Already a dozen molecules share binding to the Src homology (SH) 3 domains of human Nck, an SH3-SH3-SH3-SH2 adapter protein. It was reasoned there may be multiple gene members of Nck to accommodate the large binding repertoires. Identification of novel human and mouse Nck genes are reported and they have been renamed as the Nckalpha and Nckbeta genes (including the human Nckalpha, human Nckbeta, mouse Nckalpha, and mouse Nckbeta genes). Nckalpha and Nckbeta share 68% amino acid identity, whereas the two Nckalpha and two Nckbeta across the species show 96% identity to each other. The human Nckbeta gene is mapped to 2q12, whereas the human Nckalpha gene has previously been mapped at 3q21. Antibodies specifically against Nckalpha and Nckbeta detect Nckalpha and Nckbeta with an identical molecular mass in the same cells of various origins. Ectopically expressed Nckbeta, but not its SH2 domain mutant, strongly inhibits epidermal growth factor- and platelet-derived growth factor-stimulated DNA synthesis. Consistently, epidermal growth factor receptor and platelet-derived growth factor receptor preferentially interact with Nckbeta over Nckalpha in vitro. This study indicates that Nck proteins constitute a multiple gene family and that each gene may have its own signaling specificity. Because previous anti-Nck (human Nckalpha) antibodies cross-react with Nckbeta, reassessment of those studies with specific Nck genes would be necessary (Chen, 1998).

Adapter proteins made up of Src homology (SH) domains mediate multiple cellular signaling events initiated by receptor protein tyrosine kinases. Grb4 is an adapter protein closely related to but distinct from Nck, that is made up of three SH3 domains and one SH2 domain. Northern analysis indicated that both genes are expressed in multiple tissues. Both Nck and Grb4 proteins can associate with receptor tyrosine kinases and the SH3-binding proteins PAK, Sos1, and PRK2, and both Nck and Grb4 synergize with v-Abl and Sos to induce gene expression via the transcription factor Elk-1. Although neither protein is transforming on its own, both Nck and Grb4 cooperate with v-Abl to transform NIH 3T3 cells and influence the morphology and anchorage-dependent growth of wild type Ras-transformed cells. Nck and Grb4 therefore appear to be functionally redundant (Braverman, 1999).

The NCK adaptor proteins are composed entirely of SH3 and SH2 domains and serve as protein interaction bridges for several receptors during signal transduction events. This study reports the molecular and genetic analysis of the C. elegans nck-1 gene. C. elegans nck-1 encodes two isoforms: NCK-1A and a shorter isoform that lacks the first SH3 domain, NCK-1B. C. elegans nck-1 mutants exhibit defects in axon guidance and neuronal cell position, as well as defects in the excretory canal cell, gonad, and male mating. NCK-1 is broadly expressed in neurons and epithelial cells with NCK-1B being the most abundant isoform. NCK-1A and NCK-1B share a similar expression pattern in parts of the nervous system, but also have independent expression patterns in other tissues. Interestingly, NCK-1B is localized to the nuclei of many cells. Genetic rescue experiments show that NCK-1 functions cell autonomously and, in general, either NCK-1A or NCK-1B is sufficient to function in axon guidance. However, there appears to be specific roles for each isoform, for example NCK-1B is required for HSN cell migration while NCK-1A is required for efficient male mating. Genetic epistasis experiments show that NCK-1 functions redundantly with the LAR Receptor Tyrosine Phosphatase, PTP-3, and the Netrin receptor UNC-40 (Mohamed, 2011).

Nck is constitutively phosphorylated on serine in resting NIH 3T3 cells. Platelet-derived growth factor (PDGF) treatment leads to increased Nck phosphorylation on both tyrosine and serine. Nck is also phosphorylated on tyrosine in epidermal growth factor (EGF)-treated A431 cells and in v-Src-transformed NIH 3T3 cells. Multiple sites of serine phosphorylation are detected in Nck from resting cells, and no novel sites were found upon PDGF or EGF treatment. A single major tyrosine phosphorylation site is found in Nck in both PDGF- and EGF-treated cells and in v-Src-transformed cells. This same tyrosine is phosphorylated in vitro by purified PDGF and EGF receptors and also by pp60c-src. The phosphorylation of Nck and PLC-gamma 1 was compared in several cell lines transformed by oncogenes with different modes of transformation. Although PLC-gamma 1 and Nck have significant amino acid identity, particularly in their SH3 regions, and both associate with growth factor receptors in a ligand-dependent manner, they were not always phosphorylated on tyrosine in a coincident manner (Meisenhelder, 1992).

