RasGRP1 Functions in Developing T Cells
The small GTPase Ras acts as a molecular switch, cycling between GDP-bound “off” and a GTP-bound “on” conformations, and serves to link signals from cell surface receptors to intracellular effector pathways. Ras activation can be modulated by its own intrinsic GTPase activity, converting GTP to GDP, and guanine nucleotide exchange. However, the Ras’ rates of GTP hydrolysis and nucleotide exchange are very low unless paired with catalytic proteins. Guanine nucleotide exchange factors (Ras GEFs) control the activation of Ras by catalyzing GDP release from Ras and facilitating its association with more prevalent cellular GTP. Conversely, Ras GTPase-activating proteins (Ras GAPs) accelerate GTP hydrolysis reaction converting Ras-GTP to its inactive GDP-bound form. Tight control of Ras activity is essential for regulating cell activation, proliferation, differentiation, and apoptotic programs in multiple cell types. By associating with various effector proteins, activated Ras initiates signaling through multiple downstream pathways such as the mitogen-activated protein kinase (MAPK) cascade.
T-cell receptor (TCR) stimulation results in the rapid activation of the small GTPase Ras whose signals are essential for the development of T cells in the thymus (Alberola-Ila and Hernández-Hoyos 2003). Taking cues from Ras studies on non-lymphocytes, the Ras GEF SOS (Son of Sevenless) has been postulated to regulate Ras upon TCR activation. SOS proteins are ubiquitously expressed and their functions are modulated through their association with the adaptor GRB2 (growth factor receptor-bound protein 2). Moreover, the SH2 domain of GRB2 targets SOS to phosphorylated tyrosine residues of surface receptors and adaptor proteins.
According to the SOS-based model, the following sequence of events leads to Ras activation in T cells: TCR ligation activating the Src protein tyrosine kinases (PTKs) LCK (lymphoid cell kinase) and Fyn leading to the phosphorylation of CD3s ITAMs (immunoreceptor tyrosine-based activation motifs), these phosphorylated ITAMs mobilizing the Syk PTK ZAP-70 (zeta-associated protein – 70 kDa) by way of ZAP-70s SH2 domain and becoming activated through the action of Src PTKs, activated ZAP-70 phosphorylating multiple tyrosine residues on the docking adaptor transmembrane protein LAT (Linker for Activated T cells) and phosphorylated LAT recruiting the GRB2/SOS complex in close proximity of plasma membrane–bound Ras and facilitating its displacement of GDP. However, a SOS based model of Ras activation cannot explain at least two T-cell phenomena: (1) Why phorbol esters or their analogs activate Ras? and (2) Why PKC inhibitors dampen Ras activation?
The cloning of RasGRP1 identified the first of a novel class of Ras guanyl nucleotide-releasing proteins that possessed calcium- and diacylglycerol (DAG)-responsive elements and elucidated a critical mechanism by which TCR signal transduction and phorbol ester stimulation is linked to the activation of the Ras-MAPK cascade in T cells (Stone 2011). TCR signaling incorporates RasGRP1 function through the mobilization and enzymatic activities of PLCγ1. Similarly to GRB2/SOS, PLCγ1 is recruited to phosphorylated LAT through its SH2 domain and becomes phosphorylated by Tec family protein kinases. Subsequently, the action of activated PLCγ1 converts PIP2 (phosphatidylinositol 4, 5 bisphosphate) into IP3 (inositol 3, 4, 5 triphosphate) and DAG, a second messenger previously thought to solely activate PKC through its DAG-binding C1 domain. However, DAG also causes RasGRP1 to become membrane localized through its own DAG-binding C1 domain. Furthermore, DAG indirectly impacts RasGRP1 as activated PKC regulates RasGRP1 activity through its phosphorylation (Roose et al. 2005). As a consequence, the integration of RasGRP1 into T-cell signaling provides an explanation for the well-documented activation of Ras by DAG analogs and phorbol esters such as PMA (phorbol myristate acetate). In addition, the integration of RasGRP1 also provides a mechanism for why PKC inhibitors block Ras activation.
