Experimental Hematology
Volume 30, Issue 10 , Pages 1089-1106, October 2002

Ras and relatives—job sharing and networking keep an old family together

  • Annette Ehrhardt

      Affiliations

    • The Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia, Canada
    • ,
  • Götz R.A Ehrhardt

      Affiliations

    • Comprehensive Cancer Centre, University of Alabama, Birmingham, Ala., USA
    • ,
  • Xuecui Guo

      Affiliations

    • The Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia, Canada
    • ,
  • John W Schrader

      Affiliations

    • Corresponding Author InformationOffprint requests to: John W. Schrader, M.B., Ph.D., The Biomedical Research Centre, University of British Columbia, Vancouver V6T 1Z3, Canada
    • The Biomedical Research Centre, University of British Columbia, Vancouver, British Columbia, Canada

Article Outline

Abstract 

Many members of the Ras superfamily of GTPases have been implicated in the regulation of hematopoietic cells, with roles in growth, survival, differentiation, cytokine production, chemotaxis, vesicle-trafficking, and phagocytosis. The well-known p21 Ras proteins H-Ras, N-Ras, K-Ras 4A, and K-Ras 4B are also frequently mutated in human cancer and leukemia. Besides the four p21 Ras proteins, the Ras subfamily of the Ras superfamily includes R-Ras, TC21 (R-Ras2), M-Ras (R-Ras3), Rap1A, Rap1B, Rap2A, Rap2B, RalA, and RalB. They exhibit remarkable overall amino acid identities, especially in the regions interacting with the guanine nucleotide exchange factors that catalyze their activation. In addition, there is considerable sharing of various downstream effectors through which they transmit signals and of GTPase activating proteins that downregulate their activity, resulting in overlap in their regulation and effector function. Relatively little is known about the physiological functions of individual Ras family members, although the presence of well-conserved orthologs in Caenorhabditis elegans suggests that their individual roles are both specific and vital. The structural and functional similarities have meant that commonly used research tools fail to discriminate between the different family members, and functions previously attributed to one family member may be shared with other members of the Ras family. Here we discuss similarities and differences in activation, effector usage, and functions of different members of the Ras subfamily. We also review the possibility that the differential localization of Ras proteins in different parts of the cell membrane may govern their responses to activation of cell surface receptors.

 

The history of the Ras family of proteins dates back almost five decades to observations that viruses could cause tumor formation in mice and rats 1, 2. The viral genes responsible were called ras, for rat sarcoma, and turned out to be mutated versions of genes that encode enzymes with intrinsic GTPase activity. Thus, Ras proteins function as molecular switches determined by whether they are bound to guanine diphosphate (GDP) (“off” position) or guanine triphosphate (GTP) (“on” position). Inactive, GDP-bound Ras proteins are activated by interaction with members of a large structurally diverse class of proteins termed guanine-nucleotide exchange factors (GEFs), which catalyze the release of GDP. The lost GDP is then rapidly replaced by the more abundant GTP [3]. This exchange of GDP for GTP results in an allosteric change in two key regions of the GTPase termed Switch I and Switch II [4]. Switch I is part of the so-called effector loop and enables the binding of a variety of different effector proteins when Ras is in its GTP-bound configuration. Various GTPase activating proteins (GAPs) also bind to Ras proteins in their GTP-bound state. They act as negative regulators by greatly enhancing the low intrinsic GTPase activity of the Ras proteins, resulting in hydrolysis of GTP to GDP and causing an allosteric change of the Ras to the inactive off state.

The Ras superfamily of proteins now includes over 150 small GTPases (distinguished from the large, heterotrimeric GTPases, the G-proteins). It comprises six subfamilies, the Ras, Rho, Ran, Rab, Arf, and Kir/Rem/Rad subfamilies (Fig. 1). In this review, we will focus on the Ras subfamily, which contains 13 members that fall into five subgroups as defined by the presence of Caenorhabditis elegans (C. elegans) orthologs. The first group includes the first Ras proteins discovered, H-Ras and K-Ras. Together with N-Ras, these comprise the p21 Ras or classical Ras proteins. K-Ras occurs in two alternatively spliced forms, termed K-Ras 4A and K-Ras 4B. The p21 Ras proteins are closely related and exhibit about 85% sequence identity. Members of the other four subgroups M-Ras, R-Ras, Rap, and Ral all share 40%–50% amino acid identity with p21 Ras. For historical reasons, however, some of these proteins go by the name of Ras, while others are termed Rap or Ral. Nevertheless, as shown in Figure 1, all five subgroups fall on one branch of the Ras superfamily tree.

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  • Figure 1. 

    Phylogenetic tree of Ras family members. Human (no suffix) and C. elegans (Ce suffix) Ras sequences were obtained from the NCBI and WormBase (www.wormbase.org) databases and aligned using the ClustalW program at the European Bioinformatics Institute (www.ebi.ac. uk/clustalw). The alignment was then analyzed with PHYLIP at www. genebee.msu.su/services/phtree_full.html with the max/min factor for the cluster algorithm set to 255 and to 8 for the topological algorithm.

In addition, several Ras-like GTPases have been described and included in the Ras subfamily despite the lack of characteristic features of Ras proteins, such as prenylation signals (Rin and Rit), or typical effector domains (Rheb, Rhes, Dexras1, NOEY2, and κB-Ras1/2) [5]. These proteins have not yet been well characterized.

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Post-translational modifications regulate trafficking and localization in membranes 

Ras subfamily members exhibit a carboxy-terminal CaaX motif, where a cysteine is followed by two aliphatic residues and one random amino acid. This motif is a target for processing by enzymes, which results in the addition of a carboxy-terminal prenyl group. This group, either a farnesyl or a geranylgeranyl moiety, is involved in anchoring Ras to membranes [6]. Inhibitors of the enzymes involved in this prenylation process show promise as inhibitors of Ras function by blocking its localization to the plasma membrane (reviewed in [7]). However, prenylation alone is insufficient for functional anchorage of Ras proteins into the plasma membrane [8]. Additional molecular signals are required. The nature of these signals also dictates the route by which Ras proteins reach the plasma membrane and whether they localize to lipid rafts or to the disordered membrane. For example, the carboxy-termini of H-Ras, N-Ras, and K-Ras 4A include cysteine residues that undergo further lipid modification by attachment of palmitoyl moieties that extend far into the plasma membrane 8, 9. The palmitoylated carboxy-termini of H-Ras and N-Ras also dictate their transport to the plasma membrane via the Golgi 10, 11. In contrast, the alternative carboxy-terminal exon of K-Ras 4B lacks sites for palmitoylation but exhibits multiple basic residues that are thought to stabilize membrane localization by interacting with negatively charged head groups of membrane phospholipids. There is evidence that the polybasic carboxy-terminus of K-Ras 4B dictates a route to the membrane that bypasses the Golgi but which may involve binding of K-Ras 4B to tubulin 11, 12 (Figure 2). M-Ras, which has a polybasic carboxy-terminus like K-Ras 4B, also associates with tubulin (Quadroni and Schrader, unpublished observations).

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  • Figure 2. 

    Differential trafficking and membrane localization of Ras proteins with palmitoylated or polybasic carboxy-termini. As shown in references 6, 8, 9, 10, 11, 12, 13, 14, prenylation occurs in the cytoplasm followed by proteolysis of the last three amino acids on Ras proteins (indicated by -aaX) on the endoplasmic reticulum (ER) membrane. After methylesterfication (indicated by +CH3), isoforms with stretches of basic residues near their C-termini take a relatively undefined route to the disordered plasma membrane that may involve microtubules (indicated by tubulin?). Other isoforms that are palmitoylated on cysteine residues near their C-termini traffic through the Golgi to lipid raft areas by the conventional exocytic pathway. Receptors present in lipid rafts or in the disordered membrane may hypothetically activate Ras proteins colocalized in these areas preferentially (indicated by ? on the arrows from receptors to Ras molecules).

