Ras is phosphorylated on a conserved tyrosine at position 32 within the switch I region via Src kinase. This phosphorylation inhibits the binding of effector Raf while promoting the engagement of GTPase-activating protein (GAP) and GTP hydrolysis. Here we identify SHP2 as the ubiquitously expressed tyrosine phosphatase that preferentially binds to and dephosphorylates Ras to increase its association with Raf and activate downstream proliferative Ras/ERK/MAPK signalling. In comparison to normal astrocytes, SHP2 activity is elevated in astrocytes isolated from glioblastoma multiforme (GBM)-prone H-Ras(12V) knock-in mice as well as in glioma cell lines and patient-derived GBM specimens exhibiting hyperactive Ras. Pharmacologic inhibition of SHP2 activity attenuates cell proliferation, soft-agar colony formation and orthotopic GBM growth in NOD/SCID mice and decelerates the progression of low-grade astrocytoma to GBM in a spontaneous transgenic glioma mouse model. These results identify SHP2 as a direct activator of Ras and a potential therapeutic target for cancers driven by a previously ‘undruggable' oncogenic or hyperactive Ras.
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...
Caveolin-1 (CAV1) is an essential structural constituent of caveolae, specialized lipid raft microdomains on the cell membrane involved in endocytosis and signal transduction, which are inexplicably deregulated and are associated with aggressiveness in numerous cancers. Here we identify CAV1 as a direct transcriptional target of oxygenlabile hypoxia-inducible factor 1 and 2 that accentuates the formation of caveolae, leading to increased dimerization of EGF receptor within the confined surface area of caveolae and its subsequent phosphorylation in the absence of ligand. Hypoxia-inducible factordependent up-regulation of CAV1 enhanced the oncogenic potential of tumor cells by increasing the cell proliferative, migratory, and invasive capacities. These results support a concept in which a crisis in oxygen availability or a tumor exhibiting hypoxic signature triggers caveolae formation that bypasses the requirement for ligand engagement to initiate receptor activation and the critical downstream adaptive signaling during a period when ligands required to activate these receptors are limited or are not yet available.
Capicua (CIC) is a transcriptional repressor that counteracts activation of genes downstream of receptor tyrosine kinase (RTK)/Ras/ERK signaling. It is well-established that tumorigenesis, especially in glioblastoma (GBM), is attributed to hyperactive RTK/Ras/ERK signaling. While CIC is mutated in other tumors, here we show that CIC has a tumor suppressive function in GBM through an alternative mechanism. We find that CIC protein levels are negligible in GBM due to continuous proteasome-mediated degradation, which is mediated by the E3 ligase PJA1 and show that this occurs through binding of CIC to its DNA target and phosphorylation on residue S173. PJA1 knockdown increased CIC stability and extended survival using in-vivo models of GBM. Deletion of the ERK binding site resulted in stabilization of CIC and increased therapeutic efficacy of ERK inhibition in GBM models. Our results provide a rationale to target CIC degradation in Ras/ERK-driven tumors, including GBM, to increase efficacy of ERK inhibitors.
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