Ras-MAPK signaling downstream of the pre-TCR and TCR is critical for two developmental checkpoints as thymocytes undergo an ordered series of maturation steps within the thymic microarchitecture (Alberola-Ila and Hernández-Hoyos 2003). Thymocyte development is most often tracked through the variable expression of the cell surface markers CD4 and CD8. After productive rearrangement of the TCRß chain and pairing with the pre-TCRα, pre-TCR expression by the most immature CD4- CD8- double-negative (DN) thymocytes drives ligand-independent Ras-MAPK signaling at the first developmental checkpoint and differentiation into CD4+ CD8+ double-positive (DP) thymocytes. Accompanying rearrangement and expression of the TCRα chain, TCR-dependent Ras-MAPK signaling at the second developmental checkpoint (called “thymocyte selection”) becomes contingent upon the recognition of self-antigens (i.e., self-peptides presented in the context of self-MHC molecules). The intensity of TCR interaction with self-antigens on thymic cortical epithelial cells and bone marrow–derived cells is presumed to determine the strength of signal and the fate of the developing DP thymocyte. According to the strength of signal hypothesis, cells that do not recognize self-antigens fail to receive TCR signaling resulting in death by neglect; cells that recognize self-antigens robustly receive strong TCR signaling dying via active apoptosis (negative selection); and cells that recognize self-antigens weakly receive moderate TCR signaling differentiating into mature CD4+ CD8- or CD4- CD8+ single-positive (SP) T cells (positive selection).
DP thymocytes discriminate graded TCR-dependent Ras-MAPK signals and translate them into a cell fate decision (Alberola-Ila and Hernández-Hoyos 2003). TCR-induced Ras signaling results in the activation of three distinct families of MAPKs: ERK (extracellular signal-regulated kinases), JNK (c-Jun N-terminal kinases), and p38. The activation of the MAPKs plays qualitatively and quantitatively distinct roles in thymocyte selection: ERK has been most often paired with positive selection whereas JNK and p38 are associated with negative selection. How might different MAPKs selectively pair and become activated upon TCR-induced Ras signaling was not clear. To elucidate the role of RasGRP1 in T-cell development, analyses of RasGRP1 −/− mice revealed a near-normal number of DN and DP thymocytes but a severe deficiency in mature thymocytes, suggesting a block in thymocyte selection (Stone 2011). Furthermore, RasGRP1 −/− thymocytes failed to activate both Ras and ERK upon phorbol ester stimulation. In addition, one-month old RasGRP1 −/− mice had very few splenic T cells. As a consequence of the T-cell phenotype present in RasGRP1 −/− mice along with the governing role that TCR signaling plays in T-cell development, RasGRP1 was hypothesized to link Ras activation with TCR signaling.
To examine the role of RasGRP1 in thymocyte selection, two lines of RasGRP1 −/− TCR transgenic mice were generated to determine the effect of RasGRP1 under conditions of defined TCR signaling strength (Priatel et al. 2002). Results from these experiments indicated that positive selection, particularly a weakly selecting TCR, and TCR-induced ERK activation are critically dependent on RasGRP1. By contrast, RasGRP1-deficiency had no effect on negative selection or JNK and p38 MAPK activation. These conclusions were consistent with complementary findings from another study investigating Grb2 +/− mice (Gong et al. 2001). Halving of the amount of GRB2/SOS led to decreased Ras, JNK, and p38 activation and impaired negative selection. However, ERK activation and positive selection were unaffected by Grb2 haploinsufficiency, suggesting that the RasGRP1-ERK pathway may have a lower threshold of activation than GRB2/SOS. The rationale for TCR signaling to employ two different Ras GEFs may be to subject Ras to differential regulation or to pair it with a unique subset of effectors. Based on the findings from the above studies, a hypothesis was formulated that the Ras GEFs RasGRP1 and GRB2/SOS may serve to selectively pair Ras activation with differential MAPKs pathways. According to this model, DP thymocytes expressing a positively selecting TCR will activate Ras and solely the MAPK ERK via RasGRP1 whereas DP thymocytes expressing a negatively selecting TCR will activate Ras and the full range of MAPKs (ERK, JNK, and p38) via the use of both GRB2/SOS and RasGRP1 pathways.
