RasGEFN

Guanine nucleotide exchange factor for Ras-like GTPases; N-terminal motif

• SMART accession number: SM00229
• Description: A subset of guanine nucleotide exchange factor for Ras-like small GTPases appear to possess this domain N-terminal to the RasGef (Cdc25-like) domain. The recent crystal structureof Sos shows that this domain is alpha-helical and plays a "purely structural role" (Nature 394, 337-343).
• Interpro abstract (IPR000651):

  The crystal structure of the GEF region of human Sos1 complexes with Ras has been solved [ (PUBMED:9690470) ]. The structure consists of two distinct alpha helical structural domains: the N-terminal domain which seems to have a purely structural role and the C-terminal domain which is sufficient for catalytic activity and contains all residues that interact with Ras. A main feature of the catalytic domain is the protrusion of a helical hairpin important for the nucleotide-exchange mechanism. The N-terminal domain is likely to be important for the stability and correct placement of the hairpin structure.

  This entry represents a domain found in several GEF for Ras-like small GTPases which lies N-terminal to the RasGef (Cdc25-like) domain.

  The N-terminal domain of guanine nucleotide exchange factor (GEF) for Ras-like GTPases is also called REM domain (Ras exchanger motif). REM contacts the GTPase and is assumed to participate in the catalytic activity of the exchange factor. Proteins with the REM domain include Sos1 and Sos2, which relay signals from tyrosine-kinase mediated signalling to Ras, RasGRP1-4, RasGRF1,2, CNrasGEF, and RAP-specific nucleotide exchange factors, to name a few [ (PUBMED:18541156) (PUBMED:18236214) (PUBMED:7786285) (PUBMED:8259209) (PUBMED:8479541) (PUBMED:16452984) (PUBMED:12628188) (PUBMED:9690470) ].

There are 13658 RasGEFN domains in 12887 proteins in SMART's nrdb database.

Cellular role (predicted cellular role)

Cellular role: signalling

Literature (relevant references for this domain)

Primary literature is listed below; Automatically-derived, secondary literature is also avaliable.

• Schultz J, Milpetz F, Bork P, Ponting CP
• SMART, a simple modular architecture research tool: identification of signaling domains.
• Proc Natl Acad Sci U S A. 1998; 95: 5857-64

Accurate multiple alignments of 86 domains that occur in signaling proteins have been constructed and used to provide a Web-based tool (SMART: simple modular architecture research tool) that allows rapid identification and annotation of signaling domain sequences. The majority of signaling proteins are multidomain in character with a considerable variety of domain combinations known. Comparison with established databases showed that 25% of our domain set could not be deduced from SwissProt and 41% could not be annotated by Pfam. SMART is able to determine the modular architectures of single sequences or genomes; application to the entire yeast genome revealed that at least 6.7% of its genes contain one or more signaling domains, approximately 350 greater than previously annotated. The process of constructing SMART predicted (i) novel domain homologues in unexpected locations such as band 4.1-homologous domains in focal adhesion kinases; (ii) previously unknown domain families, including a citron-homology domain; (iii) putative functions of domain families after identification of additional family members, for example, a ubiquitin-binding role for ubiquitin-associated domains (UBA); (iv) cellular roles for proteins, such predicted DEATH domains in netrin receptors further implicating these molecules in axonal guidance; (v) signaling domains in known disease genes such as SPRY domains in both marenostrin/pyrin and Midline 1; (vi) domains in unexpected phylogenetic contexts such as diacylglycerol kinase homologues in yeast and bacteria; and (vii) likely protein misclassifications exemplified by a predicted pleckstrin homology domain in a Candida albicans protein, previously described as an integrin.

• Baouz S, Jacquet E, Bernardi A, Parmeggiani A
• The N-terminal moiety of CDC25(Mm), a GDP/GTP exchange factor of Ras proteins, controls the activity of the catalytic domain. Modulation by calmodulin and calpain.
• J Biol Chem. 1997; 272: 6671-6

This work describes the in vitro properties of full-length CDC25(Mm) (1262 amino acid residues), a GDP/GTP exchange factor (GEF) of H-ras p21. CDC25(Mm), isolated as a recombinant protein in Escherichia coli and purified by various chromatographic methods, could stimulate the H-ras p21.GDP dissociation rate; however, its specific activity was 25 times lower than that of the isolated catalytic domain comprising the last C-terminal 285 residues (C-CDC25(Mm285)) and 5 times lower than the activity of the C-terminal half-molecule (631 residues). This reveals a negative regulation of the catalytic domain by other domains of the molecule. Accordingly, the GEF activity of CDC25(Mm) was increased severalfold by the Ca2+-dependent protease calpain that cleaves around a PEST-like region (residues 798-853), producing C-terminal fragments of 43-56 kDa. In agreement with the presence of an IQ motif on CDC25(Mm) (residues 202-229), calmodulin interacted functionally with the exchange factor. Depending on the calmodulin concentration an inhibition up to 50% of the CDC25(Mm)-induced nucleotide exchange activity on H-ras p21 was observed, an effect requiring Ca2+ ions. Calmodulin also inhibited C-CDC25(Mm285) but with a approximately 100 times higher IC50 than in the case of CDC25(Mm) ( approximately 10 &mgr;M versus 0.1 microM, respectively). Together, these results emphasize the role of the other domains of CDC25(Mm) in controlling the activity of the catalytic domain and support the involvement of calmodulin and calpain in the in vivo regulation of the CDC25(Mm) activity.

