Functional Cooperation between the Proteins Nck and ADAP Is Fundamental for Actin Reorganization

Abstract

T cell antigen receptor (TCR) activation triggers profound changes in the actin cytoskeleton. In addition to controlling cellular shape and polarity, this process regulates vital T cell responses, such as T cell adhesion, motility, and proliferation. These depend on the recruitment of the signaling proteins Nck and Wiskott-Aldrich syndrome protein (WASp) to the site of TCR activation and on the functional properties of the adapter proteins linker for activation of T cells (LAT) and SH2-domain-containing leukocyte protein of 76 kDa (SLP76). We now demonstrate that Nck is necessary but insufficient for the recruitment of WASp. We show that two pathways lead to SLP76-dependent actin rearrangement. One requires the SLP76 acidic domain, crucial to association with the Nck SH2 domain, and another requires the SLP76 SH2 domain, essential for interaction with the adhesion-and degranulation-promoting adapter protein ADAP. Functional cooperation between Nck and ADAP mediates SLP76-WASp interactions and actin rearrangement. We also reveal the molecular mechanism linking ADAP to actin reorganization.T cell activation triggers multiple molecular events, including the activation of protein tyrosine kinases (PTKs), formation of multiprotein signaling complexes, and activation of enzymes and transcription factors (25,37,47). Cytoskeletal actin reorganization is also dependent on these events initiated at the T cell-antigen-presenting cell (APC) interface, the immunological synapse (IS). Interference with actin dynamics results in an impaired immune response and can induce T cell anergy (40).We and others (2,4,8,11,12,16) have demonstrated complex molecular events linking T cell antigen receptor (TCR) activation to actin rearrangement. One major pathway, mediated by the activation of multiple PTKs, leads to phosphorylation of the adapter molecules linker for activation of T cells (LAT) and SH2-domain-containing leukocyte protein of 76 kDa (SLP76). Phosphorylation of SLP76 leads to recruitment of the Nck adapter molecule, which is associated with key regulators of the actin cytoskeleton Wiskott-Aldrich syndrome protein (WASp) and WAVE2.The molecular structure of SLP76 consists of an N-terminal sterile-alpha motif (SAM) (41), an acidic domain containing tyrosine residues subject to phosphorylation, a central prolinerich region, and a C-terminal SH2 domain. Phosphorylation of the tyrosines allows the interaction of SLP76 with the adapter Nck, the Rho-family GEF, VAV, and Itk, all via their SH2 domains (5,8,48,49,51). The interactions of SLP76, Nck, and VAV are essential for the activation of WASp and its recruitment to the IS (51). TCR engagement also induces the association of the SLP76 SH2 domain with the adhesion-and degranulation-promoting adapter protein (ADAP) and to the serine-threonine kinase hematopoietic progenitor kinase 1 (HPK-1) (38). In addition to SLP76, ADAP is capable of binding other proteins, and it is recruited to the IS (26, 35). The role of ADAP in integrin function has been explored (10,23,36,46); however, it...

Analysis of Phosphorylation-dependent Protein Interactions of Adhesion and Degranulation Promoting Adaptor Protein (ADAP) Reveals Novel Interaction Partners Required for Chemokine-directed T cell Migration

“…Under migratory conditions it is unlikely that such co-segregation of receptors is sustained; one possibility is that ADAP recruitment of ZAP70 prevents the pre-emptive binding of the kinase to small amounts of phosphorylated TCR. Moreover, because ADAP is directly linked to cytoskeletal dynamics (20), it is conceivable that Y571 phosphorylation is required for linking its scaffolding function at the membrane with actin regulatory events that are critical for maintaining the motility but not the adhesive mode of migrating T cells.…”

“…Moreover, ADAP is strongly tyrosine-phosphorylated upon TCR stimulation and thereby serves as a hub for SH2 domain-containing proteins such as SLP76, FYN and NCK (Fig. 1A) (17)(18)(19)(20)(21). Beside these well characterized interactions, several other SH2-domain containing binders were identified by pull-down approaches using phosphorylated peptide baits (Fig.…”

RNAi down-regulation of Nck1 adaptor protein in Jurkat T cells

“…RNAi-based gene silencing is commonly used to study gene function in T cell responses 4,[16][17][18][19] . Here we compared three different RNAi strategies, including synthetic siRNA, plasmid-and lentivirusmediated shRNA production in silencing of the Nck1 gene and their outcomes after cell activation.…”

ARAP, a Novel Adaptor Protein, Is Required for TCR Signaling and Integrin-Mediated Adhesion

“…The absence of these domain sequences may explain the differential regulation of F-actin organization between ADAP and ARAP. ADAP regulates TCR-mediated F-actin organization by mediating interactions between SLP-76 and WiskottAldrich syndrome protein together with Nck adaptor protein (41). Actin polymerization and Rac1 activation (the small GTPase that regulates actin polymerization) were unaffected in ARAPsuppressed T cells after TCR stimulation (Fig.…”