The SH2 and SH3 adapter Nck: a two-gene family and a linker between tyrosine kinases and multiple signaling networks

SH2 and SH3 adapter proteins connect cell surface tyrosine kinases to intracellular signaling networks. For instance, the SH3-SH2-SH3 adapter Grb2 links receptor tyrosine kinases to the Ras pathway. Nck, composed of three SH3 domains and one SH2 domain, represents a two-gene (alpha and beta) family in mammals. Nckalpha and Nckbeta are expressed in the same cells and appear to have distinct signaling specificity. Studies show that Nck plays a role in cell mitogenesis and morphogenesis. The former uses Ras-dependent and Ras-independent pathways. The latter appears to coordinate with the Cdc42/Rac>PAK1/WASp>actin cytoskeleton pathway. Understanding the specificity of Nckalpha and Nckbeta signal transduction will provide answers for the previously often conflicting observations.     

Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.

Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth factors stimulate the phosphorylation of Nck and its association with autophosphorylated growth factor receptors. A panel of platelet-derived growth factor (PDGF) receptor mutations at tyrosine residues has been used to identify the Nck binding site. Here we show that mutation at Tyr-751 of the PDGF beta-receptor eliminates Nck binding both in vitro and in living cells. Moreover, the Y751F PDGF receptor mutant failed to mediate PDGF-stimulated phosphorylation of Nck in intact cells. A phosphorylated Tyr-751 is also required for binding of phosphatidylinositol-3 kinase to the PDGF receptor. Hence, the SH2 domains of p85 and Nck share a binding site in the PDGF receptor. Competition experiments with different phosphopeptides derived from the PDGF receptor suggest that binding of Nck and p85 is influenced by different residues around Tyr-751. Thus, a single tyrosine autophosphorylation site is able to link the PDGF receptor to two distinct SH2 domain-containing signaling molecules.     

YXXL motifs in SH2-Bbeta are phosphorylated by JAK2, JAK1, and platelet-derived growth factor receptor and are required for membrane ruffling

SH2-Bbeta binds to the activated form of JAK2 and various receptor tyrosine kinases. It is a potent stimulator of JAK2, is required for growth hormone (GH)-induced membrane ruffling, and increases mitogenesis stimulated by platelet-derived growth factor (PDGF) and insulin-like growth factor I. Its domain structure suggests that SH2-Bbeta may act as an adapter protein to recruit downstream signaling proteins to kinase.SH2-Bbeta complexes. SH2-Bbeta is tyrosyl-phosphorylated in response to GH and interferon-gamma, stimulators of JAK2, as well as in response to PDGF and nerve growth factor. To begin to elucidate the role of tyrosyl phosphorylation in the function of SH2-Bbeta, we used phosphopeptide mapping, mutagenesis, and a phosphotyrosine-specific antibody to identify Tyr-439 and Tyr-494 in SH2-Bbeta as targets of JAK2 both in vitro and in intact cells. SH2-Bbeta lacking Tyr-439 and Tyr-494 inhibits GH-induced membrane ruffling but still activates JAK2. We provide evidence that JAK1, like JAK2, phosphorylates Tyr-439 and Tyr-494 in SH2-Bbeta and that PDGF receptor phosphorylates SH2-Bbeta on Tyr-439. Therefore, phosphorylated Tyr-439 and/or Tyr-494 in SH2-Bbeta may provide a binding site for one or more proteins linking cytokine receptor.JAK2 complexes and/or receptor tyrosine kinases to the actin cytoskeleton.     

The phosphotyrosine peptide binding specificity of Nck1 and Nck2 Src homology 2 domains

Nck proteins are essential Src homology (SH) 2 and SH3 domain-bearing adapters that modulate actin cytoskeleton dynamics by linking proline-rich effector molecules to tyrosine kinases or phosphorylated signaling intermediates. Two mammalian pathogens, enteropathogenic Escherichia coli and vaccinia virus, exploit Nck as part of their infection strategy. Conflicting data indicate potential differences in the recognition specificities of the SH2 domains of the isoproteins Nck1 (Nckalpha) and Nck2 (Nckbeta and Grb4). We have characterized the binding specificities of both SH2 domains and find them to be essentially indistinguishable. Crystal structures of both domains in complex with phosphopeptides derived from the enteropathogenic E. coli protein Tir concur in identifying highly conserved, specific recognition of the phosphopeptide. Differential peptide recognition can therefore not account for the preference of either Nck in particular signaling pathways. Binding studies using sequentially mutated, high affinity phosphopeptides establish the sequence variability tolerated in peptide recognition. Based on this binding motif, we identify potential new binding partners of Nck1 and Nck2 and confirm this experimentally for the Arf-GAP GIT1.     