The differences in the carboxy-termini of Ras proteins dictate their insertion into different parts of the plasma membrane. Data from experiments where cholesterol-rich membrane domains were disrupted, as well as sucrose density gradient and electron microscopy data, indicate that H-Ras resides in lipid rafts whereas K-Ras 4B is excluded from rafts and instead localizes to the disordered plasma membrane 13, 14. M-Ras also localizes in disordered membrane, through a mechanism involving its polybasic carboxy-terminus (Ehrhardt and Schrader, unpublished observations). Lipid rafts are thought to constitute signaling platforms for receptors that preferentially reside in these microdomains or are translocated into them after ligand binding. The localization of H-Ras to lipid rafts was shown to be essential for its activation of downstream effectors, such as Raf-1 and PI-3 kinase 13, 15. Thus, differences in localization of Ras proteins may regulate proximity to different types of receptors and GEFs, which may result in differences in susceptibility to activation by particular stimuli. For example, it might be predicted that palmitoylated Ras proteins, such as H-Ras or N-Ras that are located in rafts, would likely be activated by stimulation of the B cell or T cell antigen receptors (BCR, TCR), or the receptor for IgE (FcϵR1), which are known to translocate to rafts upon activation 16, 17, 18. Likewise, Ras proteins with stretches of poly-basic amino acids near their carboxy-termini would be predicted to preferentially become activated by non–raft-associated receptors. In case of stimulation by growth factors the nature of the carboxy-termini of Ras proteins can indeed dictate their susceptibility to activation by different stimuli (Ehrhardt and Schrader, unpublished observations). However, similarities in membrane localization are not always predictive of similar susceptibilities to activation by a given extracellular stimulus (Ehrhardt and Schrader, unpublished observations) suggesting roles for other factors, such as differential sensitivity to GEFs activated by a stimulus. One important caveat to conclusions based on purported differences in localization of proteins in rafts relates to the limitations and variations intrinsic to the variety of methods used to define and study rafts [19]. This might explain why TC21, Rap1, and Rab5 were all found in detergent-resistant raft fractions from mouse lung tissue although Rap1 and Rab5 both lack palmitoylatable cysteines at their carboxy-termini [20]. Moreover, in many cases the relationship of proteins with rafts is dynamic, with evidence for the movement of activated receptors such as the BCR into rafts, and of activated H-Ras out of rafts [14].

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Regulation of Ras subfamily GTPases 

GEFs and their specificity 

One simple answer to the question of why there are so many similar members of the Ras subfamily would be that they would be activated by different GEFs that were themselves activated by different stimuli. Indeed there are many GEFs exhibiting a variety of domains that enable response to and integration of a large variety of signals (Fig. 3). However, with few exceptions, most GEFs act on multiple members of the Ras subfamily, in some instances having GEF activity on members of the Ras superfamily outside the Ras subgroup, such as Rho proteins. Nevertheless, there is emerging evidence for considerable fine-specificity, with some GEFs even able to discriminate between members of the closely related p21 Ras group. Moreover, it is conceivable that differences within the Ras family in susceptibility to a particular GEF may be obscured by the experimental techniques based on over-expression, which for example may result in non-physiological localization of Ras proteins or GEFs. The activation of different Ras subfamily proteins by the various GEFs is summarized in Table 1.

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  • Figure 3. 

    Schematic representation of Ras family guanine nucleotide exchange factors with their domain organization. RasGEF, domain with homology to the catalytic domain of CDC25 exhibiting GEF activity toward Ras subfamily proteins. RhoGEF, domain with homology to the catalytic domain of Dbl exhibiting GEF activity towards Rho subfamily proteins. PH, pleckstrin homology. P, poly-proline–rich sequences, representing SH3 (Src homology 3) domain binding sites. IQ, calmodulin binding. DAG, diacylglycerol binding. DEP, domain found in Dishevelled (Dvl) proteins. RA, Ras association. PDZ, domain found in PSD95, Dlg, ZO1 proteins. SH2, Src homology 2 domain, phosphotyrosine binding. X,Y: phospholipase catalytic domains. C2, calcium-dependent lipid binding domain. a, RasGRP4 features only one EF hand. b, Epac1 lacks the N-terminal cNMP binding domain present in Epac2. c, RA-GEF II has a longer N- but shorter C-terminus than RA-GEF I. d, the cNMP domains of RA-GEF I/II do not interact with cAMP or cGMP. e, Rgl3/RPM is the only protein of the RalGDS/Rgl families with a proline-rich region.

Table 1. Summary of Ras family substrates for GEFs
H-RasN-RasK-Ras 4AK-Ras 4BM-RasR-RasTC21RapRal
Sos1+++++−−
Sos2+
RasGRF1+− −+(+)+
RasGRF2+ −
RasGRP1++ +(+)++
RasGRP2−+ +−(+)(+)(+)
CalDAG-GEFl−− −−(+)(+)+−
RasGRP3+ (+)+++−
RasGRP4+ −
Epac1/2− −−−+
MR-GEF− −−+−
RA-GEF I +−
RA-GEF II−− −− +−
C3G −(+)(+)+
PLCϵ− −− +−
RalGDS, Rgl1/2/3− +
AND-34− + ++
smgGDS−−−++ ++

+, good activation; (+), weak activation; −, no activation.

All references are in the text.

With one single exception discussed below, all GEFs activate Ras proteins through one or more catalytic domains homologous with the minimal Ras activation domain of CDC25, the yeast GEF that activates Saccharomyces cerevisiae (S. cerevisiae) RAS. A crystallographic structure of the interaction of the CDC25 domain of Sos1 and H-Ras has revealed the details of this interaction [4]. As shown in Figure 3, the various GEFs exhibit a large range of additional structural features that enable them to respond to many different signals. These signals include the phosphorylation of proteins and lipids, calcium fluxes, and the generation of cyclic nucleotides or diacylglycerol (DAG). Some GEFs, like Sos1/2 or RasGRF1/2, have domains with homology to the catalytic domain of Dbl, which exhibit GEF activity toward Rho subfamily proteins, in addition to CDC25-like domains and thus act as GEFs for members of both the Ras and Rho subfamilies, potentially integrating their activation.

RasGRF1 and RasGRF2 21, 22, 23, 24 differ in specificity as RasGRF1 appears to selectively activate H-Ras but not N-Ras or K-Ras 4B [25]. It also acts on M-Ras and R-Ras [26]. RasGRF2 activates H-Ras but not R-Ras [27], although additional Ras proteins precipitated by Y13-259 (see below) may also be activated [24]. RasGRF1/2 contain an IQ domain, which binds calmodulin. However, while calmodulin-binding appears to be required for activation of Erk, it was shown not to modulate Ras activation [28]. Mammalian Sos1 and Sos2 are orthologues of the Drosophilia melanogaster (D. melanogaster) gene product son-of-sevenless, which functions upstream of Ras in R7 photoreceptor cell differentiation 29, 30. Sos1/2 contain pleckstrin homology (PH) domains, which interact with membrane lipids, and catalytic domains for activation of both Ras proteins and members of the Rho family. Within the Ras subfamily, Sos1 is active on H-Ras, N-Ras, K-Ras 4A and 4B, and M-Ras, but not on R-Ras or TC21 26, 31. Sos2 activates H-Ras [32].

