Essential role of the adaptor protein Nck1 in Jurkat T cell activation and function

Abstract

The non-catalytic region of tyrosine kinase (Nck) is proposed to play an essential role in T cell activation. However, evidence based on functional and biochemical studies has brought into question the critical function of Nck. Therefore, the aim of the present work was to investigate the role of Nck in T cell activation. To study this, the human Jurkat T cell line was used as a model for human T lymphocytes. The short interfering (si) RNA targeting Nck1 gene was used with electroporation to knock-down Nck1 protein expression in Jurkat T cells. Primary human CD4 T cells were also transfected with the siRNA of Nck1. The results showed that decreased Nck1 protein expression did not affect the apoptosis of the transfected Jurkat T cells compared with control siRNA-transfected cells and non-transfected cells. Upon CD3ε/CD28 stimulation, knock-down of Nck1 in Jurkat T cells caused a decrease in CD69 expression and in interleukin (IL)-2 secretion. Similarly, knock-down of Nck1 in primary CD4 T cells also caused decreased CD69 expression. However, no significant alterations of CD69 and IL-2 expression were found upon phytohaemagglutinin (PHA)/phorbol myristate acetate (PMA) stimulation. Knock-down of Nck1 had no effect on the proliferation of Jurkat T cells stimulated with either PHA or anti-T cell receptor (TCR) monoclonal antibody (C305). The reduced Nck1 expression in Jurkat cells was also associated with a reduced phosphorylation of extracellular regulated kinase (Erk)1 and Erk2 proteins upon CD3ε/CD28 stimulation. In conclusion, the decreased Nck1 protein in Jurkat T cells resulted in an impairment of TCR-CD3-mediated activation involving a defective Erk phosphorylation pathway.

Fig. 1

Expression of Nck1 protein was reduced after Nck1-specific short interfering (si)RNA transfection. E6-1 Jurkat T cells (a) and primary CD4 T cells (b) were transfected with Nck1-specific or negative control siRNA for 48 h. Cell extracts were subjected to Western blot analysis with anti-Nck1 monoclonal antibody (mAb) and anti-β-actin mAb. (a) Lane 1, blank control; lane 2, transfection with 100 pmol Nck1 siRNA; lane 3, transfection with 50 pmol Nck1 siRNA; lane 4, transfection with 100 pmol non-specific siRNA negative control; lane 5, transfection with 50 pmol non-specific siRNA negative control. (b) Lane 1, untransfected CD4 T cells; lane 2, negative control siRNA; lane 3, si RNA Nck1.

Fig. 2

Nck1 knock-down did not induce apoptosis in Jurkat T cells. E6-1 Jurkat T cells were transfected with Nck1-specific short interfering (si)RNA (a), untransfected (b) or transfected with non-specific siRNA negative control (c). Jurkat T cells were also stimulated with anti-T cell receptor (TCR) monoclonal antibody (mAb) and there was no difference in the % apoptosis of T cell receptor (TCR)-stimulated Jurkat cells transfected with Nck1-specific siRNA (d), untransfected (e) or transfected with non-specific siRNA negative control (f). The lower left zone showed the viable cells (annexinV^-/PI^-), the upper left zone represented necrosis cells (annexinV^-/PI⁺), the lower right zone showed early apoptosis (annexinV⁺/PI^-) and the upper right zone showed the late apoptosis (annexinV⁺/PI⁺). The combination of the upper and lower left zones represented the total apoptotic cells. Shown are mean percentage ± standard deviation of apoptotic cells from three experiments.

Fig. 3

Nck1 was not required for PHA- and IL-2-induced Jurkat T cell proliferation. Nck1 short interfering (si)RNA-transfected or negative control siRNA-transfected Jurkat T cells were unstimulated (□), stimulated with phytohaemagglutinin (PHA) (), anti-T cell receptor (TCR) antibody () or interleukin (IL)-2 () for 24 h and proliferation measured using 5-bromo-2′-deoxyuridine (BrdU). Values are presented as mean ± standard deviation from three experiments. OD: optical density read at 450 nm.

Fig. 4

Nck1-regulated expression of CD69 in T cell receptor (TCR)–CD3-mediated activation. Jurkat T cells and primary human CD4 T cells were stimulated with anti-CD3ε/anti-CD28 antibodies (a,c) or phytohaemagglutinin (PHA)/phorbol myristate acetate (PMA) (b,d) for 24 h. Histograms show CD69 expression of untransfected Jurkat T cells and CD4 T cells. Dotted line, unstimulated; bold solid line, negative control short interfering (si)RNA-transfected; grey histogram, Nck1 siRNA-transfected. Each cell population was stained with anti-CD69 phycoerythrin (PE) and isotype control antibodies (solid line) and was analysed by flow cytometry for CD69 expression.

Fig. 5

CD3 expression of Nck1 knock-down Jurkat cells. Jurkat T cells were transfected with Nck1 short interfering (si)RNA or negative control siRNA.

Fig. 6

Nck1 was required for interleukin (IL)-2 production by Jurkat T cells. Negative control short interfering (si)RNA-transfected (□) or Nck1 siRNA-transfected () Jurkat T cells were stimulated with anti-CD3ε/anti-CD28 antibodies (a) or phytohaemagglutinin (PHA)/phorbol myristate acetate (PMA) (b) for 24 h. Results represent mean ± standard deviation of three experiments. *P < 0·05.

Fig. 7

Extracellular regulated kinase (ERK) phosphorylation was decreased in Nck1 knock-down Jurkat cells. Jurkat T cells were treated with specific Nck1 short interfering (si)RNA (Nck1) or control siRNA (Ctl). After 48 h of cultivation, cells were subsequently stimulated with 10 µg/ml anti-CD3ε monoclonal antibody (mAb) plus 10 µg/ml anti-CD28 mAb or with 6 µg/ml phytohaemagglutinin (PHA) plus 1 ng/ml phorbol myristate acetate (PMA) or none (−) for 10 min at 37°C. Cytoplasmic proteins were immunoblotted with anti-phospho-Erk1/2 (Thr202/Tyr204, Thr185/Tyr187) antibody.