Nckbeta adapter controls neuritogenesis by maintaining the cellular paxillin level

The SH2/SH3 adapter Nck has an evolutionarily conserved role in neurons, linking the cell surface signals to actin cytoskeleton-mediated responses. The mechanism, however, remains poorly understood. We have investigated the role of Nck/Nckalpha/Nck1 versus Grb4/Nckbeta/Nck2 side-by-side in the process of mammalian neuritogenesis. Here we show that permanent genetic silencing of Nckbeta, but not Nckalpha, completely blocked nerve growth factor-induced neurite outgrowth in PC12 cells and dramatically disrupted the axon and dendrite tree in primary rat cortical neurons. By screening for changes among the components reportedly present in complex with Nck, we found that the steady-state level of paxillin was significantly reduced in Nckbeta knockdown, but not Nckalpha knockdown, neurons. Interestingly, Nckbeta knockdown did not affect the paxillin level in glial cells and several other cell types of various tissue origins. Genetic silencing of paxillin blocked neuritogenesis, just like Nckbeta knockdown. Reintroducing a nondegradable Nckbeta into Nckbeta short interfering RNA-expressing PC12 cells rescued paxillin from down-regulation and allowed the resumption of neuritogenesis. Forced expression of paxillin in Nckbeta knockdown PC12 also rescued its capacity for neuritogenesis. Finally, Nckbeta, but not Nckalpha, binds strongly to paxillin and treatment of the neurons with proteosome inhibitors prevented paxillin down-regulation in Nckbeta knockdown neurons. Thus, Nckbeta maintains paxillin stability during neuritogenesis.     

Membrane ruffling requires coordination between type Ialpha phosphatidylinositol phosphate kinase and Rac signaling

Membrane ruffle formation requires remodeling of cortical actin filaments, a process dependent upon the small G-protein Rac. Growth factors stimulate actin remodeling and membrane ruffling by integration of signaling pathways that regulate actin-binding proteins. Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates the activity of many actin-binding proteins and is produced by the type I phosphatidylinositol phosphate kinases (PIPKIs). Here we show in MG-63 cells that only the PIPKIalpha isoform is localized to platelet-derived growth factor (PDGF)-induced membrane ruffles. Further, expression of kinase dead PIPKIalpha, which acts as a dominant negative mutant, blocked membrane ruffling, suggesting that PIPKIalpha and PIP2 participate in ruffling. To explore this, PIPKIalpha was overexpressed in serum-starved cells and stimulated with PDGF. In serum-starved cells, PIPKIalpha expression did not stimulate actin remodeling, but when these cells were stimulated with PDGF, actin rapidly reorganized into foci but not membrane ruffles. PIPKIalpha-mediated formation of actin foci was independent of both Rac1 and phosphatidylinositol 3-kinase activities. Significantly, coexpression of dominant active Rac1 with PIPKIalpha in PDGF-stimulated cells resulted in membrane ruffling. The PDGF- and Rac1-stimulated ruffling was inhibited by expression of kinase-dead PIPKIalpha. Combined, these data support a model where the localized production of PIP2 by PIPKIalpha is necessary for actin remodeling, whereas formation of membrane ruffles required Rac signaling.     

Macrophage/microglia-specific protein Iba1 enhances membrane ruffling and Rac activation via phospholipase C-gamma -dependent pathway