Nck is widely distributed in rat tissues, with an especially high level of expression in testes. The expression levels of Nck remain unchanged during the development of rat brain. Stimulation of A431 cells with epidermal growth factor elicits the tight association of Nck with the epidermal growth factor receptor and phosphorylation of Nck on both serine and tyrosine residues. The phosphorylation of Nck is also enhanced in response to stimulation of the nerve growth factor receptor in PC12 cells, the T-cell receptor complex in Jurkat cells, the membrane immunoglobulin M in Daudi cells, and the low-affinity immunoglobulin G receptor (Fc gamma RII) in U937 cells. The phosphorylation of Nck was also enhanced following treatment of A431 cells with phorbol 12-myristate 13-acetate or forskolin. These results suggest that Nck is a target for a variety of protein kinases that might modulate the postulated role of Nck as an adaptor for the physical and functional coordination of signaling proteins (Park, 1992).

Hepatocyte growth factor (HGF), a mesenchyme derived growth factor, promotes cell growth, cell motility, and morphogenesis in a variety of epithelial cells. The diverse responses are transduced across the cell membrane by the met/HGF receptor, a product of c-met protooncogene. The met/HGF receptor recruits a variety of second messenger molecules which relay the diverse intracellular responses of HGF. HGF autophosphorylates and activates met/HGF receptor. The activated met/HGF receptor then physically associates with and activates phospholipase C-gamma (PLC-gamma). Furthermore, upon ligand stimulation, tyrosine-autophosphorylated met/HGF receptor also activates Nck oncogene product. Taken together, these results suggest that the receptor activation leads to formation of a complex in which PLC-gamma and Nck oncogene product co-exist with the activated met/HGF receptor, and that the Nck oncogene product is an important component of HGF signaling in Calu-1 and A549 cells (Kochhar, 1996).

Eph family receptor tyrosine kinases signal axonal guidance, neuronal bundling, and angiogenesis; yet the signaling systems that couple these receptors to targeting and cell-cell assembly responses are incompletely defined. Functional links to regulators of cytoskeletal structure are anticipated based on receptor mediated cell-cell aggregation and migratory responses. Two-hybrid interaction cloning was used to identify EphB1-interactive proteins. Six independent cDNAs encoding the SH2 domain of the adapter protein, Nck, were recovered in a screen of a murine embryonic library. The EphB1 subdomain that binds Nck and its Drosophila homolog, DOCK, map to the juxtamembrane region. Within this subdomain, Tyr594 is required for Nck binding. In P19 embryonal carcinoma cells, activation of EphB1 (ELK) by its ligand, ephrin-B1/Fc, recruites Nck to native receptor complexes and activates c-Jun kinase (JNK/SAPK). Transient overexpression of mutant EphB1 receptors (Y594F) blocks Nck recruitment to EphB1, attenuates downstream JNK activation, and blocks cell attachment responses. These findings identify Nck as an important intermediary linking EphB1 signaling to JNK (Stein, 1998).

Signaling proteins such as phospholipase C-gamma (PLC-gamma) or GTPase-activating protein (GAP) of ras contain conserved regions of approximately 100 amino acids termed src homology 2 (SH2) domains. SH2 domains have been shown to be responsible for mediating association between signaling proteins and tyrosine-phosphorylated proteins, including growth factor receptors. Nck is an ubiquitously expressed protein consisting exclusively of one SH2 and three SH3 domains. Epidermal growth factor or platelet-derived growth factor stimulation of intact human or murine cells leads to phosphorylation of Nck protein on tyrosine, serine, and threonine residues. Similar stimulation of Nck phosphorylation is detected upon activation of rat basophilic leukemia RBL-2H3 cells by cross-linking of the high-affinity immunoglobulin E receptors (Fc epsilon RI). Ligand-activated, tyrosine-autophosphorylated platelet-derived growth factor or epidermal growth factor receptors are coimmunoprecipitated with anti-Nck antibodies, and the association with either receptor molecule is mediated by the SH2 domain of Nck. Addition of phorbol ester is also able to stimulate Nck phosphorylation on serine residues. However, growth factor-induced serine/threonine phosphorylation of Nck is not mediated by protein kinase C. Interestingly, approximately fivefold overexpression of Nck in NIH 3T3 cells results in formation of oncogenic foci. These results show that Nck is an oncogenic protein and a common target for the action of different surface receptors. Nck probably functions as an adaptor protein that links surface receptors with tyrosine kinase activity to downstream signaling pathways involved in the control of cell proliferation (Li, 1992).