The RasGRP family of GEFs is defined by the presence of calcium- and DAG-binding domains and has four members. RasGRP1 (also referred to as CalDAG-GEF II) activates H-Ras, N-Ras, K-Ras 4B, R-Ras, TC21, and weakly M-Ras 26, 33. In contrast, RasGRP2 activates N-Ras and K-Ras 4B, but not H-Ras. It also efficiently activates Rap after stimulation with TPA and calcium, whereas the GEF activity toward N-Ras is partially inhibited by the presence of calcium [34]. CalDAG-GEF I is an alternatively spliced form of RasGRP2 that lacks amino-terminal myristoylation and palmitoylation and may thus localize differently in membrane domains. CalDAG-GEF I has been shown to activate Rap1 and very weakly R-Ras in vivo and in vitro 26, 34, 35. CalDAG-GEF I promotes nucleotide exchange on N-Ras in vitro, but neither H-Ras, N-Ras nor K-Ras 4B are activated by CalDAG-GEF I in vivo 34, 36. RasGRP3 (also known as CalDAG-GEF III) activates H-Ras, R-Ras, Rap1A, and Rap2A, but not RalA 35, 37. A fourth member of the RasGRP family, RasGRP4, can activate H-Ras in vitro, an action that is inhibited by the presence of calcium, but does not activate Rap1 38, 39. While the expression of the various GEFs in different types of hematopoietic cells has not been studied exhaustively, the expression pattern of members of the RasGRP family in these cells seems to be more restrictive than that of Sos1. RasGRP1 is only found in T and B cells, and there is a possibility that RasGRP3 may be a B cell–specific GEF (James Stone, personal communication). RasGRP4 is expressed in mast cells but not in lymphocytes [38] but was also found in leukemic blasts in a patient with acute myeloid leukemia (AML) and myeloid cell lines [39].

Some GEFs appear to activate only Rap. Two exchange factors that are regulated by cAMP, Epac1, and Epac2 (cAMP-GEFII/I) activated Rap1A but not H-Ras or R-Ras [40]. MR-GEF, RA-GEF I (PDZ-GEF), and RA-GEF II also seem to selectively act on Rap, although there are conflicting data on the ability of RA-GEF I to activate H-Ras 41, 42. MR-GEF and RA-GEF II appear to bind specifically to GTP-bound M-Ras and are thus potentially specific effectors of M-Ras. Recently, a novel isoform of phospholipase C, PLCϵ, has been shown to specifically activate Rap1A but not Rap2A, H-Ras, R-Ras, M-Ras, RalA, Rit, Rin, or Rheb [43]. By virtue of one of its two Ras association (RA) domains, PLCϵ also functions as a downstream effector of activated Ras.

Another family of GEFs appears to be exclusively active on Ral proteins. Interestingly, all of these RalGEFs are effectors of other members of the Ras subfamily and are discussed later. The potential links between p21 Ras and its activation of GEFs for other members of the Ras subfamily are shown in Figure 4.

Several exchange factors exhibit broader specificity within the Ras superfamily. AND-34 contains a RasGEF domain with relatively low homology to other CDC25-like domains and was shown to activate RalA, Rap1A, and R-Ras but not H-Ras [44]. C3G was found in a search for Crk-interacting proteins [45]. It activates Rap1/2, R-Ras, TC21, and the Cdc42-related protein, TC10 26, 46.

A protein termed smgGDS is the GEF with the broadest specificity. It was the first mammalian exchange factor to be identified [47]. However, in that it consists almost entirely of a series of Armadillo repeats and lacks a CDC25-like domain altogether, smgGDS is structurally unrelated to any other GEF. It acts not only on many members of the Ras subfamily like K-Ras 4B, Rap1A/B, and Ral but also on members of the Rho family like RhoA, Cdc42, and Rac [48]. Intriguingly, smgGDS fails to activate H-Ras, N-Ras, or K-Ras 4A, leading to the suggestion that it requires a polybasic carboxy-terminus on its substrate. Indeed, RhoA was no longer activated when its carboxy-terminus was replaced with that of H-Ras; however, replacing the carboxy-terminus of H-Ras with the polybasic carboxy-terminus of RhoA did not confer susceptibility to activation by smgGDS [49], indicating that basic residues on substrates are necessary but not sufficient for smgGDS exchange activity. However, smgGDS is able to activate mutants of M-Ras that lack the carboxy-terminus (Korherr and Schrader, unpublished observations). Another unusual feature of smgGDS is its ability to bind to dominant active Ras proteins in yeast two-hybrid assays [49], which is not characteristic of GEFs [3] but may indicate that smgGDS possibly also functions as a scaffold.

There are recent insights into the molecular features that determine the specificity of GEFs for particular Ras family members. Differences in the helix 3 region of H-Ras and R-Ras (residues 91-103 in H-Ras) appear to account for the ability of RasGRF1 but not Sos1 to act on R-Ras [50]. The ability of RasGRF1 to activate H-Ras but not K-Ras 4B appears to depend on residues in the carboxy-terminus [25]. Another factor affecting GEF specificity may be the nature of the prenyl groups. R-Ras is usually not activated by RasGRF2, but when the most carboxy-terminal residue is changed from leucine to serine (as in H-Ras), R-Ras is then farnesylated instead of geranylgeranylated and becomes more responsive to RasGRF2 [27].

GTPase activating proteins 

GAPs negatively regulate GTPases by catalyzing the hydrolysis of bound GTP through a mechanism involving insertion of an arginine side-chain into the active site of Ras [51]. This results in an allosteric change of Ras into its inactive configuration. p120 RasGAP was the first protein shown to bind to Ras in a GTP-dependent fashion and was initially classified as an effector of Ras [52]. However, while portions of p120 RasGAP can mimic some aspects of Ras signaling, functional Ras is sometimes required in addition 53, 54. Certainly p120 RasGAP is known to associate with p190 RhoGAP, thereby regulating activity of Rho proteins 55, 56, 57. p120 RasGAP was also shown to associate with BCR/Abl in chronic myelogeneous leukemia (CML) cell lines and with Lck 58, 59. Four other GAPs, the product of the neurofibromatosis gene Nf1, GAP1m and its close homolog GAPIII, and GAP1(IP4BP) (also known as R-Ras GAP), have been described 60, 61, 62, 63. Both GAP1m and GAP1(IP4BP) can be regulated via binding to inositol phosphates. GAP1m may interconnect the heterotrimeric and monomeric G proteins by directly binding to Gα12 64, 65. All GAPs with the exception of R-Ras GAP exhibit activity toward p21 Ras, M-Ras, R-Ras, and TC21 but not Rap1A [26]. GAP1(IP4BP) is only active on R-Ras and Rap1A [64].

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Ras effectors 

Activation of members of the Ras subfamily leads to their interaction with a variety of downstream effector proteins (Figure 4). These effectors may be broadly grouped into GEFs for other Ras superfamily members, protein or lipid kinases, GAPs, and a still expanding group of other effectors with poorly characterized functions.

GEFs as effectors of the Ras subfamily 

Interest in the Ral GTPases RalA and RalB increased when GEFs for these proteins were found to be effectors of Ras. These RalGEFs include RalGDS, Rgl, Rlf/Rgl2, RPM/Rgl3, the PH-domain containing RalGPS/RalGEF2, and Rgr, the GEF portion of the Rsc fusion protein 66, 67, 68, 69, 70, 71, 72, 73, 74. Through their Ras association (RA) domains, these RalGEFs interact with various activated members of the Ras subfamily including Ras, Rap, and Rit. RalGPS proteins have not yet been shown to actually bind GTP-Ras. Activated TC21 was shown to interact with RalGDS and Rgl, but there are conflicting data on whether this association can mediate activation of RalA 75, 76. The RA domains of RalGEFs are required for Ras-mediated activation of Ral, placing Ral downstream of Ras. However, activation of Ral that is independent of Ras but dependent on calcium has also been demonstrated [77]. Unlike RalGDS and Rlf, RPM/Rgl3 appears to function as a negative regulator of the p21 Ras path ([71]; see below).

