CD28 and Grb-2, relative to Gads or Grap, preferentially co-operate with Vav1 in the activation of NFAT/AP-1 transcription

Abstract

The co-receptor CD28 binds to several intracellular proteins including PI3 kinase, Grb-2, Gads and ITK. Grb-2 and PI3 kinase binding has been mapped to the pYMNM motif within the cytoplasmic tail of CD28 and has been shown to play a role in co-stimulation. In this study, we demonstrate that amongst the Grb-2 family adapter proteins, CD28 precipitated Grb-2 and specifically co-operated in the up-regulation of NFAT/AP-1 transcription. By contrast, Gads and Grap either failed or only weakly collaborated with CD28 ligation. Further, the loss of Grb-2 binding interferes with the ability of Vav1 to co-operate with CD28. Anti-CD28 ligation alone was capable for co-operating with Grb-2 or Grb-2-Vav1. Our findings define a pathway involving CD28 binding to Grb-2 and its co-operativity with Vav1 in the regulation of T-cell co-stimulation.

Fig. 1

CD28 binding to PI3 K, Grb-2 and Vav1. (A) Jurkat T-cells were either left untreated (lane 1) or stimulated with anti-CD28 (4 μg/ml) and rabbit anti-hamster (2 μg/ml) antibodies (lanes 2–5). Cells were washed and solubilized in Triton X-100 lysis buffer containing protease and phosphatase inhibitors. Anti-CD28 immunoprecipitates and lysates were separated by SDS–PAGE gel, transferred to nitrocellulose and immunoblotted with anit-p85 (upper panel) or anti-Grb-2 (lower panel) antibodies. Lane 6 shows the position of p85 (upper panel) and Grb-2 (lower panel) in cell lysates. Cell lysates blotted for p85 (upper panel) and Grb-2 (lower panel) served as loading controls. (B, upper panel): Jurkat cells were lysed, immunoprecipitated with anti-Vav1 (lane 1) or anti-Grb-2 (lane 2) antibodies and blotted with anti-Vav1. Lower panel: Jurkat cells were lysed, immunoprecipitated with rabbit anti-mouse (lane 1) or anti-CD28 (lane 2) antibodies and blotted for associated Vav1 with anti-Vav1 mAb. Lane 3 shows the position of Vav1 in cell lysates.

Fig. 2

Grb-2 specifically co-operates with CD28 in the up-regulation of NFAT/AP-1 transcription, (A) Jurkat cells were transfected with mock, Grb-2, Gads and Grap together with a reporter plasmid containing the luciferase gene linked to 3XNFAT/AP-1. NFAT/AP-1 activation was determined as a measure of luciferase activity after anti-CD3, anti-CD28 and anti-CD3/CD28 stimulation for 6 h. Expression of the individual proteins are shown in the upper right panel. (B) Grb-2 co-operates with Vav1 in the up-regulation of NFAT/AP-1 activity. Jurkat cells were transfected with mock, Vav1, Vav1/Grb-2, Vav1/Gads and Vav1/Grap together with a reporter plasmid containing the luciferase gene linked to 3XNFAT/AP-1 and stimulated with anti-CD3, anti-CD28 and anti-CD3/CD28 antibodies. Six hours later, luciferase activity was determined. Expression of the individual proteins are shown in the upper right panel. Lower right histogram shows the relative intensity of the expressed proteins.

Fig. 3

Defective Vav GEF activity (VavL213Q) markedly inhibited co-operativity in CD28 induced NFAT/AP-1 activity, (A) Jurkat cells were transfected with either mock, Grb-2, Gads and Vav1 alone or together with Vav1 or with VavL213Q and VavL213Q and Grb-2 in addition with the reporter plasmid containing the luciferase gene linked to 3XNFAT/AP-1. Luciferase activity was measured after anti-CD3, anti-CD28 and anti-CD3/CD28 stimulation for 6 h. Expression of the individual plasmids are shown in the upper right panel. (B) Loss of Grb-2 binding to CD28 abrogates the co-operativity with Vav1 in the up-regulation of NFAT/AP-1 activity. Jurkat cells were transfected with either mock, Grb-2, CD28-193 and Vav1 alone or together with Vav1 and CD28 or CD28-193 in addition with the reporter plasmid containing the luciferase gene linked to 3XNFAT/AP-1. Luciferase activity was measured after anti-CD3, anti-CD28 and anti-CD3/CD28 stimulation for 6 h. Expression of the individual proteins are shown in the lower right panel. Upper right panel: Jurkat cells were transfected with either mock, CD28 wild type, CD28-193 mutant alone or together with Vav1 in addition with the reporter plasmid containing the luciferase gene linked to 3XNFAT/AP-1. Luciferase activity was measured as described above.