Iba1 is a macrophage/microglia-specific calcium-binding protein that is involved in RacGTPase-dependent membrane ruffling and phagocytosis. In this study, we introduced Iba1 into Swiss 3T3 fibroblasts and demonstrated the enhancement of platelet-derived growth factor (PDGF)-induced membrane ruffling and chemotaxis. Wortmannin treatment did not completely suppressed this enhanced membrane ruffling in Iba1-expressing cells, whereas it did in Iba1-nonexpressing cells, suggesting that the enhancement is mediated through a phosphatidylinositol 3-kinase (PI3K)-independent signaling pathway. Porcine aorta endothelial cells transfected with expression constructs of Iba1 and PDGF receptor add-back mutants were used to analyze the signaling pathway responsible for the Iba1-induced enhancement of membrane ruffling. In the absence of Iba1 expression, PDGF did not induced membrane ruffling in cells expressing the Tyr-1021 receptor mutant, which is capable of activating phospholipase C-gamma (PLC-gamma) but not PI3K. In contrast, in the presence of Iba1 expression, membrane ruffling was formed in cells expressing the Tyr-1021 mutant. In addition, Rac was shown to be activated during membrane ruffling in cells expressing Iba1 and the Tyr-1021 mutant. Furthermore, dominant negative forms of PLC-gamma completely suppressed PDGF-induced Iba1-dependent membrane ruffling and Rac activation. These results indicate the existence of a novel signaling pathway where PLC-gamma activates Rac in a manner dependent on Iba1.     

Nck adapters are involved in the formation of dorsal ruffles, cell migration, and Rho signaling downstream of the platelet-derived growth factor beta receptor

The SH3 and SH2 domain-containing adapter proteins Nck1 and Nck2 are known to function downstream of activated tyrosine kinase receptors, such as the platelet-derived growth factor (PDGF) receptors. The SH2 domain of Nck1 binds to phosphorylated tyrosine residue 751 in PDGFbeta receptor and has been suggested to have a role in the PDGF-induced mobilization of the actin filament system. Because Tyr-751 is a site for additional receptor interactors, it has been difficult to discriminate the signaling from Nck from signaling via other molecules. For this reason we have used mouse embryonic fibroblasts derived from mice in which the genes for Nck1 and Nck2 have been inactivated by gene targeting (knock-out (KO) cells). The mutant cells had a reduced ability to form edge ruffles in response to PDGF, and the presence of Nck was obligatory for the formation of dorsal ruffles. In addition, the KO cells had a reduced chemotactic and migratory potential. Importantly, KO cells had reduced cell attachment properties and a reduced ability to form focal adhesions in response to serum stimulation. Moreover, signaling involving the Rho GTPases was defective in KO cells. In summary, our observations suggest that the Nck adapters are needed for signaling to Rho GTPases and actin dynamics downstream of the PDGFbeta receptor.     

Localization and Down-Regulating Role of the Protein Tyrosine Phosphatase PTP2C in Membrane Ruffles of PDGF-Stimulated Cells

PTP2C (also known as Syp/SH-PTP2/PTP1D) is a soluble protein tyrosine phosphatase present in most cell types. It interacts directly with activated PDGF receptor via its SH2 domains, which results in its phosphorylation on tyrosine residue(s). The phosphorylated PTP2C in turn binds to the SH2 domain of GRB2, serving as an adaptor in the transduction of mitogenic signals from the growth factor receptor to the Ras and MAP kinase signaling pathways. We investigated the interaction of PTP2C with the PDGF receptor by examining the localization of both proteins after PDGF stimulation of 293 cells which stably express the human PDGF receptor. In resting cells, transiently expressed PTP2C was distributed throughout the cytoplasm. Upon stimulation with PDGF, PTP2C was translocated from the cytoplasm to membrane ruffles. Immunofluorescence examination revealed that PTP2C colocalized with actin, the PDGF receptors, and hyper-tyrosine-phosphorylated protein(s). Neither deletion of the SH2 domains nor point mutations at either the catalytic site or the major phosphorylation site affected membrane ruffling or the localization of PTP2C to the ruffles of PDGF-stimulated cells. However, the expression of a catalytically inactive mutant PTP2C substantially prolonged ruffling activity following PDGF stimulation. These results suggest that PTP2C is involved in the down-regulation of the membrane ruffling pathway, and in contrast to its positive function in the MAP kinase pathway, the phosphatase activity negatively regulates ruffling activity.     

