Grb2 is a negative modulator of the intrinsic Ras-GEF activity of hSos1.

hSos1 is a Ras guanine-nucleotide exchange factor. It was suggested that the carboxyl-terminal region of hSos1 down-regulates hSos1 functionality and that the intrinsic guanine-nucleotide exchange activity of this protein may be different before and after stimulation of tyrosine kinase receptors. Using different myristoylated hSos1 full-length and carboxyl-terminal truncated mutants, we show that Grb2 function accounts not only for recruitment of hSos1 to the plasma membrane but also for modulation of hSos1 activity. Our results demonstrate that the first two canonical Grb2 binding sites, inside the carboxyl-terminal region of hSos1, are responsible for this regulation. Following different approaches, such as displacement of Grb2 from the hSos1-Grb2 complex or depletion of Grb2 levels by small interfering RNA, we found that the full-length Grb2 proteins mediate negative regulation of the intrinsic Ras guanine-nucleotide exchange activity of hSos1.

The histone acetyltransferases CBP/p300 are degraded in NIH 3T3 cells by activation of Ras signalling pathway.

The CBP [CREB (cAMP-response-element-binding protein)-binding protein]/p300 acetyltransferases function as transcriptional co-activators and play critical roles in cell differentiation and proliferation. Accumulating evidence shows that alterations of the CBP/p300 protein levels are linked to human tumours. In the present study, we show that the levels of the CBP/p300 co-activators are decreased dramatically by continuous PDGF (platelet-derived growth factor) and Ras signalling pathway activation in NIH 3T3 fibroblasts. This effect occurs by reducing the expression levels of the CBP/p300 genes. In addition, CBP and p300 are degraded by the 26 S proteasome pathway leading to an overall decrease in the levels of the CBP/p300 proteins. Furthermore, we provide evidence that Mdm2 (murine double minute 2), in the presence of active H-Ras or N-Ras, induces CBP/p300 degradation in NIH 3T3 cells. These findings support a novel mechanism for modulating other signalling transduction pathways that require these common co-activators.

The P34G mutation reduces the transforming activity of K-Ras and N-Ras in NIH 3T3 cells but not of H-Ras.

Ras proteins (H-, N-, and K-Ras) operate as molecular switches in signal transduction cascades controlling cell proliferation, differentiation, or apoptosis. The interaction of Ras with its effectors is mediated by the effector-binding loop, but different data about Ras location to plasma membrane subdomains and new roles for some docking/scaffold proteins point to signaling specificities of the different Ras proteins. To investigate the molecular mechanisms for these specificities, we compared an effector loop mutation (P34G) of three Ras isoforms (H-, N-, and K-Ras4B) for their biological and biochemical properties. Although this mutation diminished the capacity of Ras proteins to activate the Raf/ERK and the phosphatidylinositol 3-kinase/AKT pathways, the H-Ras V12G34 mutant retained the ability to cause morphological transformation of NIH 3T3 fibroblasts, whereas both the N-Ras V12G34 and the K-Ras4B V12G34 mutants were defective in this biological activity. On the other hand, although both the N-Ras V12G34 and the K-Ras4B V12G34 mutants failed to promote activation of the Ral-GDS/Ral A/PLD and the Ras/Rac pathways, the H-Ras V12G34 mutant retained the ability to activate these signaling pathways. Interestingly, the P34G mutation reduced specifically the N-Ras and K-Ras4B in vitro binding affinity to Ral-GDS, but not in the case of H-Ras. Thus, independently of Ras location to membrane subdomains, there are marked differences among Ras proteins in the sensitivity to an identical mutation (P34G) affecting the highly conserved effector-binding loop.