• Byrne JL, Paterson HF, Marshall CJ
• p21Ras activation by the guanine nucleotide exchange factor Sos, requires the Sos/Grb2 interaction and a second ligand-dependent signal involving the Sos N-terminus.
• Oncogene. 1996; 13: 2055-65

It has been suggested that a key event in growth factor-induced p21Ras activation by the guanine nucleotide exchange factor Sos, is the recruitment of Sos to the plasma membrane by its interaction with the adaptor protein Grb2. However, other evidence argues that the sub cellular localisation of Sos is independent of Grb2, and that the Sos/Grb2 interaction can be dispensed with for p21Ras activation. To clarify the role of the Sos/Grb2 interaction in ligand-stimulated p21Ras activation, we have utilised the observation that overexpression of the Sos C-terminal domain can effectively inhibit p21Ras-dependent signalling in three different mammalian systems. We have shown that concurrent expression of Grb2, but not SH2 or SH3 domain mutants of Grb2, or the alternative adaptor protein Nck, can rescue this inhibitory effect of the C-terminus. This shows that the Grb2/Sos interaction is required to mediate growth factor-dependent activation of p21Ras, and requires the presence of intact SH2 and SH3 domains of Grb2. This approach was also used for a functional analysis of Sos which revealed that growth factor dependent signals are transmitted through both the N-terminal and C-terminal domains.

• Quilliam LA, Khosravi-Far R, Huff SY, Der CJ
• Guanine nucleotide exchange factors: activators of the Ras superfamily of proteins.
• Bioessays. 1995; 17: 395-404

Ras proteins function as critical relay switches that regulate diverse signaling pathways between cell surface receptors and the nucleus. Over the past 2-3 years researchers have identified many components of these pathways that mediate Ras activation and effector function. Among these proteins are several guanine nucleotide exchange factors (GEFs), which are responsible for directly interacting with and activating Ras in response to extracellular stimuli. Analogous GEFs regulate Ras-related proteins that serve other diverse cellular functions. In particular, a growing family of proteins (Dbl homology proteins) has recently been identified, which may function as GEFs for the Rho family of Ras-related proteins. This review summarizes our current knowledge of the structure, biochemistry and biology of Ras and Rho family GEFs. Additionally, we describe mechanisms of GEF activation of Ras in signal transduction and address the potential that deregulated GEFs might contribute to malignant transformation through chronic Ras protein activation.

• Boguski MS, McCormick F
• Proteins regulating Ras and its relatives.
• Nature. 1993; 366: 643-54

GTPases of the Ras superfamily regulate many aspects of cell growth, differentiation and action. Their functions depend on their ability to alternate between inactive and active forms, and on their cellular localization. Numerous proteins affecting the GTPase activity, nucleotide exchange rates and membrane localization of Ras superfamily members have now been identified. Many of these proteins are much larger and more complex than their targets, containing multiple domains capable of interacting with an intricate network of cellular enzymes and structures.

• Lai CC, Boguski M, Broek D, Powers S
• Influence of guanine nucleotides on complex formation between Ras and CDC25 proteins.
• Mol Cell Biol. 1993; 13: 1345-52

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.

• Li N et al.
• Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling.
• Nature. 1993; 363: 85-8

Many of the actions of receptor tyrosine kinases are mediated by the protein Ras, including the activation of various downstream serine/threonine kinases and the stimulation of growth and differentiation. The human protein Grb2 binds to ligand-activated growth factor receptors and downstream effector proteins through its Src-homology (SH) domains SH2 and SH3, respectively, and like its homologue from Caenorhabditis elegans, Sem-5, apparently forms part of a highly conserved pathway by which these receptors can control Ras activity. Here we show that the SH3 domains of Grb2 bind to the carboxy-terminal part of hSos1, the human homologue of the Drosophila guanine-nucleotide-releasing factor for Ras, which is essential for control of Ras activity by epidermal growth factor receptor and sevenless. Moreover, a synthetic 10-amino-acid peptide containing the sequence PPVPPR specifically blocks the interaction. These results indicate that the Grb2/hSos1 complex couples activated EGF receptor to Ras signalling.

