Abstract
BackgroundSignalling by the T cell antigen receptor (TCR) results in the activation of T lymphocytes. Nck1 and Nck2 are two highly related adaptor proteins downstream of the TCR that each contains three SH3 and one SH2 domains. Their individual functions and the roles of their SH3 domains in human T cells remain mostly unknown.ResultsUsing specific shRNA we down-regulated the expression of Nck1 or Nck2 to approximately 10% each in Jurkat T cells. We found that down-regulation of Nck1 impaired TCR-induced phosphorylation of the kinases Erk and MEK, activation of the AP-1 and NFAT transcription factors and subsequently, IL-2 and CD69 expression. In sharp contrast, down-regulation of Nck2 hardly impacts these activation read-outs. Thus, in contrast to Nck2, Nck1 is a positive regulator for TCR-induced stimulation of the Erk pathway. Mutation of the third SH3 domain of Nck1 showed that this domain was required for this activity. Further, TCR-induced NFAT activity was reduced in both Nck1 and Nck2 knock-down cells, showing that both isoforms are involved in NFAT activation. Lastly, we show that neither Nck isoform is upstream of p38 phosphorylation or Ca2+influx.ConclusionsIn conclusion, Nck1 and Nck2 have non-redundant roles in human T cell activation in contrast to murine T cells.
Highlights
Signalling by the T cell antigen receptor (TCR) results in the activation of T lymphocytes
To knockdown either Nck1 or Nck2, Jurkat T cells were transfected with a plasmid encoding either Nck1- or Nck2-specific short hairpin RNA or an empty control plasmid
Individual clones were screened by analyzing Nck1, Nck2 and TCR expression
Summary
Signalling by the T cell antigen receptor (TCR) results in the activation of T lymphocytes. Binding of the TCR to peptide-loaded major histocompatibility complexes (pMHCs) presented on the surface of antigen presenting cells (APCs) initiates the formation of intracellular protein signalling complexes [2] The formation of these complexes is required for T cell activation and is driven by adaptor proteins in order to activate downstream pathways, such as the Ras-mitogen-activated protein kinase (MAPK), protein kinase C (PKC) and Ca2+-mediated. SH3.1 of Nck binds directly to a PRS in the cytoplasmic tail of CD3ε exposed upon antigen-induced conformational changes within the TCR complex This interaction is required for IL-2 production and synapse formation [9]. The guanine nucleotide exchange factor SOS is required for activation of the Ras-Raf-MEK-Erk pathway [15]
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