The Ras binary switch: an ideal processor for decoding complex Ca2+ signals?

2003 ◽  
Vol 31 (5) ◽  
pp. 966-969 ◽  
Author(s):  
S.A. Walker ◽  
P.J. Lockyer ◽  
P.J. Cullen
Keyword(s):  
Intracellular Calcium ◽  
Calcium Concentration ◽  
Intracellular Calcium Concentration ◽  
Nucleotide Exchange ◽  
Ras Proteins ◽  
Complex Calcium ◽  
Calcium Signals ◽  
Surface Receptors ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

Activation of cell-surface receptors often leads to changes in intracellular calcium concentration ([Ca2+]i). Receptor-generated calcium transients are often seen as repetitive spikes of elevated intracellular calcium concentration ([Ca2+]i), whose frequency varies according to the amplitude of the receptor stimuli. This suggests a requirement for molecular decoders, capable of interpreting such complex calcium signals into the correct physiological response. Ras proteins are binary molecular switches controlling a plethora of cellular responses. Whether Ras is in its active GTP-bound, or inactive GDP-bound, form is determined by the activity of guanine nucleotide exchange factors (GEFs) and GTPase-activating protein (GAPs). Calcium-regulated GEFs and GAPs have been identified, some with an exquisite sensitivity to [Ca2+]i, implicating a potential role of complex calcium signals in regulating Ras.

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

1994 ◽  
Vol 14 (2) ◽  
pp. 1104-1112
Author(s):  
R D Mosteller ◽  
J Han ◽  
D Broek
Keyword(s):  
Random Mutagenesis ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
Ras Proteins ◽  
Ras Protein ◽  
Guanine Nucleotide Exchange ◽  
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.

Role of the Vps9-domain protein RgfA inDictyosteliumchemotaxis and development

2013 ◽  
Vol 59 (1) ◽  
pp. 22-27
Author(s):  
Jeffrey A. Hadwiger
Keyword(s):  
Cell Growth ◽  
Heterologous Expression ◽  
Rab Proteins ◽  
Nucleotide Exchange ◽  
Surface Receptors ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Genes Encoding ◽  
Redundant Functions ◽  
External Signals

Proteins with a Vps9 domain function as guanine nucleotide exchange factors for Rab proteins and can mediate the uptake of cell surface receptors or other molecules through endocytosis. However, genes encoding these proteins have not been previously studied in cells with robust chemotactic capabilities. Several genes encoding Vps9 domains were identified in the genome of Dictyostelium discoideum, and one of the genes, designated as rgfA (DDB_G0272038), was examined for functions in cell growth, development, and chemotaxis. The rgfA gene was expressed during vegetative growth and throughout development, but disruption of this gene resulted in no major alterations in cell growth, macropinocytosis, developmental morphology, or chemotactic movement. However, heterologous expression of RgfA resulted in a delay of developmental morphogenesis and impaired chemotaxis of cells to folate but did not affect macropinocytosis. These results suggest that RgfA might share redundant functions with other Dictyostelium Vps9-domain proteins and that heterologous expression of this protein can alter processes that depend on the reception of external signals.

scholarly journals Influence of guanine nucleotides on complex formation between Ras and CDC25 proteins.

1993 ◽  
Vol 13 (3) ◽  
pp. 1345-1352 ◽  
Author(s):  
C C Lai ◽  
M Boguski ◽  
D Broek ◽  
S Powers
Keyword(s):  
Fusion Protein ◽  
Guanine Nucleotides ◽  
Free Form ◽  
Physical Interaction ◽  
Nucleotide Exchange ◽  
Ras Proteins ◽  
Positive Role ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

The Saccharomyces cerevisiae CDC25 gene and closely homologous genes in other eukaryotes encode guanine nucleotide exchange factors for Ras proteins. We have determined the minimal region of the budding yeast CDC25 gene capable of activity in vivo. The region required for full biological activity is approximately 450 residues and contains two segments homologous to other proteins: one found in both Ras-specific exchange factors and the more distant Bud5 and Lte1 proteins, and a smaller segment of 48 amino acids found only in the Ras-specific exchange factors. When expressed in Escherichia coli as a fusion protein, this region of CDC25 was found to be a potent catalyst of GDP-GTP exchange on yeast Ras2 as well as human p21H-ras but inactive in promoting exchange on the Ras-related proteins Ypt1 and Rsr1. The CDC25 fusion protein catalyzed replacement of GDP-bound to Ras2 with GTP (activation) more efficiently than that of the reverse reaction of replacement of GTP for GDP (deactivation), consistent with prior genetic analysis of CDC25 which indicated a positive role in the activation of Ras. To more directly study the physical interaction of CDC25 and Ras proteins, we developed a protein-protein binding assay. We determined that CDC25 binds tightly to Ras2 protein only in the absence of guanine nucleotides. This higher affinity of CDC25 for the nucleotide-free form than for either the GDP- or GTP-bound form suggests that CDC25 catalyzes exchange of guanine nucleotides bound to Ras proteins by stabilization of the transitory nucleotide-free state.