RasGRP1 Functions in Other Blood Cells
RasGRP1 was originally envisioned to have very restricted expression, transcripts being detected solely in T cells and some neuronal cell lineages (Stone 2011). More recent studies have found that RasGRP1 has a much broader tissue distribution than previously thought. RasGRP1 is also expressed in B cells and is presumed to couple the B-cell antigen receptor (BCR) to Ras-ERK signaling in an analogous fashion to the way it functions in TCR signal transduction (Coughlin et al. 2005). However, RasGRP1 function in BCR signaling appears to be partially masked through the coexpression of RasGRP3 and the sharing of some redundant functions with this RasGRP family member. Studies using the immature B cell line WEHI-231 have linked a RasGRP1-pathway that is ERK-independent to BCR-induced apoptosis (Guilbault and Kay 2004). Analyses of human NK (natural killer) cells using RNA interference have demonstrated that RasGRP1 regulates ITAM-dependent cytokine production and NK cell cytotoxicity (Lee et al. 2009). In addition, RasGRP1 knockdown in NK cells was found to result in dampened Ras, ERK, and JNK activation.
RasGRP1 is also expressed by mast cells and signals downstream of the high affinity IgE receptor FceR1 (Liu et al. 2007). Moreover, FceR1 degranulation and cytokine production were greatly reduced in RasGRP1 −/− mast cells relative to wild type and RasGRP1 −/− mice failed to elicit anaphylactic allergic reactions. Interestingly, RasGRP1 in mast cells was found to link FceR1-mediated Ras signaling to PI3K (phosphatidyl inositol 3-kinase) pathway rather than to ERK activation. By contrast, a concentrated effort to establish a connection between RasGRP1 signaling and PI3K activation in lymphocytes has been unsuccessful (Stone 2011). In addition, RasGRP1 expression and function has been recently described outside of the hematopoietic system, although those studies are described elsewhere (Stone 2011).
RasGRP1 Activity and Subcellular Localization
An exquisite investigation monitoring endogenous signaling molecules has suggested that subcellular compartmentalization of the Ras/RasGRP1/ERK pathway plays a key role in developing thymocytes undergoing selection (Daniels et al. 2006). Using OT-1 TCR transgenic preselection CD4+CD8+ thymocytes and specific peptides that span the boundary of positive and negative selection, it was revealed that negatively selecting peptides targeted Ras, Raf-1, and RasGRP1 to the plasma membrane, whereas these molecules colocalized to endomembranes when thymocytes were stimulated with peptides mediating positive selection. Notwithstanding, as this study determined the localization of total Ras rather than active Ras (i.e., Ras-GTP), it is conceivable that sufficient signaling necessary to mediate positive selection is initiated via trace amounts of RasGRP1 and Ras that is situated at the plasma membrane.
A recent study utilizing novel high affinity probes for Ras-GTP imaged in live Jurkat T cells was capable of discerning the accumulation of endogenous Ras-GTP solely at the plasma membrane (Rubio et al. 2010). In addition, the failure of a palmitoylation-defective mutant of N-Ras that is restricted to endomembranes to become activated upon TCR stimulation further asserts that plasma membrane localization is required for Ras activation. Future studies will require sophisticated tools like the one discussed above and high-resolution microscopic imaging to settle the debate over the localization of active RasGRP1 and Ras-GTP.
