Identification of residues of the H-ras protein critical for functional interaction with guanine nucleotide exchange factors.

Ras proteins are activated in vivo by guanine nucleotide exchange factors encoded by genes homologous to the CDC25 gene of Saccharomyces cerevisiae. We have taken a combined genetic and biochemical approach to probe the sites on Ras proteins important for interaction with such exchange factors and to further probe the mechanism of CDC25-catalyzed GDP-GTP exchange. Random mutagenesis coupled with genetic selection in S. cerevisiae was used to generate second-site mutations within human H-ras-ala15 which could suppress the ability of the Ala-15 substitution to block CDC25 function. We transferred these second-site suppressor mutations to normal H-ras and oncogenic H-rasVal-12 to test whether they induced a general loss of function or whether they selectively affected CDC25 interaction. Four highly selective mutations were discovered, and they affected the surface-located amino acid residues 62, 63, 67, and 69. Two lines of evidence suggested that these residues may be involved in binding to CDC25: (i) using the yeast two-hybrid system, we demonstrated that these mutants cannot bind CDC25 under conditions where the wild-type H-Ras protein can; (ii) we demonstrated that the binding to H-Ras of monoclonal antibody Y13-259, whose epitope has been mapped to residues 63, 65, 66, 67, 70, and 73, is blocked by the mouse sos1 and yeast CDC25 gene products. We also present evidence that the mechanism by which CDC25 catalyzes exchange is more involved than simply catalyzing the release of bound nucleotide and passively allowing nucleotides to rebind. Most critically, a complex of Ras and CDC25 protein, unlike free Fas protein, possesses significantly greater affinity for GTP than for GDP. Furthermore, the Ras CDC25 complex is more readily dissociated into free subunits by GTP than it is by GDP. Both of these results suggest a function for CDC25 in promoting the selective exchange of GTP for GDP.

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Oncogenic KRAS: Signaling and Drug Resistance

Cancers ◽

10.3390/cancers13225599 ◽

2021 ◽

Vol 13 (22) ◽

pp. 5599

Author(s):

Hyeon Jin Kim ◽

Han Na Lee ◽

Mi Suk Jeong ◽

Se Bok Jang

Keyword(s):

Recent Progress ◽

Human Cancer ◽

Guanine Nucleotide Exchange Factors ◽

Physiological Signals ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Ras Proteins ◽

Ras Protein ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factors

RAS proteins play a role in many physiological signals transduction processes, including cell growth, division, and survival. The Ras protein has amino acids 188-189 and functions as GTPase. These proteins are switch molecules that cycle between inactive GDP-bound and active GTP-bound by guanine nucleotide exchange factors (GEFs). KRAS is one of the Ras superfamily isoforms (N-RAS, H-RAS, and K-RAS) that frequently mutate in cancer. The mutation of KRAS is essentially performing the transformation in humans. Since most RAS proteins belong to GTPase, mutated and GTP-bound active RAS is found in many cancers. Despite KRAS being an important molecule in mostly human cancer, including pancreatic and breast, numerous efforts in years past have persisted in cancer therapy targeting KRAS mutant. This review summarizes the biological characteristics of these proteins and the recent progress in the exploration of KRAS-targeted anticancer, leading to new insight.

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Essential Role of Vav Family Guanine Nucleotide Exchange Factors in EphA Receptor-Mediated Angiogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.02215-05 ◽

2006 ◽

Vol 26 (13) ◽

pp. 4830-4842 ◽

Cited By ~ 84

Author(s):

Sonja G. Hunter ◽

Guanglei Zhuang ◽

Dana Brantley-Sieders ◽

Wojciech Swat ◽

Christopher W. Cowan ◽

...

Keyword(s):

Guanine Nucleotide Exchange Factors ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factors ◽

Rac1 Gtpase ◽

Rho Family ◽

Epha Receptor

ABSTRACT Angiogenesis, the process by which new blood vessels are formed from preexisting vasculature, is critical for vascular remodeling during development and contributes to the pathogenesis of diseases such as cancer. Prior studies from our laboratory demonstrate that the EphA2 receptor tyrosine kinase is a key regulator of angiogenesis in vivo. The EphA receptor-mediated angiogenic response is dependent on activation of Rho family GTPase Rac1 and is regulated by phosphatidylinositol 3-kinase. Here we report the identification of Vav2 and Vav3 as guanine nucleotide exchange factors (GEFs) that link the EphA2 receptor to Rho family GTPase activation and angiogenesis. Ephrin-A1 stimulation recruits the binding of Vav proteins to the activated EphA2 receptor. The induced association of EphA receptor and Vav proteins modulates the activity of Vav GEFs, leading to activation of Rac1 GTPase. Overexpression of either Vav2 or Vav3 in primary microvascular endothelial cells promotes Rac1 activation, cell migration, and assembly in response to ephrin-A1 stimulation. Conversely, loss of Vav2 and Vav3 GEFs inhibits Rac1 activation and ephrin-A1-induced angiogenic responses both in vitro and in vivo. In addition, embryonic fibroblasts derived from Vav2−/− Vav3−/− mice fail to spread on an ephrin-A1-coated surface and exhibit a significant decrease in the formation of ephrin-A1-induced lamellipodia and filopodia. These findings suggest that Vav GEFs serve as a molecular link between EphA2 receptors and the actin cytoskeleton and provide an important mechanism for EphA2-mediated angiogenesis.

