Two distinct regions of the CD28 intracytoplasmic domain are involved in the tyrosine phosphorylation of Vav and GTPase activating protein- associated p62 protein

T cell activation requires both specific recognition of the peptide-MHC complex by the TCR and additional signals delivered by costimulatory receptors. We have identified rainbow trout sequences similar to CD28 (rbtCD28) and CTLA4 (rbtCTLA4). rbtCD28 and rbtCTLA4 are composed of an extracellular Ig-superfamily V domain, a transmembrane region, and a cytoplasmic tail. The presence of a conserved ligand binding site within the V domain of both molecules suggests that these receptors likely recognize the fish homologues of the B7 family. The mRNA expression pattern of rbtCD28 and rbtCTLA4 in naive trout is reminiscent to that reported in humans and mice, because rbtCTLA4 expression within trout leukocytes was quickly up-regulated following PHA stimulation and virus infection. The cytoplasmic tail of rbtCD28 possesses a typical motif that is conserved in mammalian costimulatory receptors for signaling purposes. A chimeric receptor made of the extracellular domain of human CD28 fused to the cytoplasmic tail of rbtCD28 promoted TCR-induced IL-2 production in a human T cell line, indicating that rbtCD28 is indeed a positive costimulator. The cytoplasmic tail of rbtCTLA4 lacked obvious signaling motifs and accordingly failed to signal when fused to the huCD28 extracellular domain. Interestingly, rbtCTLA4 and rbtCD28 are not positioned on the same chromosome and thus do not belong to a unique costimulatory cluster as in mammals. Finally, our results raise questions about the origin and evolution of positive and negative costimulation in vertebrate immune systems.

Section: Discussionmentioning

confidence: 99%

Costimulatory Receptors in a Teleost Fish: Typical CD28, Elusive CTLA4

Bernard

Riteau

Hansen

et al. 2006

“…33, and the present work). Under physiological conditions of CD28 ligation, Vav-1 membrane recruitment/phosphorylation, an event shown to be sustained and effective (24,25), is likely to contribute to cortical actin rearrangements through a high local enrichment of activated Rac-1. Indeed, recent observations have indicated that Vav-1 as well as Rho family proteins are concentrated beneath the T cell area contacting the APC (53) 5 , where intense changes in membrane morphology and dynamics take place even before intracellular Ca 2ϩ increase (62)(63)(64).…”

Section: Discussionmentioning

confidence: 99%

“…Toward this end, a Jurkat clone variant CH7C17 (37) that had lost expression of CD28 (CD28 Neg) was transfected with wild-type CD28 (CD28 WT) or a truncated mutant of it (CD28 Del.30) lacking the sequence coding for the last 30 residues of the intracellular tail of the protein (44). CD28 Del.30 protein does not promote Vav-1 tyrosine phosphorylation when expressed in a T cell hybridoma (25) nor activation of the IL-2 gene when coengaged with the TCR (44) and similar results were obtained with CH7C17 cells reconstituted with this mutant (our unpublished data). Cell surface expression of CD28 in CD28 WT and CD28 Del.30 cells was similar (Fig.…”

Section: The Signaling Motifs Of the Cd28 Intracellular Region Are Rementioning

confidence: 99%

“…Indeed, CD28 and the TCR have a number of signaling elements in common (e.g., Src and Tec family protein tyrosine kinases, PI3K, Vav-1, Cbl, and perhaps the adapter Grb-2) (21,22), though some others, like ZAP-70, LAT, and SLP-76, are activated uniquely through the TCR (23,24). One potential candidate contributing to enhance TCR signaling by CD28 may be Vav-1, which is inducibly phosphorylated upon CD28 engagement (24,25). Vav-1 phosphorylation has been correlated with up-regulation of its exchange activity (through its Dbl-like, DH domain) for the Rho GTPase family and preferentially for Rac-1 (26,27).…”

mentioning

confidence: 99%

See 1 more Smart Citation

CD28 Utilizes Vav-1 to Enhance TCR-Proximal Signaling and NF-AT Activation

Michel

Mangino

Attal-Bonnefoy

et al. 2000

The mechanism through which CD28 costimulation potentiates TCR-driven gene expression is still not clearly defined. Vav-1, an exchange factor for Rho GTPases thought to regulate, mainly through Rac-1, various signaling components leading to cytokine gene expression, is tyrosine phosphorylated upon CD28 engagement. Here, we provide evidence for a key role of Vav-1 in CD28-mediated signaling. Overexpression of Vav-1 in Jurkat cells in combination with CD28 ligation strongly reduced the concentration of staphylococcus enterotoxin E/MHC required for TCR-induced NF-AT activation. Surprisingly, upon Vav-1 overexpression CD28 ligation sufficed to activate NF-AT in the absence of TCR engagement. This effect was not mediated by overexpression of ZAP-70 nor of SLP-76 but necessitated the intracellular tail of CD28, the intactness of the TCR-proximal signaling cascade, the Src-homology domain 2 (SH2) domain of Vav-1, and SLP-76 phosphorylation, an event which was favored by Vav-1 itself. Cells overexpressing Vav-1 formed lamellipodia and microspikes reminiscent of Rac-1 and Cdc42 activation, respectively, for which the SH2 domain of Vav-1 was dispensable. Together, these data suggest that CD28 engagement activates Vav-1 to boost TCR signals through a synergistic cooperation between Vav-1 and SLP-76 and probably via cortical actin changes to facilitate the organization of a signaling zone.

“…Some of these clones represent sequences not present in the GenBank database and are currently the subject of further investigation. In addition to these novel sequences, three previously characterized clones were obtained that encoded SLP-76 (47), the GTPase-activating protein p62 dok (48,49) and RACK-1. GST and GST-GRID were incubated with lysates from either resting or activated Jurkat cells.…”

Section: Grid Associates With Other Signaling Proteinsmentioning

confidence: 99%

GRID: A Novel Grb-2-Related Adapter Protein That Interacts with the Activated T Cell Costimulatory Receptor CD28

Ellis¹,

Ashman²,

Burden³

et al. 2000

Adapter proteins such as Grb2 play a central role in the formation of signaling complexes through their association with multiple protein binding partners. These interactions are mediated by specialized domains such as the well-characterized Src homology SH2 and SH3 motifs. Using yeast three-hybrid technology, we have identified a novel adapter protein, expressed predominantly in T lymphocytes, that associates with the activated form of the costimulatory receptor, CD28. The protein is a member of the Grb2 family of adapter proteins and contains an SH3-SH2-SH3 domain structure. A unique glutamine/proline-rich domain (insert domain) of unknown function is situated between the SH2 and N-terminal SH3 domains. We term this protein GRID for Grb2-related protein with insert domain. GRID coimmunoprecipitates with CD28 from Jurkat cell lysates following activation of CD28. Using mutants of CD28 and GRID, we demonstrate that interaction between the proteins is dependent on phosphorylation of CD28 at tyrosine 173 and integrity of the GRID SH2 domain, although there are also subsidiary stabilizing contacts between the PXXP motifs of CD28 and the GRID C-terminal SH3 domain. In addition to CD28, GRID interacts with a number of other T cell signaling proteins, including SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa), p62dok, and RACK-1 (receptor for activated protein kinase C-1). These findings suggest that GRID functions as an adapter protein in the CD28-mediated costimulatory pathway in T cells.