• Skolnik EY et al.
• The function of GRB2 in linking the insulin receptor to Ras signaling pathways.
• Science. 1993; 260: 1953-5

Insulin-induced activation of extracellular signal-regulated kinases [ERKs, also known as mitogen-activated protein (MAP) kinases] is mediated by Ras. Insulin activates Ras primarily by increasing the rate of guanine nucleotide-releasing activity. Here, we show that insulin-induced activation of ERKs was enhanced by stable overexpression of growth factor receptor-bound protein 2 (GRB2) but not by overexpression of GRB2 proteins with point mutations in the Src homology 2 and 3 domains. Moreover, a dominant negative form of Ras (with Ser17 substituted with Asn) blocked insulin-induced activation of ERKs in cells that overexpressed GRB2. GRB2 overexpression led to increased formation of a complex between the guanine nucleotide-releasing factor Sos (the product of the mammalian homolog of son of sevenless gene) and GRB2. In response to insulin stimulation, this complex bound to tyrosine-phosphorylated IRS-1 (insulin receptor substrate-1) and Shc. In contrast to the activated epidermal growth factor receptor that binds the GRB2-Sos complex directly, activation of the insulin receptor results in the interaction of GRB2-Sos with IRS-1 and Shc, thus linking the insulin receptor to Ras signaling pathways.

Metabolism (metabolic pathways involving proteins which contain this domain)

% proteins involved	KEGG pathway ID	Description
10.93	map04010	MAPK signaling pathway
5.81	map04510	Focal adhesion
5.81	map04910	Insulin signaling pathway
5.81	map05211	Renal cell carcinoma
5.35	map05210	Colorectal cancer
3.95	map04664	Fc epsilon RI signaling pathway
3.95	map05213	Endometrial cancer
3.95	map04912	GnRH signaling pathway
3.95	map04630	Jak-STAT signaling pathway
3.95	map05220	Chronic myeloid leukemia
3.95	map04320	Dorso-ventral axis formation
3.95	map05215	Prostate cancer
3.95	map04012	ErbB signaling pathway
3.95	map05214	Glioma
3.95	map04810	Regulation of actin cytoskeleton
3.95	map04540	Gap junction
3.95	map04660	T cell receptor signaling pathway
3.95	map05221	Acute myeloid leukemia
3.95	map05223	Non-small cell lung cancer
3.95	map04650	Natural killer cell mediated cytotoxicity
3.26	map04670	Leukocyte transendothelial migration
1.63	map04720	Long-term potentiation
1.40	map05212	Pancreatic cancer
0.70	map04111	Cell cycle - yeast

This information is based on mapping of SMART genomic protein database to KEGG orthologous groups. Percentage points are related to the number of proteins with RasGEFN domain which could be assigned to a KEGG orthologous group, and not all proteins containing RasGEFN domain. Please note that proteins can be included in multiple pathways, ie. the numbers above will not always add up to 100%.

Structure (3D structures containing this domain)

3D Structures of RasGEFN domains in PDB

PDB code	Title
1bkd	COMPLEX OF HUMAN H-RAS WITH HUMAN SOS-1
1nvu	Structural evidence for feedback activation by RasGTP of the Ras-specific nucleotide exchange factor SOS
1nvv	Structural evidence for feedback activation by RasGTP of the Ras-specific nucleotide exchange factor SOS
1nvw	Structural evidence for feedback activation by RasGTP of the Ras-specific nucleotide exchange factor SOS
1nvx	Structural evidence for feedback activation by RasGTP of the Ras-specific nucleotide exchange factor SOS
1xd2	Crystal Structure of a ternary Ras:SOS:Ras*GDP complex
1xd4	Crystal structure of the DH-PH-cat module of Son of Sevenless (SOS)
1xdv	Experimentally Phased Structure of Human the Son of Sevenless protein at 4.1 Ang.
2byv	Structure of the cAMP responsive exchange factor Epac2 in its auto- inhibited state
2ii0	Crystal Structure of catalytic domain of Son of sevenless (Rem-Cdc25) in the absence of Ras
3cf6	Structure of Epac2 in complex with cyclic-AMP and Rap
3ksy	Crystal structure of the Histone domain, DH-PH unit, and catalytic unit of the Ras activator Son of Sevenless (SOS)
4f7z	Conformational dynamics of exchange protein directly activated by cAMP
4jgw	The conformation of a docking site for SH3 domains is pre-selected in the Guanine Nucleotide Exchange Factor Rlf
4l9m	Autoinhibited state of the Ras-specific exchange factor RasGRP1
4mgi	4MGI
4mgk	4MGK
4mgy	4MGY
4mgz	4MGZ
4mh0	4MH0
4nyi	Approach for Targeting Ras with Small Molecules that Activate SOS-Mediated Nucleotide Exchange
4nyj	Approach for Targeting Ras with Small Molecules that Activate SOS-Mediated Nucleotide Exchange
4nym	Approach for Targeting Ras with Small Molecules that Activate SOS-Mediated Nucleotide Exchange
4uru	4URU
4urv	4URV
4urw	4URW
4urx	4URX
4ury	4URY
4urz	4URZ
4us0	4US0
4us1	4US1
4us2	4US2
5cm8	5CM8
5cm9	5CM9

Links (links to other resources describing this domain)

• PFAM: RasGEFN
• INTERPRO: IPR000651