The Y13-238 monoclonal antibody reacts with human, mouse, and rat v-H-ras (within amino acids 120-138) and does not react with v-K-ras. v-H-ras binds GTP/GDP and has intrinsic GTPase activity. Ras proteins alternate between an inactive form bound to GDP and an active form bound to GTP, activated by a guanine nucleotide-exchange factor (GEF) and inactivated by a GTPase-activating protein (GAP). Under normal conditions, Ras family members influence cell growth and differentiation events. Mutations in the Ras family of proto-oncogenes are very common, being found in 20% to 30% of all human tumors. Ras point mutations are the single most common abnormality of human proto-oncogenes. The Y13-238 antibody does not neutralize ras GTPase binding and hydrolysis in vivo or in vitro.

InVivoMAb anti-mouse/human/rat v-H-Ras (Clone: Y13-238)

 Dhillon, A. S., et al. (2009). “The C-terminus of Raf-1 acts as a 14-3-3-dependent activation switch.” Cell Signal 21(11): 1645-1651. PubMed

The Raf-1 protein kinase is a major activator of the ERK MAPK pathway, which links signaling by a variety of cell surface receptors to the regulation of cell proliferation, survival, differentiation and migration. Signaling by Raf-1 is regulated by a complex and poorly understood interplay between phosphorylation events and protein-protein interactions. One important mode of Raf-1 regulation involves the phosphorylation-dependent binding of 14-3-3 proteins. Here, we have examined the mechanism whereby the C-terminal 14-3-3 binding site of Raf-1, S621, controls the activation of MEK-ERK signaling. We show that phosphorylation of S621 turns over rapidly and is enriched in the activated pool of endogenous Raf-1. The phosphorylation on this site can be mediated by Raf-1 itself but also by other kinase(s). Mutations that prevent the binding of 14-3-3 proteins to S621 render Raf-1 inactive by specifically disrupting its capacity to bind to ATP, and not by gross conformational alteration as indicated by intact MEK binding. Phosphorylation of S621 correlates with the inhibition of Raf-1 catalytic activity in vitro, but 14-3-3 proteins can completely reverse this inhibition. Our findings suggest that 14-3-3 proteins function as critical cofactors in Raf-1 activation, which induce and maintain the protein in a state that is competent for both ATP binding and MEK phosphorylation.

Liao, J., et al. (2006). “Growth factor-dependent AKT activation and cell migration requires the function of c-K(B)-Ras versus other cellular ras isoforms.” J Biol Chem 281(40): 29730-29738. PubMed

K-Ras-negative fibroblasts are defective in their steady-state expression of MMP-2. This occurs through c-K(B)-Ras dependent regulation of basal levels of AKT activity. In this report, we have extended those studies to demonstrate that in the absence of K-Ras expression, PDGF-BB fails to induce significant AKT activation, although this was not the case in N-Ras-negative cells. This phenotype was directly linked to PDGF-dependent cell migration. All of the independently immortalized K-Ras-negative cells failed to migrate upon the addition of PDGF. Only ectopic expression of c-K(B)-Ras, not c-K(A)-Ras nor oncogenic N-Ras, could restore both PDGF-dependent AKT activation and cell migration. Since most Ras binding partners can interact with all Ras isoforms, the specificity of PDGF-dependent activation of AKT and enhanced cell migration suggests that these outcomes are likely to be regulated through a c-K(B)-Ras-specific binding partner. Others have published that of the four Ras isoforms, only K(B)-Ras can form a stable complex with calmodulin (CaM). Along those lines, we provide evidence that 1) PDGF addition results in increased levels of a complex between c-K(B)-Ras and CaM and 2) the biological outcomes that are strictly dependent on c-K(B)-Ras (AKT activation and cell migration) are blocked by CaM antagonists. The PDGF-dependent activation of ERK is unaffected by the absence of K(B)-Ras and presence of CaM antagonists. This is the first example of a linkage between a specific biological outcome, cell migration, and the activity of a single Ras isoform, c-K(B)-Ras.

Light, Y., et al. (2002). “14-3-3 antagonizes Ras-mediated Raf-1 recruitment to the plasma membrane to maintain signaling fidelity.” Mol Cell Biol 22(14): 4984-4996. PubMed

We have investigated the role that S259 phosphorylation, S621 phosphorylation, and 14-3-3 binding play in regulating Raf-1 activity. We show that 14-3-3 binding, rather than Raf-1 phosphorylation, is required for the correct regulation of kinase activity. Phosphorylation of S621 is not required for activity, but 14-3-3 binding is essential. When 14-3-3 binding to conserved region 2 (CR2) was disrupted, Raf-1 basal kinase activity was elevated and it could be further activated by (V12,G37)Ras, (V23)TC21, and (V38)R-Ras. Disruption of 14-3-3 binding at CR2 did not recover binding of Raf-1 to (V12,G37)Ras but allowed more efficient recruitment of Raf-1 to the plasma membrane and stimulated its phosphorylation on S338. Finally, (V12)Ras, but not (V12,G37)Ras, displaced 14-3-3 from full-length Raf-1 and the Raf-1 bound to Ras. GTP was still phosphorylated on S259. Our data suggest that stable association of Raf-1 with the plasma membrane requires Ras-mediated displacement of 14-3-3 from CR2. Small G proteins that cannot displace 14-3-3 fail to recruit Raf-1 to the membrane efficiently and so fail to stimulate kinase activity.