Inducible clustering of membrane-targeted SH3 domains of the adaptor protein Nck triggers localized actin polymerization

BACKGROUND: SH2/SH3 adaptor proteins play a critical role in tyrosine kinase signaling pathways, regulating essential cell functions by increasing the local concentration or altering the subcellular localization of downstream effectors. The SH2 domain of the Nck adaptor can bind tyrosine-phosphorylated proteins, while its SH3 domains can modulate actin polymerization by interacting with effectors such as WASp/Scar family proteins. Although several studies have implicated Nck in regulating actin polymerization, its role in living cells is not well understood. RESULTS: We used an antibody-based system to experimentally modulate the local concentration of Nck SH3 domains on the plasma membrane of living cells. Clustering of fusion proteins containing all three Nck SH3 domains induced localized polymerization of actin, including the formation of actin tails and spots, accompanied by general cytoskeletal rearrangements. All three Nck SH3 domains were required, as clustering of individual SH3 domains or a combination of the two N-terminal Nck SH3 domains failed to promote significant local polymerization of actin in vivo. Changes in actin dynamics induced by Nck SH3 domain clustering required the recruitment of N-WASp, but not WAVE1, and were unaffected by downregulation of Cdc42. CONCLUSIONS: We show that high local concentrations of Nck SH3 domains are sufficient to stimulate localized, Cdc42-independent actin polymerization in living cells. This study provides strong evidence of a pivotal role for Nck in directly coupling ligand-induced tyrosine phosphorylation at the plasma membrane to localized changes in organization of the actin cytoskeleton through a signaling pathway that requires N-WASp.     

SH2-B is required for growth hormone-induced actin reorganization

The Src homology-2 (SH2) domain-containing protein SH2-Bbeta is a substrate of the growth hormone (GH) receptor-associated tyrosine kinase JAK2. Here we tested whether SH2-Bbeta is involved in GH regulation of the actin cytoskeleton. Based on cell fractionation and confocal microscopy, we find SH2-Bbeta present at the plasma membrane and in the cytosol. SH2-Bbeta colocalized with filamentous actin in GH and platelet-derived growth factor (PDGF)-induced membrane ruffles. To test if SH2-Bbeta is required for actin reorganization, we transiently overexpressed wild-type or mutant SH2-Bbeta in 3T3-F442A cells and assayed for GH- and PDGF-induced membrane ruffling and fluid phase pinocytosis. Overexpression of wild-type SH2-Bbeta enhanced ruffling and pinocytosis produced by submaximal GH but not submaximal PDGF. Point mutant SH2-Bbeta (R555E) and truncation mutant DeltaC555, both lacking a functional SH2 domain, inhibited membrane ruffling and pinocytosis induced by GH and PDGF. Mutant DeltaN504, which possesses a functional SH2 domain and enhances JAK2 kinase activity in overexpression systems, also inhibited GH-stimulated membrane ruffling. DeltaN504 failed to inhibit GH-induced nuclear localization of Stat5B, indicating JAK2 is active in these cells. Taken together, these results show that SH2-Bbeta is required for GH-induced actin reorganization by a mechanism discrete from the action of SH2-Bbeta as a stimulator of JAK2 kinase activity.     

In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase.

We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

3BP-1, an SH3 domain binding protein, has GAP activity for Rac and inhibits growth factor-induced membrane ruffling in fibroblasts.

The SH3 binding protein, 3BP-1, was originally cloned as a partial cDNA from an expression library using the Abl SH3 domain as a probe. In addition to an SH3 binding domain, 3BP-1 displayed homology to a class of GTPase activating proteins (GAPs) active against Rac and Rho proteins. We report here a full length cDNA of 3BP-1 which extends the homology to GAP proteins previously noted. 3BP-1 functions in vitro as a GAP with a specificity for Rac-related G proteins. Microinjection of the 3BP-1 protein into serum-starved fibroblasts produces an inhibition of platelet-derived growth factor (PDGF)-induced membrane ruffling mediated by Rac. Co-injection of 3BP-1 with an activated Rac mutant that is unresponsive to GAPs, counter-acts this inhibition. 3BP-1 does not show in vitro activity towards Rho and, in agreement with this finding, microinjection of 3BP-1 into fibroblasts has no effect on lysophosphatidic acid (LPA)-induced stress fiber assembly mediated by Rho. Thus 3BP-1 is a new and specific Rac GAP that can act in cells to counter Rac-mediated membrane ruffling. How its SH3 binding site interacts with its GAP activity remains to be understood.     

Wiskott-Aldrich syndrome protein is associated with the adapter protein Grb2 and the epidermal growth factor receptor in living cells.