Two proteins MR-GEF and RA-GEF II that bind selectively to GTP-bound M-Ras and are thus potential effectors of M-Ras are known to function as RapGEFs. MR-GEF is specifically expressed in the brain and in vitro activates Rap1A but not RalA or H-Ras [41]. However, over-expression of activated M-Ras has no influence on total cellular levels of Rap1-GTP levels and co-transfection of activated M-Ras with MR-GEF actually decreases total cellular levels of GTP-Rap [41]. RA-GEF II is widely expressed and binds specifically to active M-Ras but not H-Ras, N-Ras, R-Ras, Rap2A, RalA, Rit, Rin, or Rheb [42]. Over-expression of activated M-Ras and RA-GEF II results in translocation of Rap1A to the plasma membrane where it is activated. However, this was again associated with decreases in total cellular GTP-Rap1A [42]. Thus, the effect on Rap1A of the recruitment of MR-GEF or RA-GEF II by activated M-Ras appears to be complex and potentially involves increases in Rap1A activity at the plasma membrane but decreases in the cytoplasm. Another newly identified Rap1A-specific GEF is PLCϵ, which acts as an effector of H-Ras and TC21 [76]. Over-expression of H-Ras Q61L stimulated PLCϵ-mediated hydrolysis of PI(4,5)P2 in a calcium-dependent manner [78].

The SH2 domain-containing protein RIN1 interacts with activated H-Ras and was shown to block Ras-induced transformation, possibly by competing with Ras for Raf-1 [79]. By virtue of its Vps9p-like RabGEF domain, RIN1 functions as a GEF for Rab5A and potentiates Ras-mediated endocytosis [80].

Protein and lipid kinases 

The best-characterized signal transduction pathway downstream of p21 Ras is the one that leads to activation of members of the mitogen-activated protein (MAP) kinase family and in particular of Erk1/2. The first step in initiating the cascade leading to Erk activation is binding of p21 Ras to members of the Raf family of serine/threonine kinases, Raf-1, A-Raf, and B-Raf 81, 82. The precise mechanism through which binding of Raf to activated p21 Ras results in activation of Raf is still not completely understood. Recent evidence suggests that Ras participates directly in the activation of its downstream effectors and does not simply mediate membrane localization. Thus, there is some evidence that Raf-1 may be recruited to the plasma membrane by phosphatidic acid rather than by its interaction with Ras, but its activation still requires activated Ras [83].

Raf-1 is most efficiently activated by activated K-Ras 4B, followed by activated K-Ras 4A, N-Ras, and H-Ras [84]. R-Ras and M-Ras are very poor activators of the Raf-Erk pathway 85, 86, 87. There is conflicting evidence on the ability of TC21 to activate Raf and Erk 88, 89, 90. Erk activity was increased in cells transformed by activated TC21, although this was shown to be an indirect effect [91]. Activation of B-Raf (but not Raf-1) by Rap1 was reported to lead to sustained activation of Erk after stimulation of PC12 cells with NGF [92].

The serine/threonine kinase MEKK-1 was originally identified as an upstream regulator of MEK1/2 and Erk1/2, but was later shown to more potently activate the stress-activated MAP kinases JNK and p38 93, 94. Experiments with dominant active and dominant-negative mutants of p21 Ras demonstrated that MEKK-1 probably functions downstream of p21 Ras [95]. Consistent with this, MEKK-1 binds directly to the effector domain of activated p21 Ras [96]. MEKK-1 is also part of the 700 kDa IKK complex that is involved in the activation of NFκB [97].

Phosphatidylinositol (PI)-3 kinase is another effector of Ras subfamily members and has kinase activity against both lipids and proteins. Its catalytic p110 subunit interacts directly with GTP-bound Ras [98]. By catalyzing the production of 3′ phosphorylated phosphatidylinositols, PI-3 kinase targets proteins containing PH domains to the plasma membrane where they can be activated (i.e., by phosphorylation) and mediate their respective effects. These proteins include the kinases Akt and Btk (reviewed in [99]). Activated R-Ras and M-Ras may increase the activity of PI-3 kinase more efficiently than H-Ras, which in turn is more efficient than K-Ras 4B 85, 100, 101. PI-3 kinase is likely to be one of the major effectors for M-Ras or R-Ras, as neither are efficient at activating the MAP kinases Erk, JNK and p38 85, 87, 101. The increases in activity of PI-3 kinase induced by growth factors and cytokines are not entirely dependent on activation of Ras family members. Interleukin (IL)-4 stimulation does not result in activation of Ras but does result in increased activity of PI-3 kinase via IRS-2 102, 103.

Ras subfamily proteins are also able to mediate activation of Rho subfamily proteins via PI-3 kinase. PI(3,4,5)P3 produced by PI-3 kinase can bind to the PH domains of Sos1/2, Vav, and Tiam-1, all of which stimulate nucleotide exchange on Rac 104, 105, 106. However, activated H-Ras or M-Ras may also stimulate activation of Rac through mechanisms that do not depend on PI-3 kinase activity (Grill and Schrader, unpublished observations). Rac1 is more efficiently activated by K-Ras 4B compared to H-Ras, leading to increased cell motility of K-Ras 4B G12V-expressing cells 84, 107.

Although the protein kinase PKCζ was reported to interact with GTP-bound Ras [108], other studies failed to confirm this interaction. Recent reports suggest that PKCζ probably functions downstream of Ras-mediated activation of Rac, i.e., to mediate cytoskeletal reorganization or transcriptional upregulation of cyclin D1 109, 110.

Effectors with negative regulatory functions 

We and others have recently identified an effector of M-Ras, H-Ras, and Rit that shares homology with RalGDS, Rgl1, and Rgl2 (Rlf) and was termed RPM or Rgl3 70, 71. This protein functions as an exchange factor for RalA and RalB, but unlike Rgl1 and Rgl2, it does not synergize with Ras to activate Elk-1 and inhibits growth of transformed fibroblasts, indicating a potential role for RPM/Rgl3 as a tumor suppressor.

RIN1, at least when overexpressed, exhibits a negative regulatory effect on p21 Ras action that may be due to its competition for p21 Ras with Raf [79]. RIN1 exhibits a Vps9p RabGEF domain and activates Rab5 to stimulate receptor-mediated endocytosis [80]. 14-3-3 binding negatively regulates RIN1 [111].

Nore1 was identified in a yeast two-hybrid screen as a protein that interacts with GTP-bound H-Ras. The presence of a DAG-binding site and of several potential SH3-domain binding sites suggests that this protein is an adapter protein [112]. Nore1 also efficiently interacts with activated M-Ras and K-Ras 4B 71, 113. Recently, Nore1 and three other highly homologous proteins, RasSF1A, RasSF1C, and C. elegans T24F1.3, were shown to interact with the pro-apoptotic kinase, MST1. The recruitment of Nore1-MST1 complexes by activated K-Ras 4B or H-Ras provided a possible explanation for the apoptotic effects observed after over-expression of these activated Ras proteins in Jurkat or NIH3T3 cells [114]. Despite the presence of domains homologous to the RA domain of Nore1, RasSF1C was shown to interact with activated H-Ras only when RasSF1C was very highly overexpressed [115]. A second study demonstrated that neither RasSF1A or -C nor T24F1.3 are able to interact with several different Ras proteins directly in a yeast 2-hybrid assay, although RasSF1A and -C can form heterodimers with Nore1 and thus may be recruited to activated p21 Ras or M-Ras this way [113]. RasSF1C was found to induce apoptosis of Ras-transformed cells in a caspase-dependent manner [115].

