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RasGRP: the missing link for Ras activation in thymocytes
News & Comment
TRENDS in Immunology Vol.22 No.2 February 2001
69
Journal Club
RasGRP: the missing link for Ras activation in thymocytes Positive selection of MHC-restricted T cells in the thymus requires the activation of the Ras–Raf–Mek–Erk pathway. This was first shown by analysis of Ras and Mek dominant negative mutants, which resulted in profoundly impaired positive selection. Until recently, however, it was unclear exactly how T-cell receptor (TCR) signaling activates Ras in thymocytes. Ras is a small GTPase that resides at the plasma membrane because it is lipid modified. Its activation can be regulated in both a positive manner [by guanine nucleotide exchange factors (GEFs)] or in a negative manner [by GTPase-activating proteins (GAPs)]. The Son of sevenless (Sos) GEF is recruited to the TCR activation complex by interacting with Grb2 and LAT. Thus, for some time, Sos recruitment to the plasma membrane was assumed to be the mechanism of Ras activation in T cells. Nevertheless, this model did not explain
why, in T cells, Ras activation is dependent on PLCγ1 and can be efficiently stimulated by phorbol esters. Recently, a GEF termed RasGRP was identified. This molecule contains a diacylglycerol (DAG)-binding domain. As DAG is a product of PLCγ1 activation, RasGRP could provide a link between TCR ligation and Ras activation. Dower and colleagues1 now report analysis of RasGRP-deficient mice. Erk activation by anti-CD3 or phorbol esters was completely absent in thymocytes from these mice, implying that RasGRP is indeed an important mediator of Ras activation by the TCR. Not surprisingly, a profound thymic phenotype was observed, namely impaired positive selection. Although RasGRP deficiency had little effect on the differentiation of doublenegative cells to double-positive cells, it resulted in a near-complete absence of mature ‘single-positive’ cells, both in the thymus and in peripheral lymphoid organs.
RasGRP-deficient thymocytes were also unable to proliferate in response to anti-CD3, although this might be secondary to the defect in positive selection. Nevertheless, the results indicate that RasGRP provides a nonredundant link between TCR ligation and Ras activation in thymocytes. Further experiments will be necessary to determine if Ras activation in mature T cells is wired similarly. An interesting aspect of the early work using Ras or MEK dominant negative mutants was the lack of an effect on negative selection, despite a profound effect on positive selection. Thus, it will be interesting to learn whether RasGRP deficient mice show impaired negative selection. 1 Dower, N.A. et al. (2000) RasGRP is essential for mouse thymocyte differentiation and TCR signaling. Nat. Immunol. 1, 317–321
Kristin Hogquist e-mail: [email protected]
Double break-point in the match for the mutator Mammalian B cells use heretical genetic mechanisms to generate an astonishingly diverse array of antibody molecules. In fact, the creation of a single antibody molecule involves three separate events that permanently alter genomic DNA. The variable (V) regions of the immunoglobulin gene are created when smaller gene segments are recombined, the constant (C) regions of the immunoglobulin genes can be replaced or ‘switched’ at the gene level and single base pair changes can appear within the recombined antibody variable region genes in a process referred to as somatic hypermutation. Our understanding of each of these DNA-altering events is still incomplete, but somatic hypermutation, which is responsible for the dramatic increase in affinity characteristic of a memory response to a pathogen, remains particularly mysterious. Investigators have hunted for the elusive ‘somatic mutase’ for years with little success. Two recent papers1,2, however, have clarified this effort by reaching the relatively unexpected conclusion that, like VDJ recombination and class switching, somatic mutation depends on double stranded breaks (DSBs).
Both groups of investigators exploited a clever variant of the polymerase chain reaction [ligation-mediated polymerase chain reaction (LM-PCR)], to reveal double stranded breaks in cells undergoing somatic mutation. By joining known pieces of DNA to double-stranded break-points and then amplifying DNA between the break and up- or down-stream sequences, the investigators were able to show that these break-point dependent PCR products are present preferentially within cells capable of undergoing somatic mutation (i.e. germinal center B cells). Sequencing the PCR products also revealed that the DSBs were found predominantly near sequences defined as ‘hot spots’ for somatic hypermutation. Notably, both sets of investigators found that the upstream (5′) side of a doublestranded break proved more accessible to ligation than the 3′ side of the DSB, hinting at some as yet uncharacterized asymmetry in the process by which the DSB is generated. Interestingly, although both groups speculate that error-prone repair processes are ultimately responsible for the characteristic mutations, they draw different conclusions about which repair mechanism
is responsible. Bross and colleagues1 propose that the introduction of doublestrand breaks is repaired imperfectly by a nonhomologus DNA end-joining (NHEJ) system. Although Papavasiliou and Schatz2 do not rule out a role for NHEJ, they show that DSBs are found preferentially in cells in the S and G2 phases of the cell cycle, suggesting that homologous recombination, which uses sister chromatids as templates, might be more centrally involved. With these break-through observations, somatic hypermutation is no longer an orphan process. It can now join VDJ recombination and class switching as a double-strand break-dependent event. It remains to be seen what else, if anything, these processes have in common. 1 Bross, L. et al. (2000) DNA Double strand breaks in immunoglobulin genes undergoing somatic hypermutation. Immunity 13, 589–597 2 Papavasiliou, F.N. and Schatz, D.G. (2000) Cell cycle regulated DNA double-strand breaks in somatic hypermutation of immunoglobulin genes. Nature 408, 216–221
Judith A. Owen and Jennifer A. Punt* *e-mail: [email protected]
http://immunology.trends.com 1471-4906/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.