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

1994 ◽  
Vol 14 (2) ◽  
pp. 1104-1112 ◽  
Author(s):  
R D Mosteller ◽  
J Han ◽  
D Broek
Keyword(s):  
Random Mutagenesis ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
Ras Proteins ◽  
Ras Protein ◽  
Guanine Nucleotide Exchange ◽  
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.

scholarly journals Focus on Cdc42 in Breast Cancer: New Insights, Target Therapy Development and Non-Coding RNAs

Cells ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 146 ◽  
Author(s):  
Yu Zhang ◽  
Jun Li ◽  
Xing-Ning Lai ◽  
Xue-Qiao Jiao ◽  
Jun-Ping Xiong ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Malignant Tumors ◽  
Target Therapy ◽  
Nucleotide Exchange ◽  
Surface Receptors ◽  
Cellular Processes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Non Coding Rnas ◽  
Guanosine Triphosphatase

Breast cancer is the most common malignant tumors in females. Although the conventional treatment has demonstrated a certain effect, some limitations still exist. The Rho guanosine triphosphatase (GTPase) Cdc42 (Cell division control protein 42 homolog) is often upregulated by some cell surface receptors and oncogenes in breast cancer. Cdc42 switches from inactive guanosine diphosphate (GDP)-bound to active GTP-bound though guanine-nucleotide-exchange factors (GEFs), results in activation of signaling cascades that regulate various cellular processes such as cytoskeletal changes, proliferation and polarity establishment. Targeting Cdc42 also provides a strategy for precise breast cancer therapy. In addition, Cdc42 is a potential target for several types of non-coding RNAs including microRNAs and lncRNAs. These non-coding RNAs is extensively involved in Cdc42-induced tumor processes, while many of them are aberrantly expressed. Here, we focus on the role of Cdc42 in cell morphogenesis, proliferation, motility, angiogenesis and survival, introduce the Cdc42-targeted non-coding RNAs, as well as present current development of effective Cdc42-targeted inhibitors in breast cancer.

The GAP1 family of GTPase-activating proteins: spatial and temporal regulators of small GTPase signalling

2006 ◽  
Vol 34 (5) ◽  
pp. 846-850 ◽  
Author(s):  
S. Yarwood ◽  
D. Bouyoucef-Cherchalli ◽  
P.J. Cullen ◽  
S. Kupzig
Keyword(s):  
Control Cell ◽  
Guanine Nucleotide Exchange Factors ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Ras Proteins ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Gtpase Activating Proteins ◽  
Oncogenic Mutations ◽  
Hydrolysis Of

Ras proteins are binary switches that, by cycling between inactive GDP-bound and active GTP-bound conformations, regulate multiple cellular signalling pathways including those that control cell growth, differentiation and survival. Approximately 30% of all human tumours express Ras-containing oncogenic mutations that lock the protein into a constitutively active conformation. The activation status of Ras is regulated by two groups of proteins: GEFs (guanine nucleotide-exchange factors) bind to Ras and enhance the exchange of GDP for GTP, thereby activating it, whereas GAPs (GTPase-activating proteins) inactivate Ras by binding to the GTP-bound form and enhancing the hydrolysis of the bound nucleotide back to GDP. In this review, we focus on a group of key regulators of Ras inactivation, the GAP1 family of Ras-GAPs. The members of this family are GAP1m, GAP1IP4BP, CAPRI (Ca2+-promoted Ras inactivator) and RASAL (Ras-GTPase-activating-like protein) and, as we will discuss, they are emerging as important modulators of Ras and small GTPase signalling that are subject to regulation by a diverse array of events and second messenger signals.

scholarly journals Oncogenic KRAS: Signaling and Drug Resistance

Cancers ◽  
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.

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

2001 ◽  
Vol 276 (50) ◽  
pp. 47248-47256 ◽  
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.

scholarly journals RAS, wanted dead or alive: Advances in targeting RAS mutant cancers

2020 ◽  
Vol 13 (624) ◽  
pp. eaay6013 ◽  
Author(s):  
Clint A. Stalnecker ◽  
Channing J. Der
Keyword(s):  
Guanine Nucleotide Exchange Factors ◽  
Ras Signaling ◽  
Nucleotide Exchange ◽  
Ras Proteins ◽  
Oncogenic Ras ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Ras Activation ◽  
Downstream Effectors ◽  
And Function

Oncogenic RAS proteins, which are mutated in approximately 24% of all human cancers, have earned a well-deserved reputation as being “undruggable.” However, several studies have challenged that reputation. With the first small molecules that directly target one oncogenic RAS mutant (G12C) undergoing clinical evaluation, there have been substantial advances in finding anti-RAS therapeutic strategies. Furthermore, new insights have come from the growing appreciation that neither all RAS proteins (HRAS, NRAS, and KRAS4A/KRAS4B) nor all oncogenic RAS mutations (such as at residues Gly12, Gly13, and Gln61) have the same impact on RAS signaling and function. The role of the nonmutated, wild-type RAS proteins in the context of mutant RAS is increasingly considered to be targetable, with reports of strategies that directly disrupt either the RAS interaction with activating guanine nucleotide exchange factors (GEFs) or receptor tyrosine kinase–mediated and GEF-dependent RAS activation (such as by targeting the scaffolding phosphatase SHP2). Last, the development of agents that target downstream effectors of RAS signaling has advanced substantially. In this review, we highlight some important trends in the targeting of RAS proteins in cancer.

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