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Non-Overlapping Functions of Nck1 and Nck2 Adaptor Proteins in T Cell

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Non-Overlapping Functions of Nck1 and Nck2 Adaptor Proteins in T Cell Ngoenkam et al. Cell Communication and Signaling 2014, 12:21 http://www.biosignaling.com/content/12/1/21 RESEARCH Open Access Non-overlapping functions of Nck1 and Nck2 adaptor proteins in T cell activation Jatuporn Ngoenkam1, Pussadee Paensuwan1, Kanlaya Preechanukul1, Boonruang Khamsri1, Ichaya Yiemwattana2, Esmeralda Beck-García3,4, Susana Minguet3, Wolfgang WA Schamel3 and Sutatip Pongcharoen5,6* Abstract Background: Signalling 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. Results: Using 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. Conclusions: In conclusion, Nck1 and Nck2 have non-redundant roles in human T cell activation in contrast to murine T cells. Keywords: Adaptor protein Nck, Nck1, Nck2, shRNA, Jurkat T cells, T cell receptor/TCR activation Background signalling pathways. Stimulation of these pathways results T cells play an important role in adaptive immunity. They in the activation of the transcription factors activator express a structurally unique receptor called the T cell protein-1 (AP-1), nuclear factor kappa-light-chain-enhan- antigen receptor (TCR). The TCR is composed of the vari- cer of activated B cells (NF-κB) and nuclear factor of acti- able antigen-binding TCRαβ heterodimer noncovalently vated T cells (NFAT), respectively [3]. The cooperative associated with the non-variable CD3εγ,CD3εδ and ζζ activity of these transcription factors is required for the signal transduction subunits [1]. Binding of the TCR regulation of interleukin-2 (IL-2) production [4]. to peptide-loaded major histocompatibility complexes Two highly conserved members of the cytosolic adaptor (pMHCs) presented on the surface of antigen presenting protein Nck (non-catalytic region of tyrosine kinase) are cells (APCs) initiates the formation of intracellular protein found in humans: Nck1/Nckα and Nck2/Nckβ (also signalling complexes [2]. The formation of these com- known as Grb4) [5,6]. These two proteins have a 68% plexes is required for T cell activation and is driven by similarity when the amino acid sequences are compared adaptor proteins in order to activate downstream path- [7]. Nck consists of three consecutive Src homology 3 ways, such as the Ras-mitogen-activated protein kinase (SH3) domains at N-terminus followed by a SH2 domain (MAPK), protein kinase C (PKC) and Ca2+-mediated at the C-terminus [5]. The SH2 domain of Nck binds to phosphotyrosine-containing proteins, and the SH3 do- * Correspondence: [email protected] mains bind to proline-rich sequences (PRS) within target 5Centre of Excellence in Medical Biotechnology (CEMB), Faculty of Medical proteins [7,8]. In T cells, the SH2 domain of Nck mediates Science, Naresuan University, Phitsanulok 65000, Thailand the interaction with phosphorylated SLP-76 (SH2 6Department of Medicine, Faculty of Medicine, Naresuan University, Phitsanulok 65000, Thailand domain-containing leukocyte protein of 76 kDa), while Full list of author information is available at the end of the article each of the SH3 domains interacts with distinct target © 2014 Ngoenkam et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ngoenkam et al. Cell Communication and Signaling 2014, 12:21 Page 2 of 13 http://www.biosignaling.com/content/12/1/21 proteins. For instance, SH3.1 of Nck binds directly to a acetate (PMA). Nck1-knockdown Jurkat T cells exhi- PRS in the cytoplasmic tail of CD3ε exposed upon bited decreased CD69 expression in response to CD3 antigen-induced conformational changes within the TCR stimulation (Figure 1C). In contrast, silencing of Nck2 complex. This interaction is required for IL-2 production in Jurkat T cells had no effect on CD69 expression. As a and synapse formation [9]. The SH3.2 domain of Nck in- control, a similar level of CD69 expression was observed teracts with the kinase Pak1, which is essential for regula- in Nck1- and Nck2-knockdown cells in response to tion of the reorganization of actin cytoskeleton [7,10,11] PHA plus PMA (Figure 1D). and for activation of NFAT [12]. The SH3.3 domain of Another important marker for indicating T cell activa- Nck binds to WASp to