The adapter protein Nck: role of individual SH3 and SH2 binding modules for protein interactions in T lymphocytes

Abstract

Nck is a ubiquitously expressed, primarily cytosolic adapter protein consisting of one SH2 domain and three SH3 domains. It links receptor and nonreceptor tyrosine kinases to actin cytoskeleton reorganizing proteins. In T lymphocytes, Nck is a crucial component of signaling pathways for T cell activation and effector function. It recruits actin remodeling proteins to T cell receptor (TCR)-associated activation clusters and thereby initiates changes in cell polarity and morphology. Moreover, Nck is crucial for the TCR-induced mobilization of secretory vesicles to the cytotoxic immunological synapse. To identify the interactome of Nck in human T cells, we performed a systematic screen for interaction partners in untreated or pervanadate-treated cells. We used GST fusion proteins containing full length Nck, the combined SH3 domains or the individual SH3 and SH2 domains to precipitate putative Nck interactors from cellular lysates. Protein bands were excised from gels, processed by tryptic in-gel digestion and analyzed by mass spectrometry. Using this approach, we confirmed previously established interactions (e.g., with Slp76, CD3 epsilon, WASP, and WIPF1) and identified several novel putative Nck-binding proteins. We subsequently verified the SH2 domain binding to the actin-binding protein HIP55 and to FYB/ADAP, and the SH3-mediated binding to the nuclear proteins SFPQ/NONO. Using laser scanning microscopy, we provide new evidence for a nuclear localization of Nck in human T cells. Our data highlight the fundamental role of Nck in the TCR-to-cytoskeleton crosstalk and point to yet unknown nuclear functions of Nck also in T lymphocytes.

Figures

Figure 1

Pull-down of Nck-interacting proteins. Precipitation experiments with GST fusion proteins containing full length Nck1 (FL), the SH2 domain (SH2), all three SH3 domains (SH3.1-3), the individual SH3 domains (SH3.1, SH3.2, SH3.3) and GST as control. The respective fusion proteins were used to precipitate putative Nck-associated proteins from human T cell blasts with or without pervanadate-treatment (A) or from Jurkat T cells JFL (B). After separation by SDS-PAGE and protein visualization, bands that did not appear in controls were excised and subjected to MALDI-TOF analysis. The Flamingo Pink-stained gel (A, left gel) displays a master gel combining the results of eight independent experiments using Coomassie staining, conventional silver staining and Flamingo Pink staining. The Flamingo Pink-stained gel (A, right gel) displays a master gel combining the results of two independent experiments also using Coomassie staining. The silver-stained gel (B) displays a master gel combining the results of six independent experiments using Coomassie staining, conventional silver staining and staining with Flamingo Pink or Sypro Ruby. Numbers correspond to those provided in Tables I and II.

Figure 2

Verification of the Nck/HIP55 interaction. (A) Precipitation with GST fusion proteins containing full length Nck1 (FL), all three SH3 domains (SH3.1-3), the SH2 domain (SH2) and GST as control from lysates of untreated and pervanadate-treated T cell blasts. Proteins were transferred to nitrocellulose followed by an anti-HIP55 immunoblot. (B) After immunoprecipitation (IP) of HIP55 from lysates of untreated and pervanadate-treated T cell blasts (using IgG1 as control), the proteins were transferred to nitrocellulose followed by incubation with the Nck SH2 domain and GST as control, an anti-GST mAb and HRP-conjugated secondary antibodies in a Far Western blot (FWB). (C) Myc-tagged Nck variants were transiently co-expressed with EGFP-tagged HIP55 (murine) in 293T transfectants. 18–24 h after transfection, cells were treated with pervanadate for 2 min. and lysed. Lysates were subjected to immunoprecipitation with anti-myc mAb 9B11 and separated by SDS-PAGE. Proteins were transferred to nitrocellulose followed by Western blot with anti-GFP pAb A11122 to visualize coprecipitated proteins (upper left panel). The blots were stripped and precipitated Nck was detected by Western blot using anti-Nck pAb C-19 (lower left panel). To monitor expression of EGFP-tagged HIP55 25 μg of the cellular lysates used for immunoprecipitation were subjected to SDS-PAGE and transferred to nitrocellulose followed by Western blot with anti-GFP pAb A11122 (right panel). (D) Subcellular distribution of Nck and HIP55 in cloned T cells that were either left untreated or exposed to superantigen-stimulated B-LCL (*) for 30 min. before fixation and staining with anti-HIP55 mAb and AlexaFluor488-conjugated anti-mouse antibody and anti-Nck pAb sc-19 and AlexaFluor555-conjugated anti-rabbit antibody. ProLong gold antifade mounting medium contained DAPI to visualize nuclei. Transmitted light was recorded to visualize cell shapes. Scale bars represent 10 μm.