Overexpression of Nck caused cell transformation in vitro and tumor formation in the nude mice. The mechanism of this action by Nck, however, remains unclear. Rat adrenal pheochromocytoma cell line PC12 provides a useful system for studying growth factor-regulated cell proliferation and differentiation. Serum and epidermal growth factor (EGF) stimulate proliferation, whereas nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) cause growth arrest and sympathetic neurite outgrowth in these cells. To study the function of Nck, stable clones of PC12 cells were generated overexpressing the human Nck. The overexpressed Nck causes continued proliferation of PC12 cells even in the presence of NGF and blocks both the NGF- and bFGF-induced neurite outgrowth. Anti-sense but not sense oligonucleotides to the human Nck resumes the NGF-induced differentiation, indicating the specific inhibitory effect of Nck. Interestingly, Nck does not interfere with the kinetics of NGF- and EGF-stimulated protein tyrosine phosphorylation and the mitogen-activated protein kinase (MAPK) activation, suggesting that Nck inhibits the induced PC12 cell differentiation via a MAPK-independent mechanism. This study has provided a useful system for further understanding the function of Nck (Rockow, 1996).

The SH3-SH3-SH3-SH2 adapter protein Nck links receptor tyrosine kinases, such as EGF and PDGF receptors, to downstream signaling pathways: among those pathways are p21cdc42/rac-activated kinase cascade; Sos-activated Ras signaling and the human Wiskott-Aldrich Syndrome protein (WASp)-mediated actin cytoskeleton changes. In EGF stimulated cells, Nck co-immunoprecipitates with a number of phosphotyrosine proteins including the EGF receptor. To identify the phosphotyrosine protein(s) that directly interacts with Nck and to distinguish it from indirectly associated proteins, preexisting phosphotyrosine protein complexes in the cell lysate were dissociated by heat and SDS prior to the test for binding to Nck. Nck does not directly bind to EGF receptor; instead it binds via its SH2 domain to a 62 kDa phosphotyrosine protein. The Nck-bound p62 is related to the previously identified GTPase-activating protein (GAP)-associated phosphotyrosine protein p62. Four binding patterns are reported: (1) the Nck-bound and the GAP-bound p62 proteins co-migrate with each other in SDS-PAGE; (2) SH2 domains from Nck and GAP compete for binding to p62 in vitro; (3) purified GST-Nck-SH2 binds directly to the GAP-associated p62, (under these conditions, SH2 domains from PLCgamma, PI-3 kinase, SHC, and Grb2 do not bind p62) and (4) tryptic phosphopeptide maps of the Nck- and the GAP-associated p62 proteins are identical. However, Nck and GAP do not co-immunoprecipitate with each other and apparently bind to different pools of p62. This study suggests that the GAP-associated p62 acts as an SH2 domain docking protein and mediates the interaction between Nck and EGF receptor in response to EGF stimulation (Tang, 1997).

The SH2/SH3 adapters Nck, Grb2 and Crk promote the assembly of signaling complexes by binding to tyrosine phosphorylated proteins using their SH2 domains and to proline-rich sequences on effector molecules using their SH3 domains. FGF, which activates a receptor tyrosine kinase, induces mesoderm formation in Xenopus embryos through activation of the Ras/Raf/MAPK signaling pathway. Dominant-negative mutants of Nck and Grb2, but not Crk1, can inhibit mesoderm-specific gene induction by eFGF in Xenopus animal cap explants. Dominant-negative mutants of Grb2 and Nck can inhibit eFGF-induced Erk1 activation in Xenopus animal caps, and targeting the first two SH3 domains of Nck to the membrane can activate Erk1 in the absence of eFGF. Combinations of the dominant-negative Grb2 mutants with the inhibitory Nck mutant synergistically inhibit Erk1 activation by eFGF in Xenopus animal caps, suggesting that the dominant-negative Nck and Grb2 mutants inhibit Erk1 activation by binding to different proteins. By contrast only Grb2 mutants could inhibit eFGF-induced Erk1 activation in human 293 cells, demonstrating diversity in the specific mechanisms of signaling from FGF to MAP kinases in different cells (Gupta, 1998).