Effectors with uncertain functions 

AF-6 was identified by means of the yeast 2-hybrid system and is the mammalian homologue of the D. melanogaster protein Canoe, which is functionally linked with Ras in eye development. AF-6 preferentially binds to activated Rap1 and less efficiently to H-Ras, N-Ras, K-Ras, and M-Ras 71, 116. AF-6 may physically link adhesion molecules at the plasma membrane and the cortical actin cytoskeleton [117]. Calmodulin was recently shown to specifically interact with activated K-Ras 4B but not with any other p21 Ras isoform [118]. The significance of this interaction is unknown, but it is possible that it may inhibit the interaction of other effector proteins such as Raf-1 with K-Ras 4B.

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Functions of Ras subfamily members 

Redundancy? 

There is a substantial degree of amino acid sequence identity between members of the Ras subfamily (40%–95% identity), with members of the p21 Ras subgroup sharing 85% amino acid identity. As discussed, there is also considerable overlap in the sets of GEFs that activate Ras proteins and downstream effectors that transmit their signals. Moreover, when overexpressed in a constitutively activated form, they produce similar phenotypes such as transformation. This observation led to speculations about functional redundancy within this family. However, it is becoming increasingly evident that different members of the Ras subfamily may have different biological functions that depend not only on differences in their affinities for regulators or effectors but also in their precise subcellular localization.

Problems with interpretation of the literature 

Most of the published information focuses on the function of p21 Ras. In most cases, the experiments used the monoclonal antibody Y13-259 or overexpression of dominant active or negative mutants of p21 Ras. However, it is now clear that the close structural similarities between members of the Ras family and the widespread sharing of positive and negative regulators and effectors mean that these experimental tools are inadequate to specifically assign functions to the various Ras family members. For example, while the monoclonal antibody Y13-259 is generally assumed to be specific for the four p21 Ras proteins, the epitope to which Y13-259 binds is composed of residues in Switch II that are very similar in other members of the subfamily, such as M-Ras and TC21, resulting in crossreactivity 119, 120. Microinjection of Y13-259 into cells blocks cell-cycle progression [121], but given its crossreactivity within the family, it can no longer be concluded that the observed effects were due specifically to inhibition of p21 Ras activity. Likewise, many experiments designed to assay activation of p21 Ras in response to extracellular stimuli measured the ratio of GTP to GDP in material that was immunoprecipitated from cells using Y13-259. Thus, it is unclear whether the published reports that growth factors like IL-3, steel locus factor (SLF), epidermal growth factor (EGF), or platelet-derived growth factor (PDGF) activate p21 Ras 122, 123, 124 are correct. The use of isolated Ras binding domains of effector proteins as affinity purification tools in “pull-down” assays [125], together with the development of sensitive antibodies specific for each family member, will help to provide answers to these questions.

There are similar problems with conclusions about the functions of p21 Ras that are based on the use of overexpressed dominant negative mutants of p21 Ras. The affinity for GTP of the most commonly used dominant negative version of p21 Ras, the S17N mutant, is strongly reduced, resulting in a failure to release the GEF after binding. Thus, these mutants remain bound to GEFs, thereby sequestering them and preventing them from activating other Ras molecules in the cell. G15A mutants of p21 Ras function in a similar manner but have an even higher affinity for GEFs [120]. Most published reports on the effects of such dominant negative mutants of p21 Ras have concluded that the observed effects reflect interference with activation of p21 Ras. However, this is not necessarily the case because many GEFs that act on p21 Ras and thus will be sequestrated by dominant negative S17N or G15A p21 Ras mutants also activate other members of the Ras subfamily or even Ras superfamily. For example, the ability of H-Ras S17N to sequestrate Sos1 could also directly block activation of Rac1, as Sos1 acts as a GEF for both Ras subfamily and Rho subfamily members. Thus, the demonstration that a function, for example, the development of B or T lymphocytes is blocked by transgenic expression of H-Ras S17N 126, 127, alone does not establish that H-Ras or even p21 Ras as a group is necessary for normal T- or B-cell development. Additional evidence in support of a role for p21 Ras can be obtained by demonstrating that the effect of dominant negative Ras can be reversed by overexpression of an activated Ras effector, such as Raf, as reported by Iritani et al. [127].

Another class of dominant inhibitory mutants act downstream of Ras by sequestrating effector proteins. These have two types of mutations, one that maintains them in a constitutively active state with high affinity for effectors, and a second that prevents their association with the cell membrane. This results in sequestration of effectors in the cytoplasm where they cannot activate further downstream regulators. A third set of mutations in the Switch I region (“effector loop mutants”) can restrict the range of effectors bound and thus sequestrate only a subset of all possible effectors [120]. However, as there is overlap between effector-usage by different family members, this strategy will not provide information on the role of a given Ras isoform.

Ras can also be blocked by over-expression of the Ras binding domain of an effector, but, as discussed earlier, few effectors are probably specific for a single Ras family member, limiting the value of this approach for dissecting the function of individual family members.

A second major source of information on the potential functions of Ras family members has come from experimental over-expression of constitutively active mutants of p21 Ras such as the G12V or Q61L mutants. However, this approach also has significant limitations. The effects of expression of chronically active Ras at supra-physiological levels may be quite different from those of physiological levels of activated Ras. Moreover, in physiological situations, activation of Ras may be very tightly regulated, for example, decreasing at later stages of the cell cycle. Over-expression of a particular Ras family member may cause it to act on an effector, which at physiological concentrations it activates only inefficiently or not at all. Over-expression may also result in mislocalization in the cell and lead to interaction with non-physiological effectors.

Role in development 

Targeted disruption of individual ras genes in mice has been achieved for all three p21 Ras genes. However, with one exception, the phenotypes have not been obvious. It is not clear whether this reflects true overlaps in function, perhaps involving compensatory overexpression of other family members. Neither the N-ras or H-Ras genes, individually or in combination, are essential for normal development, fertility, or hematopoiesis in mice 128, 129. However, N-Ras null fibroblasts exhibit an increased susceptibility to Fas and TNF-induced apoptosis. This phenotype was rescued by ectopic expression of N-Ras but not K-Ras, indicating a specific role for N-Ras in fibroblast survival [130]. Mice lacking functional M-Ras are also viable and fertile and show no gross abnormalities (Wang and Schrader, unpublished observations). While the development of hematopoietic cells in H-Ras-, N-Ras-, or M-Ras–deficient mice appears grossly normal, a more detailed analysis is needed to see whether there are defects in the development of subsets of cells or in their function. Mice with non-functional K-Ras genes die in utero of anemia due to apoptosis in the fetal liver microenvironment [131] or due to increased cell death in neuronal and heart tissues [132]. It remains unclear if the observed embryonic lethality reflects the predominant expression of K-Ras rather than other forms of p21 Ras during development [133] or whether it reflects specific functions of K-Ras.

Loss-of-function mutations of Rap1 in D. melanogaster result in lethality [134]. Rap1 function was shown to be required during fly embryogenesis, imaginal development, and oogenesis but seems to be dispensable for fully differentiated cells in adult flies, indicating a role for Rap1 in cell differentiation and proliferation rather than cell survival [135]. The D. melanogaster Ral protein, DRal, appears to regulate developmental cell shape changes through inhibition of the JNK pathway [136].

Stimulation and inhibition of growth 

The role of the Ras subfamily in general in transduction of mitogenic signals and proliferation is well established, although as noted, the specific roles of different members remain unclear due to problems with crossreactivity of experimental tools. Entry into the G1 phase of the cell cycle and progression through G1 is blocked by microinjection of dominant negative H-Ras S17N or monoclonal anti-Ras antibodies 121, 137, 138. H-Ras S17N blocks upregulation of cyclin D1, a positive regulator of cell cycle progression, after serum-starvation [139]. Upregulation of cyclin D1 may contribute to the transforming activities of activated Ras. H-Ras excels in the transformation of fibroblasts, whereas N-Ras was reported to be more efficient than K-Ras 4B or H-Ras in transforming the factor-dependent hematopoietic cell line TF-1 [140].

