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, CD3e, 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.

SDF1α‐induced interaction of the adapter proteins Nck and HS1 facilitates actin polymerization and migration in T cells

“…We previously identified HS1 as a potential Nck-interacting protein [33]. In a first series of experiments we verified this interaction and show that both Nck1 and Nck2 associate with HS1.…”

“…We used different Nck1-based GST fusion proteins to precipitate putative interactors from cellular lysates and identified 141 bands representing 22 individual proteins. Among others, we found the adapter protein HS1 as a putative interactor [33].In order to verify the previous findings, we extended our initial pull-down experiments using GST fusion proteins of full-length Nck1 (Nck1-GST), the combined SH3 domains (Nck1 SH3.1-3-GST), the Nck1 SH2 domain (Nck1 SH2-GST), and GST as a control (Fig. 1A).…”

“…We used different Nck1-based GST fusion proteins to precipitate putative interactors from cellular lysates and identified 141 bands representing 22 individual proteins. Among others, we found the adapter protein HS1 as a putative interactor [33].…”

“…In an approach to characterize the interactome of Nck in human T cells, we previously performed a systematic screen for interaction partners in T cells [33]. We used different Nck1-based GST fusion proteins to precipitate putative interactors from cellular lysates and identified 141 bands representing 22 individual proteins.…”

Relevance of Nck–CD3ε Interaction for T Cell Activation In Vivo

“…Nck is an src homology 2 (SH2)/SH3 adaptor protein that plays a universal role in coordinating the signaling networks critical for organizing the actin cytoskeleton, cell movement or axon guidance, connecting transmembrane receptors to multiple intracellular signaling pathways (7,8). Nck effectors include proteins that have a pivotal role in the nucleation and polymerization of the actin cytoskeleton such as the SCAR/WAVE proteins and the serine/ threonine kinase Pak1.…”