control actin polymerization [13] tion is IL-2. Therefore, the production of IL-2 was inves- and with the Son of Sevenless (SOS) [10,14]. The guanine tigated in Nck1- and Nck2-knockdown cells. Cells were nucleotide exchange factor SOS is required for activation stimulated with anti-CD3ε antibodies (OKT3) or TCR- of the Ras-Raf-MEK-Erk pathway [15]. independent stimuli (PHA/PMA). Consistent with our Double Nck1 and Nck2-knockout mice die in early previous report [17], gene-silencing of Nck1 resulted in a embryonic stage, whereas singly Nck1 or Nck2 deleted significant decrease in IL-2 production (Figure 1E). In mice do not possess any phenotype, suggesting a re- contrast, IL-2 production was normal in Jurkat cells trans- dundant function of these two molecules in mice [16]. fected with shRNA specific for Nck2 . As expected, no Recently, we have downregulated Nck1 expression in difference in IL-2 production was observed between human Jurkat T cells and primary human CD4+ T cells Nck1- and Nck2-knockdown cells upon stimulation with using siRNA, showing that Nck1 expression is required PHA/PMA (Figure 1F). Taken together, these results for TCR-induced phosphorylation of Erk1/2, IL-2 pro- showed that in contrast to Nck2, Nck1 was required for duction and CD69 expression [17]. These results suggest TCR-mediated T cell activation in Jurkat T cells. species specific differences in the role of Nck1 and Nck2. The aim of the present study was to compare the Nck1 regulated the activation of Ras-MAPK signalling roles of Nck1 and Nck2 in TCR-mediated T cell re- pathway sponses in the human system. Our results demonstrate Following engagement of TCR, the small GTPases Ras that Nck1 and Nck2 have non redundant roles in human and Rac are activated, and subsequently activate the T cell activation. Thus, Nck1 plays a more dominant role MAPK signalling cascade. There are three groups of in TCR-mediated T cell activation in the human system enzymes in the MAPK signalling cascade that are sequen- than Nck2. tially activated: MAP kinase kinase kinase (MKKK, MEKK or MAP3K), MAP kinase kinase (MKK, MEK, or MAP2K) Results and MAP kinase (MAPK). Three MAPKs are expressed in Generation of Nck1- and Nck2-knockdown Jurkat T cells T cells: Erk1/2, JNK and p38 [3]. In this present study, the functions of Nck1 and Nck2 Our previous study has shown that reduced Nck1 ex- in activation of Jurkat T-cell leukemic line E6-1 were pression is associated with impaired Erk1/2 phospho- compared. To knockdown either Nck1 or Nck2, Jurkat rylation [17]. Nck1 and Nck2 may be differently required T cells were transfected with a plasmid encoding either for MAPK signalling cascade in Jurkat cells. To test this Nck1- or Nck2-specific short hairpin RNA (shRNA) or notion, the activation of two members of MAPK pathway, an empty control plasmid. For generating the stable Erk1/2 and p38 was investigated using Jurkat T cells with Nck1- or Nck2-knockdown Jurkat T cell lines, the trans- silenced expression of Nck1 or Nck2. Reduced Erk1/2 fected cells were selected in medium containing puro- phosphorylation was found in Nck1-, but not in Nck2- mycin. The resulting bulk population was subcloned by knockdown cells (Figure 2A). To confirm that Nck1 is limiting dilution. Individual clones were screened by required for activation of the MAPK pathway via the analyzing Nck1, Nck2 and TCR expression. The selected Ras-Raf-MEK-Erk cascade, we investigated the activation clones had about 90% downregulation of endogenous of the upstream enzyme essential for Erk1/2 phosphory- Nck1 and Nck2 (Figure 1A) and a similar level of CD3 lation: MEK1/2. As expected, cells treated with shRNA expression as control cells (Figure 1B). Here, we chose specific for Nck1 exhibited impaired MEK1/2 phosphory- one clone each that will be named shNck1 and shNck2. lation (Figure 2B). Interestingly, in contrast to Erk1/2 and MEK1/2 phosphorylation, knockdown of Nck1 or Nck2 in Nck1 is required for TCR-mediated T cell activation Jurkat T cells had no discernible effects on p38 phospho- Next, we tested whether the downmodulation of Nck1 or rylation (Figure 2C). Because all these results were ob- Nck2 had an impact on TCR-induced expression of the tained from single clones of Nck1- and Nck2-knockdown early activation marker CD69.
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