PINCH is a widely expressed and evolutionarily conserved protein comprising primarily five LIM domains, which are cysteine-rich consensus sequences implicated in mediating protein-protein interactions. PINCH is a binding protein for integrin-linked kinase (ILK), an intracellular serine/threonine protein kinase that plays important roles in the cell adhesion, growth factor, and Wnt signaling pathways. The interaction between ILK and PINCH has been consistently observed under a variety of experimental conditions. They have interacted in yeast two-hybrid assays, in solution, and in solid-phase-based binding assays. Furthermore, ILK, but not vinculin or focal adhesion kinase, has been coisolated with PINCH from mammalian cells by immunoaffinity chromatography, indicating that PINCH and ILK associate with each other in vivo. The PINCH-ILK interaction is mediated by the N-terminal-most LIM domain (LIM1, residues 1 to 70) of PINCH and multiple ankyrin (ANK) repeats located within the N-terminal domain (residues 1 to 163) of ILK. Additionally, biochemical studies indicate that ILK, through the interaction with PINCH, is capable of forming a ternary complex with Nck-2, an SH2/SH3-containing adapter protein implicated in growth factor receptor kinase and small GTPase signaling pathways. PINCH is concentrated in peripheral ruffles of cells spreading on fibronectin and clusters of PINCH have been detected that are colocalized with the alpha5beta1 integrins. These results demonstrate a specific protein recognition mechanism utilizing a specific LIM domain and multiple ANK repeats and suggest that PINCH functions as an adapter protein connecting ILK and the integrins with components of growth factor receptor kinase and small GTPase signaling pathways (Tu, 1999).

Fc receptors modulate inflammatory processes, including phagocytosis, serotonin and histamine release, superoxide production, and secretion of cytokines. Aggregation of FcgammaRIIa, the low-affinity receptor for monomeric IgG, activates nonreceptor protein tyrosine kinases such as Lyn, Hck, and Syk, potentially driving the phosphorylation of the downstream adaptor proteins, including Cbl and/or Nck. FcgammaRII receptor signaling in interferon-gamma-differentiated U937 (U937IF) and HEL cells involves Cbl and Nck, suggesting that Cbl-Nck interactions may link FcgammaRII to downstream activation of Pak kinase. FcgammaRII crosslinking induces the phosphorylation of Cbl and Nck on tyrosine. AlphaCbl immunoprecipitations reveal constitutive binding of Nck and Grb2 to Cbl and FcgammaRII-inducible binding of CrkL to Cbl. The interactions of Cbl with Nck and CrkL are phosphorylation dependent. Cbl and Pak1 bind to the second SH3 domain of Nck. A specific Src inhibitor, PP1, completely abrogates the FcgammaR-induced superoxide response, correlating with a decrease in Cbl and Nck tyrosine phosphorylation. These results provide the first evidence that Src is required for FcgammaR activation of the respiratory burst in myeloid cells and suggest that Cbl-Nck, Cbl-Pak1, and Nck-Pak1 interactions may regulate this response (Izadi, 1998).

The dopamine D4 receptor is a G protein-coupled receptor (GPCR) that belongs to the dopamine D2-like receptor family. Functionally, the D2-like receptors are characterized by their ability to inhibit adenylyl cyclase. The dopamine D4 receptor as well as many other catecholaminergic receptors contain several putative SH3 binding domains. Most of these sites in the D4 receptor are located in a polymorphic repeat sequence and flanking sequences in the third intracellular loop. This region of the D4 receptor can interact with a large variety of SH3 domains of different origin. The strongest interactions were seen with the SH2-SH3 adapter proteins Grb2 and Nck. The repeat sequence itself is not essential in this interaction. The different SH3 domains in the adapter proteins interact in a cooperative fashion with two distinct sites immediately upstream and downstream from the repeat sequence. Removal of all the putative SH3 binding domains in the third intracellular loop of the dopamine D4 receptor results in a receptor that can still bind spiperone and dopamine. Dopamine cannot modulate the coupling of these mutant receptors to adenylyl cyclase and MAPK, although dopamine modulated receptor-G protein interaction appears normal. The receptor deletion mutants show strong constitutive internalization that may account for the deficiency in functional activation of second messengers. The data indicate that the D4 receptor contains SH3 binding sites and that these sites fall within a region involved in the control of receptor internalization (Oldenhof, 1998).