G of TCRb with pre-Ta produces the pre-TCR that signals the DN3 thymocytes to undergo a process termed `bselection’. The characteristic features of b-selection include commitment to the ab-T cell lineage, continued differentiation, proliferation, survival and cessation of recombination at the TCRb locus. Thymocytes that pass the b-selection checkpoint enter the CD442CD252 DN4 stage before CD4 and CD8 are upregulated to generate `double positive’ (DP) thymocytes [2]. Following a productive TCRa rearrangement and pairing with TCRb to produce a mature abTCR, the DP thymocyte is then subjected to positive and negative selection based upon the RG7666 cost specificity of the mature TCR for self-peptide MHC complexes [3]. Currently it is thought that signals downstream of the pre-TCR, Notch and CXCR4 drive DN3 thymocytes through the bselection checkpoint [4]. The signal transduction pathway downstream of the pre-TCR is thought to be similar to that of the mature ab-TCR. For example, mice deficient in Zap70 and Syk, LAT 18325633 or SLP-76 show a profound block in T cell development at the b-selection checkpoint [5?]. Additionally, it is known thatRasGRP1 Is Required for b-Selectionsignals downstream of the pre-TCR can activate the canonical Ras/Erk pathway [10]. While Notch signaling is critically important at earlier stages of DN thymocyte development (for recent review see [11]), at the b-selection checkpoint, Notch appears to cooperate with pre-TCR signals to promote survival and metabolic activity through the PI3K pathway. It was recently demonstrated that the chemokine receptor CXCR4 is required as a `co-stimulatory’ receptor for b-selection [12,13]. Again, the preTCR appears to cooperate with CXCR4 possibly through a PI3K mediated program of survival. While it is known that the Ras/Erk pathway is activated during b-selection, the identity of the upstream Ras activator was unclear. Ras is a small, lipidated G-protein whose activity is regulated by guanine nucleotide binding. Ras is allosterically activated by GTP binding while the GDP bound form is inactive. Guanine nucleotide exchange factors (GEFs) catalyse the exchange of GDP for GTP thereby activating Ras, while an intrinsic GTPase activity is enhanced by GTPase activating proteins (GAPs). Ras activation leads to the recruitment and activation of the Raf kinase that can phosphorylate and activate MEK that in turn phosphorylates and activates Erk1 and 2. Erk1 and 2 can then modulate gene expression that influences many cellular processes including proliferation, survival and lineage commitment to name but a few [14]. In lymphocytes, there are two major families of GEFs that regulate Ras activity: the son of sevenless (Sos) and Ras guanylnucleotide releasing protein (RasGRP) families. The RasGRP family consists of four ARN-810 biological activity members (RasGRP1?). RasGRP1 is expressed mainly in T and B cells while RasGRP3 is prominently expressed in B cells. Both RasGRP1 and 3 are regulated by the binding of their C1 domain to diacylglycerol and also have the potential to be regulated by PKC phosphorylation [15?8]. The role of other domains in the regulation of RasGRP activity is currently unclear. There are two Sos family members, Sos1 and Sos2. The Sos family is also regulated by recruitment to membranes through interactions with adaptor molecules such as Grb-2 and Shc. Additionally, it has recently been demonstrated that allosteric, or GTP-bound, Ras binding as well as the binding of PIP2 to the DH-PH domain of Sos increases the a.G of TCRb with pre-Ta produces the pre-TCR that signals the DN3 thymocytes to undergo a process termed `bselection’. The characteristic features of b-selection include commitment to the ab-T cell lineage, continued differentiation, proliferation, survival and cessation of recombination at the TCRb locus. Thymocytes that pass the b-selection checkpoint enter the CD442CD252 DN4 stage before CD4 and CD8 are upregulated to generate `double positive’ (DP) thymocytes [2]. Following a productive TCRa rearrangement and pairing with TCRb to produce a mature abTCR, the DP thymocyte is then subjected to positive and negative selection based upon the specificity of the mature TCR for self-peptide MHC complexes [3]. Currently it is thought that signals downstream of the pre-TCR, Notch and CXCR4 drive DN3 thymocytes through the bselection checkpoint [4]. The signal transduction pathway downstream of the pre-TCR is thought to be similar to that of the mature ab-TCR. For example, mice deficient in Zap70 and Syk, LAT 18325633 or SLP-76 show a profound block in T cell development at the b-selection checkpoint [5?]. Additionally, it is known thatRasGRP1 Is Required for b-Selectionsignals downstream of the pre-TCR can activate the canonical Ras/Erk pathway [10]. While Notch signaling is critically important at earlier stages of DN thymocyte development (for recent review see [11]), at the b-selection checkpoint, Notch appears to cooperate with pre-TCR signals to promote survival and metabolic activity through the PI3K pathway. It was recently demonstrated that the chemokine receptor CXCR4 is required as a `co-stimulatory’ receptor for b-selection [12,13]. Again, the preTCR appears to cooperate with CXCR4 possibly through a PI3K mediated program of survival. While it is known that the Ras/Erk pathway is activated during b-selection, the identity of the upstream Ras activator was unclear. Ras is a small, lipidated G-protein whose activity is regulated by guanine nucleotide binding. Ras is allosterically activated by GTP binding while the GDP bound form is inactive. Guanine nucleotide exchange factors (GEFs) catalyse the exchange of GDP for GTP thereby activating Ras, while an intrinsic GTPase activity is enhanced by GTPase activating proteins (GAPs). Ras activation leads to the recruitment and activation of the Raf kinase that can phosphorylate and activate MEK that in turn phosphorylates and activates Erk1 and 2. Erk1 and 2 can then modulate gene expression that influences many cellular processes including proliferation, survival and lineage commitment to name but a few [14]. In lymphocytes, there are two major families of GEFs that regulate Ras activity: the son of sevenless (Sos) and Ras guanylnucleotide releasing protein (RasGRP) families. The RasGRP family consists of four members (RasGRP1?). RasGRP1 is expressed mainly in T and B cells while RasGRP3 is prominently expressed in B cells. Both RasGRP1 and 3 are regulated by the binding of their C1 domain to diacylglycerol and also have the potential to be regulated by PKC phosphorylation [15?8]. The role of other domains in the regulation of RasGRP activity is currently unclear. There are two Sos family members, Sos1 and Sos2. The Sos family is also regulated by recruitment to membranes through interactions with adaptor molecules such as Grb-2 and Shc. Additionally, it has recently been demonstrated that allosteric, or GTP-bound, Ras binding as well as the binding of PIP2 to the DH-PH domain of Sos increases the a.