TRENDS in Immunology Vol.22 No.2 February 2001
69
Journal Club
RasGRP: the missing link for Ras activation in thymocytes Positive selection of MHC-restricted T cells in the thymus requires the activation of the Ras–Raf–Mek–Erk pathway. This was first shown by analysis of Ras and Mek dominant negative mutants, which resulted in profoundly impaired positive selection. Until recently, however, it was unclear exactly how T-cell receptor (TCR) signaling activates Ras in thymocytes. Ras is a small GTPase that resides at the plasma membrane because it is lipid modified. Its activation can be regulated in both a positive manner [by guanine nucleotide exchange factors (GEFs)] or in a negative manner [by GTPase-activating proteins (GAPs)]. The Son of sevenless (Sos) GEF is recruited to the TCR activation complex by interacting with Grb2 and LAT. Thus, for some time, Sos recruitment to the plasma membrane was assumed to be the mechanism of Ras activation in T cells. Nevertheless, this model did not explain
why, in T cells, Ras activation is dependent on PLCγ1 and can be efficiently stimulated by phorbol esters. Recently, a GEF termed RasGRP was identified. This molecule contains a diacylglycerol (DAG)-binding domain. As DAG is a product of PLCγ1 activation, RasGRP could provide a link between TCR ligation and Ras activation. Dower and colleagues1 now report analysis of RasGRP-deficient mice. Erk activation by anti-CD3 or phorbol esters was completely absent in thymocytes from these mice, implying that RasGRP is indeed an important mediator of Ras activation by the TCR. Not surprisingly, a profound thymic phenotype was observed, namely impaired positive selection. Although RasGRP deficiency had little effect on the differentiation of doublenegative cells to double-positive cells, it resulted in a near-complete absence of mature ‘single-positive’ cells, both in the thymus and in peripheral lymphoid organs.
RasGRP-deficient thymocytes were also unable to proliferate in response to anti-CD3, although this might be secondary to the defect in positive selection. Nevertheless, the results indicate that RasGRP provides a nonredundant link between TCR ligation and Ras activation in thymocytes. Further experiments will be necessary to determine if Ras activation in mature T cells is wired similarly. An interesting aspect of the early work using Ras or MEK dominant negative mutants was the lack of an effect on negative selection, despite a profound effect on positive selection. Thus, it will be interesting to learn whether RasGRP deficient mice show impaired negative selection. 1 Dower, N.A. et al. (2000) RasGRP is essential for mouse thymocyte differentiation and TCR signaling. Nat. Immunol. 1, 317–321
Kristin Hogquist e-mail: [email protected]
Double break-point in the match for the mutator Mammalian B cells use heretical genetic mechanisms to generate an astonishingly diverse array of antibody molecules. In fact, the creation of a single antibody molecule involves three separate events that permanently alter genomic DNA. The variable (V) regions of the immunoglobulin gene are created when smaller gene segments are recombined, the constant (C) regions of the immunoglobulin genes can be replaced or ‘switched’ at the gene level and single base pair changes can appear within the recombined antibody variable region genes in a process referred to as somatic hypermutation. Our understanding of each of these DNA-altering events is still incomplete, but somatic hypermutation, which is responsible for the dramatic increase in affinity characteristic of a memory response to a pathogen, remains particularly mysterious. Investigators have hunted for the elusive ‘somatic mutase’ for years with little success. Two recent papers1,2, however, have clarified this effort by reaching the relatively unexpected conclusion that, like VDJ recombination and class switching, somatic mutation depends on double stranded breaks (DSBs).
Both groups of investigators exploited a clever variant of the polymerase chain reaction [ligation-mediated polymerase chain reaction (LM-PCR)], to reveal double stranded breaks in cells undergoing somatic mutation. By joining known pieces of DNA to double-stranded break-points and then amplifying DNA between the break and up- or down-stream sequences, the investigators were able to show that these break-point dependent PCR products are present preferentially within cells capable of undergoing somatic mutation (i.e. germinal center B cells). Sequencing the PCR products also revealed that the DSBs were found predominantly near sequences defined as ‘hot spots’ for somatic hypermutation. Notably, both sets of investigators found that the upstream (5′) side of a doublestranded break proved more accessible to ligation than the 3′ side of the DSB, hinting at some as yet uncharacterized asymmetry in the process by which the DSB is generated. Interestingly, although both groups speculate that error-prone repair processes are ultimately responsible for the characteristic mutations, they draw different conclusions about which repair mechanism
is responsible. Bross and colleagues1 propose that the introduction of doublestrand breaks is repaired imperfectly by a nonhomologus DNA end-joining (NHEJ) system. Although Papavasiliou and Schatz2 do not rule out a role for NHEJ, they show that DSBs are found preferentially in cells in the S and G2 phases of the cell cycle, suggesting that homologous recombination, which uses sister chromatids as templates, might be more centrally involved. With these break-through observations, somatic hypermutation is no longer an orphan process. It can now join VDJ recombination and class switching as a double-strand break-dependent event. It remains to be seen what else, if anything, these processes have in common. 1 Bross, L. et al. (2000) DNA Double strand breaks in immunoglobulin genes undergoing somatic hypermutation. Immunity 13, 589–597 2 Papavasiliou, F.N. and Schatz, D.G. (2000) Cell cycle regulated DNA double-strand breaks in somatic hypermutation of immunoglobulin genes. Nature 408, 216–221
Judith A. Owen and Jennifer A. Punt* *e-mail: [email protected]
http://immunology.trends.com 1471-4906/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved.