role for Ras-GAP in cell survival has been suspected from the study of knock-out mouse embryos. In search for selective killing of tumour cells, we asked whether Ras-GAP inhibition by other means would lead to apoptosis in established cell lines. We injected a monoclonal antibody directed against the SH3 domain of Ras-GAP (mAb200) that has been shown to block Ras-GAP downstream signalling into various human normal and tumour cell lines. We show that inhibition of Ras-GAP induces apoptosis speci®cally in tumour, but not in normal cells, therefore pointing at a speci®c role for Ras-GAP in tumour cell survival. MAb200-induced apoptosis is largely prevented by coinjection of activated RhoA or Cdc42 proteins, by injection of a constitutively activated mutant of phosphoinositide 3-OH kinase (PI3-K), but not by injection of v-Raf. These results show that targeting of Ras-GAP could represent a novel anticancer approach.