Mutations in Ras GTPase and various other components of the Ras signaling pathways are among the most common genetic alterations in human cancers and also have been identified in several familial developmental syndromes. Over the past few decades it has become clear that the activity or the oncogenic potential of Ras is dependent on the nonreceptor tyrosine kinase Src to promote the Ras/Raf/MAPK pathway essential for proliferation, differentiation, and survival of eukaryotic cells. However, no direct relationship between Ras and Src has been established. We show here that Src binds to and phosphorylates GTP-, but not GDP-, loaded Ras on a conserved Y32 residue within the switch I region in vitro and that in vivo, Ras-Y32 phosphorylation markedly reduces the binding to effector Raf and concomitantly increases binding to GTPase-activating proteins and the rate of GTP hydrolysis. These results suggest that, in the context of predetermined crystallographic structures, Ras-Y32 serves as an Src-dependent keystone regulatory residue that modulates Ras GTPase activity and ensures unidirectionality to the Ras GTPase cycle.T he pioneering work of Harvey (1) and Kirsten and Mayer (2) showed that the Harvey strain murine sarcoma virus (HaMSV) and Kirsten strain murine sarcoma virus (KiMSV) sarcoma retroviruses cause rapid tumor formation in rats. The viral oncogenes, H-Ras and K-Ras, responsible for the oncogenic properties are altered versions of rat genes that encode enzymes with intrinsic guanine nucleotide binding and GTPase activity (3). The seminal discovery of mutationally activated RAS genes in human cancer in 1982 initiated an intensive research effort to understand Ras protein structure, function, and biology that continues to this day (4).The three human RAS oncogenes (H-RAS, N-RAS, and K-RAS) encode highly related (90% amino acid identity) 188-or 189-amino acid proteins. They are canonical members of a large superfamily consisting of more than 150 cellular members of small monomeric GTPase proteins, which function as molecular switches in a number of signaling pathways that regulate cell proliferation, differentiation, and apoptosis (1-3, 5, 6). As do other GTP-binding proteins, Ras cycles between the inactive GDP-and the active GTP-bound forms through conformational changes near the nucleotide-binding site localized in the switch I (amino acids 30-38) and switch II (amino acids 59-72) regions (7).Activation of the cell-surface receptor leads to the activation of Ras via guanine nucleotide-exchange factor (GEF), which binds to the Ras-GDP complex, causing dissociation of the bound GDP (8). Because GTP is present in cells at a much higher concentration than GDP, GTP binds spontaneously to the "empty" Ras molecule with the release of GEF (9, 10). Hydrolysis of GTP to GDP results from intrinsic Ras GTPase activity accelerated by GTPase-activating proteins (GAPs) that bind to and stabilize the Ras catalytic machinery, supplying additional catalytic "arginine finger" residues resulting in the inactivation of Ras an...

Deregulation of the RAS GTPase cycle due to mutations in the three RAS genes is commonly associated with cancer development. Protein tyrosine phosphatase SHP2 promotes RAF-to-MAPK signaling pathway and is an essential factor in RAS-driven oncogenesis. Despite the emergence of SHP2 inhibitors for the treatment of cancers harbouring mutant KRAS, the mechanism underlying SHP2 activation of KRAS signaling remains unclear. Here we report tyrosyl-phosphorylation of endogenous RAS and demonstrate that KRAS phosphorylation via Src on Tyr32 and Tyr64 alters the conformation of switch I and II regions, which stalls multiple steps of the GTPase cycle and impairs binding to effectors. In contrast, SHP2 dephosphorylates KRAS, a process that is required to maintain dynamic canonical KRAS GTPase cycle. Notably, Src- and SHP2-mediated regulation of KRAS activity extends to oncogenic KRAS and the inhibition of SHP2 disrupts the phosphorylation cycle, shifting the equilibrium of the GTPase cycle towards the stalled ‘dark state’.

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Members of the ErbB receptor family are associated with several cancers and appear to be providing useful targets for pharmacological therapeutics for tumours of the lung and breast. Further improvements of these therapies may be guided by a quantitative, dynamic integrative systems understanding of the complexities of ErbB dimerisation, trafficking and activation, for it is these complexities that render difficult intuiting how perturbations such as drug intervention will affect ErbB signalling activities. Towards this goal, we have developed a computational model implementing commonly accepted principles governing ErbB receptor interaction, trafficking, phosphorylation and dephosphorylation. Using this model, we are able to investigate several hypotheses regarding the compartmental localisation of dephosphorylation. Model results applied to experimental data on ErbB 1, ErbB2 and ErbB3 phosphorylation in H292 human lung carcinoma cells support a hypothesis that key dephosphorylation activity for these receptors occurs largely in an intracellular, endosomal compartment rather than at the cell surface plasma membrane. Thus, the endocytic trafficking-related compartmentalisation of dephosphorylation may define a critical aspect of the ErbB signalling response to ligand.

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