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ITAM Signaling by Vav Family Rho Guanine Nucleotide Exchange Factors Regulates Interstitial Transit Rates of Neutrophils In Vivo

PLoS ONE ◽

10.1371/journal.pone.0004652 ◽

2009 ◽

Vol 4 (2) ◽

pp. e4652 ◽

Cited By ~ 25

Author(s):

Daniel B. Graham ◽

Bernd H. Zinselmeyer ◽

Francesca Mascarenhas ◽

Ryan Delgado ◽

Mark J. Miller ◽

...

Keyword(s):

Guanine Nucleotide Exchange Factors ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factors

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New insights into the Ras onco-protein and its interactions with the Raf-1-1 kinase

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166853 ◽

1996 ◽

Vol 54 ◽

pp. 878-879

Author(s):

Sharon Campbell ◽

Helen Mott ◽

Sheng Zhong ◽

Jonelle Drugan ◽

John Carpenter

Keyword(s):

Bound States ◽

Direct Interaction ◽

Nucleotide Binding ◽

Guanine Nucleotide Exchange Factors ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Biochemical Effect ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factors

The Ras proteins are members of a large superfamily of nucleotide-binding proteins that cycle between active GTP- and inactive GDP-bound states. They are positively regulated by guanine nucleotide exchange factors (GEFs) that promote formation of the active GTP-bound state and negatively regulated by GTPase activating proteins (GAPs) that stimulate formation of the inactive GDP-complexed protein. Structural mutations that activate Ras oncogenic potential either impair GAP-stimulated GTPase activity or promote enhanced intrinsic nucleotide exchange. The net result of either biochemical effect is to favor elevated levels of Ras-GTP in vivo.We are investigating selected Ras variants containing substitutions at conserved sites thought to be important for guanine nucleotide binding, GTP hydrolysis, GTP/GDP interconversion and protein recognition, to better understand the role of these conserved amino acids in Ras-mediated signal transduction. Recent studies conducted at the U. of North Carolina will be presented, where we have characterized a novel activating mutation in Ras using multi-dimensional NMR spectroscopy and established a region involved in direct interaction with guanine nucleotide exchange factors.

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Basis for Signaling Specificity Difference between Sos and Ras-GRF Guanine Nucleotide Exchange Factors

Journal of Biological Chemistry ◽

10.1074/jbc.m107407200 ◽

2001 ◽

Vol 276 (50) ◽

pp. 47248-47256 ◽

Cited By ~ 13

Author(s):

Xuejun Tian ◽

Larry A. Feig

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Guanine Nucleotide Exchange Factors ◽

Amino Acid Sequences ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Ras Proteins ◽

Signaling Specificity ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factors

Sos and Ras-GRF are two families of guanine nucleotide exchange factors that activate Ras proteins in cells. Sos proteins are ubiquitously expressed and are activated in response to cell-surface tyrosine kinase stimulation. In contrast, Ras-GRF proteins are expressed primarily in central nervous system neurons and are activated by calcium/calmodulin binding and by phosphorylation. Although both Sos1 and Ras-GRF1 activate the Ras proteins Ha-Ras, N-Ras, and Ki-Ras, only Ras-GRF1 also activates the functionally distinct R-Ras GTPase. In this study, we determined which amino acid sequences in these exchange factors and their target GTPases are responsible for this signaling specificity difference. Analysis of chimeras and individual amino acid exchanges between Sos1 and Ras-GRF1 revealed that the critical amino acids reside within an 11-amino acid segment of their catalytic domains between the second and third structurally conserved regions (amino acids (aa) 828–838 in Sos1 and 1057–1067 in Ras-GRF1) of Ras guanine nucleotide exchange factors. In Sos1, this segment is in helix B, which is known to interact with the switch 2 region of Ha-Ras. Interestingly, a similar analysis of Ha-Ras and R-Ras chimeras did not identify the switch 2 region of Ha-Ras as encoding specificity. Instead, we found a more distal protein segment, helix 3 (aa 91–103 in Ha-Ras and 117–129 in R-Ras), which interacts instead primarily with helix K (aa 1002–1016) of Sos1. These findings suggest that specificity derives from the fact that R-Ras-specific amino acids in the region analogous to Ha-Ras helix 3 prevent a functional interaction with Sos1 indirectly, possibly by preventing an appropriate association of its switch 2 region with helix B of Sos1. Although previous studies have shown that helix B of Sos1 and helix 3 of Ha-Ras are involved in promoting nucleotide exchange on Ras proteins, this study highlights the importance of these regions in establishing signaling specificity.

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