Over the last several years it has become increasingly evident that in addition to promoting cell growth and proliferation, p21 Ras may also induce apoptosis and senescence by induction of p19ARF or of cell cycle inhibitors such as p16INK4a, p21Cip1/WAF1, and p27Kip1 (reviewed in 141, 142, 143). One mechanism of Ras-mediated growth arrest is through its induction of p19ARF, which via inactivation of Mdm2, causes accumulation of p53. Inhibition of cyclin-dependent kinases by p16INK4a, p21Cip1/WAF1, and p27Kip1 and subsequent decreases in phosphorylation of Rb proteins is another way to achieve cell cycle arrest. In normal human fibroblasts, both p53 and p16INK4a are required for Ras-induced senescence. In the absence of p19ARF, p53, or p16INK4a, overexpression of activated p21 Ras results in transformation rather than inhibition of growth 144, 145. The apparent paradoxical ability of Ras to promote both cell cycle progression and arrest is in part explained by the ability of high levels of Raf activity to induce senescence, whereas lower levels promote cell cycle progression 146, 147. Thus, the levels of activated p21 Ras may determine whether its effects are anti- or pro-apoptotic. Interestingly, H-Ras appears to be a more potent inhibitor of growth than N-Ras or K-Ras 4B in the K562 myeloid cell line [148]. Moreover, levels of H-Ras correlated with apoptosis in early-stage breast tumors [149]. Recently, another possible mechanism for Ras-induced apoptosis was proposed. Activated K-Ras 4B and, to a lesser extent, H-Ras, were shown to associate with the pro-apoptotic kinase MST1 via interaction with the Ras effector Nore1, and this association resulted in apoptosis of different cell lines [114].

Differentiation 

Ras proteins are critically involved in the development and function of hematopoietic cells. This is discussed in the context of specific cell types as below.

Cell adhesion 

Cells adhere to the extracellular matrix via integrins. During events, such as cell migration or division, cells convert from being non-adherent to being adherent and vice versa through integrin activation or deactivation. Both H-Ras and Raf-1 were found in a screen for suppressors of integrin activation, which provided an explanation for the less adhesive phenotype of Ras-transformed cells [150]. However, over-expression of H-Ras has also been reported to increase the activation of integrins and to induce adhesion, and expression of an S17N p21 Ras mutant blocked IL-3–induced adhesion and activation of β1 integrin [151]. R-Ras also stimulates integrin activation [152], an effect that was recently shown to be negatively regulated by Src [153]. Rap1 also contributes to integrin-mediated cell adhesion although the underlying mechanism is yet to be analyzed [154]. Again a dominant negative Rap mutant blocked the IL-3–induced β1 integrin–dependent adhesion [151]. However, since Rap can be activated by GEFs that are effectors of other Ras family members, it remains challenging to determine whether Rap and these other Ras proteins are acting in parallel or sequentially.

Functions of Ral 

Relatively little is known about the functions of the Ral GTPases RalA and RalB of the Ras subfamily. Constitutively active RalA is not transforming in fibroblasts but it strongly enhances the transforming activities of activated Ras and Raf-1. Moreover, dominant negative mutants of Ral strongly inhibit the transforming activities of activated mutants of Ras and Ral [155]. The mechanisms through which Ral mediates its effects are poorly understood. Active, GTP-bound Ral interacts with RalBP1 which acts as a GAP for the Rho-family GTPases Rac and Cdc42 and inhibits their activation [156]. RalBP1 is also known as cytocentrin, a protein which shuttles between the cytosol and the nucleus, where it regulates the assembly of the mitotic spindle [157]. A role for Ral in regulation of the cell cycle has been further substantiated by demonstration of its ability to upregulate expression of cyclin D1 [158]. Independent of their nucleotide binding, Ral GTPases also interact with phospholipase D1 which is implicated in vesicular trafficking 159, 160. Recent observations suggest the involvement of Ral in other cellular functions such as chemotaxis and receptor-mediated endocytosis 161, 162. Ral can be activated by growth factors such as EFG and insulin, and there is also evidence that place Ral upstream of the Src tyrosine kinase in the response to stimulation with EGF or insulin 163, 164. In platelets, Ral activation by α-thrombin or other platelet agonists is thought to be dependent on calcium mobilization and correlates with calcium-dependent activation of Rap1, but not Ras [165].

Functions of Rap 

Rap1 was originally identified in a screen for genes that reverse the phenotype of fibroblasts transformed by K-Ras [166]. This observation and other data on the antagonism between Rap1 and Ras in insulin signal transduction [167] and in T-cell anergy led to the notion that Rap antagonised Ras by competing for Raf and thus inhibiting Ras-dependent activation of Erk [168]. However, growth factor–induced activation of Rap1 does not correlate with the repression of Ras-dependent Erk activation [169]. Moreover, Rap1 was found to be important for NGF-induced differentiation of PC12 cells by the sustained activation of B-Raf and Erk by Rap1. In contrast, in the same cells, Ras was involved in proliferation through a transient Erk activation [92]. It is unlikely that the physiological activation of Rap1 by growth factors is solely concerned with the modulation of Ras signaling and the interference with Ras activation by Rap may be an artifact of over-expression.

Rap2 is 60% identical to Rap1, has the same effectors, and is regulated by the same set of GEFs (C3G, Epac, RasGRP2, RA-GEF I) and RapGAPs (rap1GAPII and SPA-1). However, Rap2 is unique in its low sensitivity to GAPs and more than half of Rap2 remains GTP-bound, leading to the proposal that Rap1 and Rap2 function, respectively, as fast and slow molecular switches. [170].

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Differentiation and function of hematopoietic cells 

As noted earlier, the vast majority of data on Ras functions have been generated by the use of dominant inhibitory or active mutants of Ras proteins or cross-reactive monoclonal antibodies. Thus, very little is known about the actual role of individual members of the Ras subfamily in functions of hematopoietic cells. In some cases, there is additional evidence on involvement of potential downstream pathways such as the MEK-Erk path. However, while it is often assumed that this implicates p21 Ras, Erk can also be activated downstream of Rap and by mechanisms independent of activation of the Ras family. Thus, while such experiments suggest that collectively members of the Ras subfamily may have critical functions in many aspects of the development and functions of hematopoietic cells, they do not conclusively establish their roles.

T cells 

Ras family proteins influence all stages in the life of a T cell, from development and antigenic activation to anergy. Several mouse models have indicated that Ras plays a critical role in positive selection of thymocytes. These include transgenic mice with thymocyte-specific expression of H-Ras S17N or dominant negative MEK-1 126, 171, as well as mice with ablation of genes for Erk1 and RasGRP1 172, 173, 174. There is evidence that the strength of Erk signaling may be crucial for differentiation along the CD4 lineage [175], although the role of Ras in activation of Erk in this commitment process is unclear as Erk can also be activated downstream of PKC [176]. The transgenic mice expressing H-Ras S17N were also used to demonstrate the importance of Ras and Erk in differentiation of naive peripheral T cells into Th2 cells. Evidence was presented that weak antigenic stimulation may not activate the Ras-MAPK pathway efficiently and may favor Th1 cell differentiation, whereas strong stimulation may be required to activate this pathway sufficiently to induce Th2 differentiation [177]. Thus, the extent to which the Ras-MAPK pathway is activated seemed to determine the lineage along which T cells differentiate.

