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p62dok negatively regulates CD2 signaling in Jurkat cells
J G Némorin1, P Laporte, G Bérubé
1Institut National de la Recherche Scientifique-Institut Armand-Frappier, Université du Québec, Laval QC, Canada.
Abstract:
p62(dok) belongs to a newly identified family of adaptor proteins. In T cells, the two members that are predominantly expressed, p56(dok) and p62(dok), are tyrosine phosphorylated upon CD2 or CD28 stimulation, but not upon CD3 ligation. Little is known about the biological role of Dok proteins in T cells. In this study, to evaluate the importance of p62(dok) in T cell function, we generated Jurkat clones overexpressing p62(dok). Our results demonstrate that overexpression of p62(dok) in Jurkat cells has a dramatic negative effect on CD2-mediated signaling. The p62(dok)-mediated inhibition affects several biochemical events initiated by CD2 ligation, such as the increase of intracellular Ca(2+), phospholipase C gamma 1 activation, and extracellular signal-regulated kinase 1/2 activation. Importantly, these cellular events are not affected in the signaling cascade induced by engagement of the CD3/TCR complex. However, both CD3- and CD2-induced NF-AT activation and IL-2 secretion are impaired in p62(dok)-overexpressing cells. In addition, we show that CD2 but not CD3 stimulation induces p62(dok) and Ras GTPase-activating protein recruitment to the plasma membrane. These results suggest that p62(dok) plays a negative role at multiple steps in the CD2 signaling pathway. We propose that p62(dok) may represent an important negative regulator in the modulation of the response mediated by the TCR.
Insights
p62(dok) adaptor protein negatively regulates T cell signaling. Overexpression impairs CD2-mediated responses, including calcium influx and kinase activation, while also affecting T cell receptor signaling pathways.
Area of Science:
- Immunology
- Molecular Biology
- Cell Signaling
Background:
- p62(dok) is a member of a novel adaptor protein family.
- p62(dok) is tyrosine phosphorylated in T cells upon CD2/CD28 stimulation, but not CD3 ligation.
- The precise biological role of Dok proteins in T cell function remains largely uncharacterized.
Purpose of the Study:
- To investigate the functional significance of p62(dok) in T cell activation.
- To elucidate the impact of p62(dok) overexpression on T cell signaling pathways.
Main Methods:
- Generation of Jurkat T cell clones overexpressing p62(dok).
- Analysis of T cell signaling events, including intracellular calcium increase, phospholipase C gamma 1 and extracellular signal-regulated kinase 1/2 activation.
- Assessment of Nuclear Factor of Activated T-cells (NF-AT) activation and Interleukin-2 (IL-2) secretion.
- Investigation of p62(dok) and Ras GTPase-activating protein recruitment to the plasma membrane.
Main Results:
- Overexpression of p62(dok) significantly inhibits CD2-mediated signaling in Jurkat cells.
- p62(dok) overexpression affects calcium influx, PLCγ1, and ERK1/2 activation downstream of CD2.
- CD3/TCR-induced signaling events remain unaffected by p62(dok) overexpression.
- Both CD2 and CD3/TCR-induced NF-AT activation and IL-2 secretion are impaired in p62(dok)-overexpressing cells.
- CD2 stimulation, but not CD3, induces p62(dok) and Ras-GAP recruitment to the plasma membrane.
Conclusions:
- p62(dok) acts as a negative regulator in multiple steps of the CD2 signaling pathway.
- p62(dok) plays a critical role in modulating T cell receptor-mediated responses.
- p62(dok) may represent a key inhibitory component in T cell activation pathways.