Figure 5

The C-terminal SH3 domain of Nck1 is necessary for an efficient Erk1/2 activation. A) Schematic presentation of Nck1 SH3.1 and SH3.3 mutant constructs. A nucleotide fragment encoding the FLAG peptide was linked in frame to the N-terminal of the Nck1 gene. N-terminal SH3 (SH3.1) and C-terminal SH3 (SH3.3) domains of Nck1 were mutated by changing tryptophan (W) residue at 38 and 229 to lysine (K), respectively. B) The reconstitution of WT Nck1, Nck1 W38K and Nck1 W229K in Nck1-knockdown cells were analyzed by immunoblotting with anti-Nck1 antibody. C-D) Nck1-knockdown cells stably expressing WT Nck1, Nck1 W38K and Nck1 W229K mutants were stimulated with plate pre-coated with 1 μg/ml anti-CD3 antibodies or 6 μg/ml PHA plus 1 ng/ml PMA for 24 h. Each cell population was stained with anti-CD69 conjugated phycoerythrin (PE) and isotype control antibody and then analysed by flow cytometry. Numbers in CD69 histogram indicate frequency of positive cells. Grey shaded histrogram and grey letter are cells transfected with empty plasmid (Mock), black bold solid line and black letter are Nck1-knockdown cells or Nck1-knockdown cells reconstituted with indicated construct plasmids, and black dotted line is isotype control staining. Data are representative of two independent experiments. E) Nck1-knockdown cells reconstituted as describe in C were left untreated or treated with soluble CD3 antibody (1 μg/ml) for 3 min. Lysates were immunoblotted with anti-phospho-Erk1/2 (Thr202/Tyr204, Thr185/Tyr187) antibody and anti-Erk1/2 antibody. Below, the quantified signal intensity of the pErk1/2 was normalized to its total kinase and this value was relative to that in the unstimulated control cells (Mock), set as 1 (black dashed line) and plotted in bar graph. Data are representative of two independent experiments.