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Modulation of the Grb2-associated protein complex in human CD4+ T cells by receptor activation
R Lahesmaa1, A Allsup, C Soderberg
1Department of Leukocyte Biology, Syntex Research, Palo Alto, CA 94303, USA.
Abstract:
A panel of human CD4+ T cell clones was utilized to dissect and analyze the biochemical consequences of activation of CD3 or CD28. To molecularly characterize receptor-activated proximal signaling events, tyrosine-phosphorylated proteins co-precipitating with a Grb2 fusion protein after receptor activation were analyzed. Ligation of CD28, but not other costimulatory molecules, induced the tyrosine phosphorylation of two previously identified Grb2 binding proteins (pp76 and pp116). A third Grb2 binding protein (pp36) was extensively tyrosine phosphophorylated in response to combined CD3 and CD28 activation, but not in response to ligation of either receptor alone. cAMP and co-ligation of CD45 affected the receptor-activated tyrosine phosphorylation of Grb2-associated proteins. Furthermore, we demonstrated that two signaling molecules, Vav and phosphatidylinositol 3'-kinase (PI(3)K), also interacted with the Grb2 protein complex. The activity of PI(3)K was required for T cell activation, because wortmannin, a PI(3)K inhibitor, blocked T cell proliferation and cytokine production induced by ligation of CD3 and CD28. In conclusion, we demonstrate that in activated human T cell clones, the composition of Grb2 protein complex is modulated by costimulatory signals and cAMP, which may be important for the regulation of intracellular signal transduction.
Insights
Costimulatory signals CD28 and cAMP modulate the Grb2 protein complex in human T cells. This influences intracellular signal transduction, affecting T cell activation, proliferation, and cytokine production.
Area of Science:
- Immunology
- Molecular Biology
- Cell Signaling
Background:
- CD4+ T cell activation is crucial for adaptive immunity.
- Proximal signaling events following T cell receptor (TCR) engagement dictate cellular responses.
- Costimulatory molecules like CD28 play a vital role in augmenting T cell activation.
Purpose of the Study:
- To analyze the biochemical consequences of CD3 and CD28 activation on human T cells.
- To identify tyrosine-phosphorylated proteins interacting with Grb2 after receptor ligation.
- To investigate the role of cAMP and CD45 in T cell signaling pathways.
Main Methods:
- Utilized human CD4+ T cell clones.
- Analyzed tyrosine-phosphorylated proteins co-precipitating with a Grb2 fusion protein.
- Investigated the effects of cAMP and CD45 co-ligation.
- Assessed the impact of phosphatidylinositol 3'-kinase (PI(3)K) inhibition using wortmannin.
Main Results:
- CD28 ligation induced tyrosine phosphorylation of Grb2 binding proteins pp76 and pp116.
- Combined CD3 and CD28 activation led to extensive tyrosine phosphorylation of pp36.
- Vav and PI(3)K were identified as interacting partners within the Grb2 protein complex.
- PI(3)K activity was essential for T cell proliferation and cytokine production.
Conclusions:
- Costimulatory signals and cAMP modulate the Grb2 protein complex composition in activated T cells.
- These modulations are critical for regulating intracellular signal transduction pathways.
- Understanding these pathways is key to controlling T cell-mediated immune responses.