Early studies on p21 Ras activation in lymphocytes suggested that 40%–50% of p21 Ras is activated after ligation of the T-cell receptor 178, 179. The accumulation of activated p21 Ras appeared to correlate with the inhibition of RasGAP activity. It was also noted that there were tyrosine kinase-dependent but PKC-independent pathways that led to Ras activation [179]. The ability of activators of PKC such as PMA to stimulate Ras activation in T cells has now been explained by discovery of the RasGRP family of exchange factors that exhibit DAG-binding domains. The role of Ras in activation of Erk downstream of the TCR has been demonstrated. Thus, Jurkat T cells overexpressing RasGRP1 were hypersensitive to TCR-stimulated Erk activation whereas T cells with homozygous RasGRP1 null alleles were unable to activate Erk after TCR ligation 174, 180. There appear to be at least two parallel mechanisms for Ras activation after T-cell (or B-cell) receptor ligation (reviewed in [181]). Ligation of antigen receptors results in activation of tyrosine kinases—mainly Lck for the TCR and Lyn for the BCR—that phosphorylate the respective receptor chains and recruit kinases, ZAP70 or Syk to the receptor. Their activation leads to recruitment of adapter proteins, LAT and SLP76 or BLNK, that in turn bind the adapter proteins Shc and Grb2, which is constitutively associated with the exchange factor Sos. A second pathway to Ras activation occurs via recruitment of PLCγ to the activated receptor complex. This leads to production of IP3 and DAG, which provides a binding site at the membrane for RasGRPs. Erk activation downstream of ligation of the T cell receptor also occurs independently of Ras via PKC [176]. A potential role for RasGRF1/2 in TCR- or BCR-stimulated activation of Ras has not yet been explored yet.

Rap1 (but not Rap2) is also activated following ligation of the TCR. Interestingly, co-stimulation of CD28 prevents this activation [182], raising the possibility that interference with the activation of Rap1 may be important for the normal response of T cells to antigen. Ligation of the TCR in the absence of costimulation of CD28 results in a state of long-term functional unresponsiveness termed anergy. Anergic T cells no longer respond to antigenic stimulation by activation of the Ras-MAP kinase pathway and fail to produce IL-2 [183]. It has been suggested that this is accounted for by increased levels of GTP-bound Rap1 in anergic T cells. Jurkat T cells that over-express activated Rap1 did not produce IL-2, mimicking an anergic phenotype, and again suggesting a role for Rap1 in negative regulation of TCR-induced IL-2 induction in anergy [184]. However, as noted earlier, the competition for Raf-1 with Ras resulting from overexpression of activated Rap1 may be an experimental artifact.

B cells 

The development, survival, activation, and apoptosis of B lymphocytes are regulated by signals from a variety of receptors, including the BCR and CD40. An important role for the Ras family in B-cell development is suggested by several studies with transgenic mice. In mice expressing an H-Ras S17N transgene under the Lck promoter, B-cell development was blocked at a very early stage (pre-proB to pro-B transition), a phenotype that was rescued by activated Raf [127]. In a second study, the H-Ras S17N transgene was driven by the VH gene promoter. In these mice, early pre-B cells did develop, but the numbers of late pre-B cells were reduced, suggesting that Ras is important for survival of pre-B cells [185]. Other evidence for a role for Ras in B cell development came from expression of an activated H-Ras on a Rag−/− background. This permitted Rag−/− B cells to progress from the pro-B stage, where they are normally arrested, to late pre-B cells [186].

Ligation of the mature B-cell receptor can result in activation, apoptosis, or tolerance, depending on the presence or absence of co-stimulation through CD40. Ligation of the BCR with anti-IgM antibodies causes an elevation of Ras-GTP levels from about 15% to 25% and coincides with the formation of complexes containing phosphorylated Shc, Grb2 and Sos1 187, 188. Moreover, H-Ras co-localized with the crosslinked BCR in “caps” [189], which probably represented the earliest evidence for the translocation of the ligand-bound BCR to lipid rafts where H-Ras is located [190]. Whether BCR ligation results in DAG-dependent activation of Ras, analogous to that seen ligation of the TCR, is not yet clear. However, in support of a role for DAG, in the chicken B cell-line DT40, the absence of Grb2 results in only a 50% reduction of Erk activation after ligation of the BCR, whereas Erk activation is almost completely abolished in the absence of PLC-γ2 [191].

Signaling downstream of the BCR is negatively regulated by co-ligation of the FcγRIIB. One component of the inhibitory action on Erk activation may involve the recruitment of RasGAP to the FcγRIIB via its association with SHIP and p62dok [192].

CD40 plays important roles in T-cell–dependent B-cell activation, survival of germinal centre B cells, differentiation to antibody-secreting cells, and isotype switching. In the mature B-cell line Daudi, ligation of CD40 was shown to result in activation of Ras proteins precipitated by the monoclonal antibody Y13-269, as well as of MEK [193]. However, crosslinking of CD40 did not result in activation of Erk in immature WEHI231 B cells [194], suggesting that p21 Ras may not be activated in this system.

B cells undergo changes in their ability to respond to antigen after they have been exposed in the periphery to self-antigens, a state termed tolerance. Antigenic stimulation of B cells rendered tolerant by exposure to high levels of antigen during development no longer resulted in activation of JNK and NFκB, although Erk and NFAT were still activated [195]. Ligation of the BCR results in activation of Rap1 via PLC-dependent production of DAG [196]. However, the role of Ras or Rap in B cell tolerance has not yet been explored.

Macrophages 

The macrophage growth factor CSF-1 is critical to macrophage survival and development. Binding of CSF-1 to its receptor, Fms, was shown to result in activation of Ras proteins, a result consistent with the recruitment of Shc, Grb2, and Sos to activated Fms [197]. A recent study suggests that CSF-1–induced activation of Ras is dependent on both the presence of the Grb2 docking site on Fms, and on Src activity [198]. Erk activation by CSF-1 appears to be partially mediated by A-Raf, but not by Raf-1, and another PI-3 kinase–dependent but Raf-independent pathway leading to Erk activation also exists [198].

The importance of Ras proteins for macrophage functions is documented in a study on transgenic mice in which the activity of Ras proteins or their downstream transcription factor targets was suppressed by expression of mutants of p120 RasGAP or Ets-2. This resulted in increased sensitivity of macrophages to the withdrawal of CSF-1 and an altered, spindle-shape morphology. The increase in expression of the protease uPA by CSF-1 was also severely reduced [199]. Antisense experiments suggested that N-Ras is required for the development of macrophages, but not granulocytes [200]. Interestingly, several hematopoietic cell lines such as 32D, U937, and FDC-P1 undergo differentiation to monocytes in response to expression of v-H-Ras or H-Ras G12D 201, 202, 203.

R-Ras, but not H-Ras, has been reported to have a role in adhesion and phagocytosis in macrophages through a mechanism involving activation of αMβ2 integrins via activation of Rap1 204, 205.

A major function for macrophages in immune responses is to produce pro-inflammatory cytokines, such as tumor necrosis factor (TNF), for example, in response to bacterial lipopolysaccharide (LPS). LPS stimulation of human monocytes was shown to activate the Ras-Erk pathway. This was important for activation of Elk-1 and Egr-1 and efficient production of TNF 206, 207. Activation of Ras induced by LPS was completely inhibited by IL-10 [206]. These observations contrast with older data from transgenic mice expressing v-H-Ras. Stimulation of splenocytes from this “oncomouse” with LPS resulted in the production of only about 50% of the amount of TNF-α made by wild-type cells [208]. We have observed that over-expression of H-Ras G12V in normal murine bone marrow cells leads to the generation of well-differentiated macrophages in the absence of exogenous factors (Guo and Schrader, unpublished observations).