RasGRP1 and Autoimmunity
T cells are a vital component of the body’s defense system and their capacity to differentiate self- from foreign-antigens is crucial to protect against both pathogenic challenge and autoimmune-mediated self-destruction. The dependence of T-cell development, T-cell function, and T-cell tolerance on TCR signaling suggests that mutations affecting TCR signal transduction may cause a multitude of deleterious health-related effects. Immunodeficiency may arise from alterations to the TCR repertoire and T-cell function. In addition, aberrant TCR signaling may promote autoimmunity by influencing central- (deletion of autoreactive T cells in the thymus) and peripheral-T-cell tolerance (T-cell anergy, activation-induced cell death [AICD] and suppression by regulatory T cells). Importantly, abnormal Ras-ERK signaling in T cells has been described in a number of autoimmune diseases in humans and animal models.
One reported consequence of decreased activation of Ras-ERK pathway in T cells is reduced DNA methyltransferase I (DNMT1) expression causing the derepression of autoimmune genes (Gorelik et al. 2007). Recently, two microRNAs, miRNA-21 and miRNA-148a, overexpressed in T cells from both patients with systemic lupus erythematosus (SLE) and lupus-prone MRL/lpr mice have been found to downmodulate DNMT1 directly and indirectly by turning down Ras-ERK signaling and targeting RasGRP1 transcripts (Pan et al. 2010). By contrast, defective RasGRP1 expression in a subset of SLE patients has been proposed to result from aberrant RNA splicing (Stone 2011). Additionally, dysregulated RasGRP1 expression has been implicated in another autoimmune disease through genome-wide association studies linking RasGRP1 variants to type 1 diabetes (Stone 2011).
The severely impaired T-cell maturation in the thymus of young RasGRP1-deficient mice is correlated with a small but activated population of peripheral T cells, particularly of the CD4 lineage (Layer et al. 2003; Priatel et al. 2007). However, with age, RasGRP1-deficient mice (on a mixed C57BL/6:129SvJ genetic background), derived through a classical gene targeting approach (RasGRP1 -/- ) and a spontaneous mouse mutant of RasGRP1 (RasGRP1 lag ; lag is an acronym representing lymphoproliferation-autoimmunity-glomerulonephritis), were found to exhibit massive lymphoproliferation and autoimmunity with similarity to SLE (Layer et al. 2003). At 5 months of age, RasGRP1 lag and RasGRP1 −/− mice displayed splenomegaly, lymphadenopathy, glomerulonephritis, lymphocytic infiltrates within many organs, elevated antinuclear antibodies (ANAs), anorexia, and lethargy. However, the penetrance of a severe autoimmune phenotype within one animal colony of RasGRP1 −/− mice disappeared after successive backcrossing of the targeted mutation onto the C57BL/6 background. C57BL/6 RasGRP1 −/− mice remained lymphopenic and free of severe autoimmune disease up to 1 year of age despite high-serum ANA levels (Priatel et al. 2007). It is possible that genetic modifiers from the 129/SvJ genetic background or environmental factors, such as distinct microfloral, may synergize with RasGRP1-deficiency to promote fulminant disease.
The lack of RasGRP1 in developing thymocytes may push the balance toward autoimmunity. It has been proposed that DP thymocytes capable of maturing into mature SP thymocytes need to express more strongly self-reactive TCRs if they lack RasGRP1 to overcome their signaling deficits (Priatel et al. 2002; Layer et al. 2003). As TCR transgenic studies have argued that RasGRP1 is not necessary for central tolerance (Priatel et al. 2002), the affinity/avidity of TCRs expressed by RasGRP1 −/− mature SP thymocytes perhaps straggle the boundary between positive and negative selection. In addition, RasGRP1 has been shown to play a critical role in the formation of natural Foxp3-expressing regulatory T cells, suggesting that impaired development or function of this lineage may contribute to disease in RasGRP1 mutant mice (Stone 2011).