Figure 3

Verification of the Nck/FYB interaction. (A) Precipitation with GST fusion proteins containing full length Nck1 (FL), all three SH3 domains (SH3.1-3), the SH2 domain (SH2), and GST as control from lysates of untreated and pervanadate-treated T cell blasts. Proteins were transferred to nitrocellulose followed by an anti-FYB immunoblot. (B) FYB was immunoprecipitated (IP) from lysates of untreated and pervanadate-treated T cell blasts (using IgG1 as control). Subsequently, the proteins were transferred to nitrocellulose followed by incubation with the Nck SH2 domain and GST as control, an anti-GST mAb and HRP-conjugated secondary antibodies in a Far Western blot. (C) Myc-tagged Nck variants were transiently co-expressed with FYB at different ratios in 293T transfectants. 18–24 h after transfection, cells were treated with pervanadate for 2 min. and lysed. Lysates were split into two aliquots, subjected to parallel immunoprecipitation with anti-Nck mAb 280C10 and anti-FYB mAb 460114 and separated by SDS-PAGE. As a control, 25 μg of cellular lysate were also subjected to SDS-PAGE. Proteins were transferred to nitrocellulose followed by Western blot with with anti-Nck pAb C-19 and anti-FYB mAb 460114 to visualize coprecipitated proteins. To monitor protein expression in transfectants, the blots were stripped and precipitated proteins were detected by Western blot as indicated. (D) Subcellular distribution of Nck and FYB in cloned T cells that were either left untreated or exposed to superantigen-stimulated B-LCL (*) for 30 min. before fixation and staining with anti-FYB mAb and AlexaFluor488-conjugated anti-mouse antibody and anti-Nck pAb sc-19 and AlexaFluor555-conjugated anti-rabbit antibody. ProLong gold antifade mounting medium contained DAPI to visualize nuclei. Transmitted light was recorded to visualize cell shapes. Scale bars represent 10 μm.

Figure 4

Verification of the Nck/SFPQ interaction. (A) After precipitation with GST fusion proteins containing full length Nck1 (FL), the SH2 domain (SH2), all three SH3 domains (SH3.1-3) and GST as a control from lysates of Jurkat T cells the proteins were transferred to nitrocellulose followed by an anti-SFPQ immunoblot. (B) SFPQ was immunoprecipitated (IP) from lysates of Jurkat T cells (using IgG1 as control) and the proteins were transferred to nitrocellulose followed by incubation with the Nck SH3.2 domain, the Nck SH3.3 domain, and GST as control, an anti-GST mAb and HRP-conjugated secondary antibodies in a Far Western blot (FWB). (C) Subcellular distribution of Nck, SFPQ (middle panel) and NONO (lower panel) in cloned T cells. Cells were fixed and stained with anti-SFPQ mAb or anti-NONO mAb and AlexaFluor488-conjugated anti-mouse antibody and anti-Nck pAb sc-19 and AlexaFluor555-conjugated anti-rabbit antibody. ProLong gold antifade mounting medium contained DAPI to visualize nuclei. Scale bars represent 10 μm.