Nitric oxide production is also characteristic to activated macrophages. Ras may regulate the activation of nitric oxide synthase (NOS)-2 by TNF-α, IL-1, or IFNγ, since overexpression of H-Ras Q61L blocks and H-Ras S17N enhances activation of this enzyme, at least in lung cancer cells [209].

Mast cells 

The role of individual members of the Ras family in mast cell development and function has not yet been intensively explored. However, several studies suggest that the development of mast cells is critically dependent on the Ras-Erk pathway. Haploinsufficiency for the RasGAP Nf1 results in hyperresponsiveness to SLF and increased mast cell numbers 210, 211. Also, loss of the transcription factors GATA-2 and microphthalmia (mi), which may be targets of Erk, results in absence of c-Kit expression and lack of mast cell development 212, 213. Signaling through c-Kit and the IL-3 receptor promotes mast cell survival and proliferation. Both receptors, as well as the IL-5 receptor, were shown to activate Ras proteins [122]. Overexpression of M-Ras Q71L in normal murine bone marrow cells leads to the generation of mast cells in the absence of exogenous growth factors (Guo and Schrader, unpublished observations).

Little is known about the role of Ras in the release of granule-associated mediators of acute inflammation, such as histamine and cytokines, from mast cells. Microinjection of activated H-Ras into rat mast cells resulted in their degranulation [214]. Ligation of the FcϵR1 on mast cells triggers the synthesis of a variety of cytokines. Ras was shown to be important in the induction of the IL-5 gene after FcϵR1 stimulation, which is regulated by the transcription factors Elk-1 and NFAT [215]. Experiments involving dominant active and negative H-Ras both suggest that these transcription factors were targets for Ras signals. Rac1 appears to be critical for NFAT activation [216]. Whether the production of TNF-α by mast cells involves the Ras-Erk pathway remains controversial. Using the MEK inhibitor PD098059, one group concluded that FcϵR1-induced production of TNF was independent of MEK activation in the MC/9 cell line [217], whereas another group using the CPII cell line found that MEK activation was required for TNF production [218]. In human mast cells, inhibition of Erk activation results in decreased production of arachadonic acid metabolites and GM-CSF [219]. Based on studies with dominant active mutants and inhibitors, the Ras-related GTPases Rac and Rho also appear to regulate secretion [220].

Megakaryocytes 

The proliferation and differentiation of megakaryocyte progenitors and the production of platelets are regulated by the c-Mpl receptor and its ligand thrombopoietin (TPO). Dominant active and negative mutants of H-Ras have been used to implicate Ras in megakaryocytic differentiation of a c-Mpl expressing erythroleukemia cell line F-36P [221]. It was also suggested that the transcription factor GATA-1 may be important for H-Ras G12V-induced differentiation of hematopoietic cell lines into megakaryocytes [222]. There is also evidence that Rap1 may play a role in megakaryocyte maturation through sustained activation of Erk through B-Raf [223].

Erythrocytes 

The role of Ras in the development of erythrocytes is controversial. When wild-type H-Ras was overexpressed in the erythroleukemic cell line TF1 or in human CD34+ cells, an increased number of erythroid progenitors and increased erythroid differentiation was observed [224]. However, when the Ras-Erk pathway was blocked by Ras antisense molecules or the MEK inhibitor PD98059, the pluripotent progenitor cell line K562 exhibited increased erythroid differentiation [225]. Moreover, introduction of activated N-Ras into human CD34+ cells resulted slower differentiation to erythrocytes [226], although it is possible that this effect was due to increased cell death.

Eosinophils 

When chicken myeloblasts and early erythroid progenitor cells are transformed by a virus containing a Gag/Myb/Ets fusion protein, these cells can be induced to differentiate into eosinophils by overexpression of activated Ras [227]. IL-5 is an important cytokine in the survival and proliferation of eosinophils. This growth factor mediate its effects in part by activation of the Ras-Erk pathway 122, 228. Eosinophils transduced with a tat-H-Ras S17N fusion protein showed decreased survival [229].

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Malignancies associated with Ras in hematopoietic cells 

Activating mutations in Ras genes are one of the most common abnormalities in hematologic malignancies. In acute myeloid leukemia (AML), about 30% of the patients have mutated N-Ras or K-Ras proteins. Mutations in these proteins are also frequently found in pre-leukemic syndromes such as myeloid dysplasia (comprehensively reviewed in 7, 230). Mouse models confirm the importance of N-Ras in myeloid leukemias. When bone marrow cells are transduced with activated N-Ras and transferred into lethally irradiated mice, these mice develop myeloid leukemias [231]. In this study, it was pointed out that a primary effect of N-Ras is to inhibit growth and that other transforming events are required for the onset of myeloid leukemia. The incidence of mutations in the N-Ras or K-Ras genes also far exceeds that of mutations in the H-Ras gene in lymphoid disorders. For example, in multiple myeloma mutations in N-Ras or K-Ras occur in 30%–40% of all cases. However, mouse bone marrow cells expressing activated H-Ras gave rise to lymphomas of pre–T-cell or pre–B-cell origin that are similar to acute lymphoblastic leukemia (ALL) in humans [232].

In some hematological disorders, mutations in Ras genes are rare. However, constitutive activation of Ras pathways is observed in chronic myeloid leukemia (CML), where the BCR/Abl fusion protein induces permanent activation of Ras which is essential for BCR/Abl-mediated transformation (reviewed in [233]). This may be caused in part by reduced p120 RasGAP activity that is observed in CML cells [234]. In BCR/Abl-transformed cells, Grb2 and Sos1 form a physical complex with BCR/Abl [235]. The interaction of BCR/Abl with Grb2 appears to be critical to its ability to activate Ras and to transform cells [236]. Erk is not activated by BCR/Abl, but a Ras/Raf-dependent pathway leads to the induction of c-myc [237], which is also required for BCR/Abl-induced transformation. Besides producing chronic activation of Ras, BCR/Abl also activates pathways downstream of PI-3 kinase and STAT5, each of which play essential roles in BCR/Abl-mediated leukemogenesis 238, 239. In chronic myelomonocytic leukemia (CMML), the fusion product of the PDGF receptor with tel may activate Ras constitutively [240]. Some patients with juvenile chronic myelogenous leukemia (JCML) have lost functional Nf1, a RasGAP. In mice, Nf1 deficiency also produces a myeloid disorder that resembles JCML [241]. The mouse and human genes for the GEF RasGRP4 are located in at sites in the genome that have been linked to asthma and leukemia. Aberrant splicing results in the production of non-functional RasGRP4 in many patients with asthma or mastocytosis [38].

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Where are we headed? 

Co-evolution of the members of the Ras subfamily over millions of years has resulted in a highly inter-regulated signaling network, the complexity of which we are only beginning to understand. The conservation of key structural features among members of the family throughout this long evolutionary history attests to the physiological significance of their extensive sharing of regulators and effector proteins. It is increasingly evident however that these structural similarities have clouded the interpretation of many experiments designed to investigate the function of individual members of the family, using monoclonal antibodies and dominant active or negative mutants. More specific tools such as the use of pull-down assays and specific antibodies to measure activation of individual Ras proteins and more careful analysis of isoform-deficient cells will be required to accurately identify the physiological roles of individual family members. Finally, it is becoming clear that the interaction of members of the Ras family with their regulators and effectors depends not only on the potential for interactions dictated by their structure, but also on the fine subcellular localization of the particular Ras protein and its potential regulators and effectors.

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Acknowledgements 

The experimental work from the authors' laboratory was supported by the Canadian Institutes of Health Research and the Canadian Arthritis Network.

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References 

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PII: S0301-472X(02)00904-9

Experimental Hematology
Volume 30, Issue 10 , Pages 1089-1106, October 2002

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