There are also a number of peripheral mechanisms by which RasGRP1-deficiency may collude with defective thymocyte development to cause disease. Firstly, the lymphopenic compartment within RasGRP1 −/− mice that results from decreased thymic output may favor oligoclonal T-cell outgrowth and generation of T-cell effectors through abundance of cytokines like IL-7 and increased availability of self-peptides/self-MHC molecules. Notably, RasGRP1 −/− T cells have a distinct TCR repertoire relative to wild type animals resulting from altered T-cell development or peripheral T-cell homeostasis (Priatel et al. 2007). Secondly, aberrant TCR signaling or TCR repertoire in mature T cells may lead to weakened immune responses, chronic infections, and proinflammatory conditions. Viral challenge experiments demonstrated that RasGRP1 −/− mice generate drastically fewer antigen-specific T cells and delayed pathogen clearance as compared to wild type mice (Priatel et al. 2007). Thirdly, the resistance to AICD exhibited by RasGRP1-deficient T cells in vitro has been postulated to enhance their pathogenicity in vivo by escaping apoptosis (Layer et al. 2003). Fourthly, the function or maintenance of regulatory T cells may be impacted by diminished IL-2 production observed for RasGRP1 −/− T-cell effectors (Layer et al. 2003; Priatel et al. 2010). Collectively, these findings suggest multiple means by which aberrant RasGRP1 signaling may enhance susceptibility to immunologic disease.
RasGRP1 and Cancer
Ras signaling regulates proliferation, differentiation and survival and activating Ras mutations are present in approximately 30% of all human cancers. As the original descriptions of RasGRP1 documented its capacity to transform rodent fibroblasts in vitro (Stone 2011), it raises the question as to whether altered RasGRP1 expression or activity can lead to tumorigenesis. To date, findings from several studies have supported this hypothesis.
The observation that the RasGRP1 is a frequent site of proviral insertion in retrovirus-induced murine T-cell lymphomas suggests that RasGRP1 can act as an oncogene (Stone 2011). Corroborating these findings, overexpression of RasGRP1 in the thymus was able to initiate thymic lymphomas in a pre-TCR/TCR-independent manner (Klinger et al. 2005). An investigation into genes able to induce acute myeloid leukemia (AML) found that RasGRP1 can act alone as a leukemic initiator/driver or act in concert with other leukemic causing genes to cause disease (Vassiliou et al. 2011). In addition, therapeutically targeting the Ras-ERK pathway using MEK inhibitors in a mouse model of AML revealed that increased RasGRP1 expression correlated with tumor resistance to the drug, although the precise mechanism of action remained unclear (Lauchle et al. 2009). Besides its association with blood cancers, RasGRP1 overexpression in murine epidermal keratinocytes led to spontaneous development of squamous cell papillomas in the absence of chemical tumor initiators (Diez et al. 2009). Consequently, RasGRP1 is relevant to tumorigenesis in hematopoietic and non-hematopoietic cells.
RasGRP1 is the prototypical member of the RasGRP family of guanyl nucleotide exchange factors whose function is to couple surface receptor signaling to the activation of the small GTPase Ras and downstream MAPK pathways. The activity of RasGRP1 is regulated through its mobilization to membranes and pairing with Ras by the second messenger DAG (diacylglycerol). RasGRP family members are composed of a REM (Ras exchange motif) and CDC25 (cell division cycle 25)-related domains, functioning in Ras recognition and catalysis of GDP exchange, and “EF-hand” and DAG-binding “C1” motifs, serving to modulate its recruitment to membranes. Initial investigations revealed that RasGRP1 links TCR signaling to the activation of Ras-ERK pathway, playing crucial roles in both T-cell development and mature T-cell function. More recent studies have found that RasGRP1 mediates critical regulation of surface receptor signaling and effector functions in B cells, NK cells, and mast cells. Perturbations in RasGRP1 function are suspected to underlie immunodeficiency, autoimmunity, and blood cell malignancies.
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