Src homology domains [i.e., Src homology domain 2 (SH2) and Src homology domain 3 (SH3)] play a critical role in linking receptor tyrosine kinases to downstream signaling networks. A well-defined function of the SH3-SH2-SH3 adapter Grb2 is to link receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), to the p21ras-signaling pathway. Grb2 has also been implicated to play a role in growth factor-regulated actin assembly and receptor endocytosis, although the underlying mechanisms remain unclear. In this study, we show that Grb2 interacts through its SH3 domains with the human Wiskott-Aldrich syndrome protein (WASp), which plays a role in regulation of the actin cytoskeleton. We find that WASp is expressed in a variety of cell types and is exclusively cytoplasmic. Although the N-terminal SH3 domain of Grb2 binds significantly stronger than the C-terminal SH3 domain to WASp, full-length Grb2 shows the strongest binding. Both phosphorylation of WASp and its interaction with Grb2, as well as with another adapter protein Nck, remain constitutive in serum-starved or epidermal growth factor-stimulated cells. WASp coimmunoprecipitates with the activated EGFR after epidermal growth factor stimulation. Purified glutathione S-transferase-full-length-Grb2 fusion protein, but not the individual domains of Grb2, enhances the association of WASp with the EGFR, suggesting that Grb2 mediates the association of WASp with EGFR. This study suggests that Grb2 translocates WASp from the cytoplasm to the plasma membrane and the Grb2-WASp complex may play a role in linking receptor tyrosine kinases to the actin cytoskeleton.     

The mechanism of cell membrane ruffling relies on a phospholipase D2 (PLD2), Grb2 and Rac2 association

Membrane ruffling is the formation of actin rich membrane protrusions, essential for cell motility. The exact mechanism of ruffling is not fully known. Using YFP and CFP fluorescent chimeras, we show for the first time a co-localization of Phospholipase D2 (PLD2) and Growth factor Receptor Bound protein-2 (Grb2) with actin-rich membrane protrusions of macrophages. Grb2 cooperates with PLD2 in enhancing membrane ruffling, whether in resting cells or in cells stimulated with the growth factor M-CSF, although in the latter an increase in dorsal ruffles was observed, consistent with receptor-ligand internalization. Cells transfected with PLD2 mutated in the PH domain (Y169F) or with Grb2 mutated in the SH2 site (R86K) negate this effect, indicating an association PLD2(Y169)-SH2-Grb2 that was confirmed by immunoprecipitation and Western blotting. The association results in enhanced PLD activity, but the lipase activity can only partially explain the formation of membrane ruffles in vivo. A third component involves the Rho-GTPase Rac2 and it is only when Rac2 is overexpressed along with PLD2 and Rac2 that a full biological effect, including actin polymerization in vivo, is obtained. The mechanism involved is, then, as follows: PLD enzymatic action, after having been increased due to the binding to Grb2-SH2 via Y169, cooperates with Rac2, and the three molecules stimulate actin polymerization and consequently, membrane ruffle formation. Since membrane ruffling precedes cell migration, the results herein provide a novel mechanism for control of membrane dynamics, crucial for the physiology of leukocytes.     

Phosphorylated YDXV motifs and Nck SH2/SH3 adaptors act cooperatively to induce actin reorganization

We have analyzed the means by which the Nck family of adaptor proteins couples adhesion proteins to actin reorganization. The nephrin adhesion protein is essential for the formation of actin-based foot processes in glomerular podocytes. The clustering of nephrin induces its tyrosine phosphorylation, Nck recruitment, and sustained localized actin polymerization. Any one of three phosphorylated (p)YDXV motifs on nephrin is sufficient to recruit Nck through its Src homology 2 (SH2) domain and induce localized actin polymerization at these clusters. Similarly, Nck SH3 mutants in which only the second or third SH3 domain is functional can mediate nephrin-induced actin polymerization. However, combining such nephrin and Nck mutants attenuates actin polymerization at nephrin-Nck clusters. We propose that the multiple Nck SH2-binding motifs on nephrin and the multiple SH3 domains of Nck act cooperatively to recruit the high local concentration of effectors at sites of nephrin activation that is required to initiate and maintain actin polymerization in vivo. We also find that YDXV motifs in the Tir protein of enteropathogenic Escherichia coli and nephrin are functionally interchangeable, indicating that Tir reorganizes the actin cytoskeleton by molecular mimicry of nephrin-like signaling. Together, these data identify pYDXV/Nck signaling as a potent and portable mechanism for physiological and pathological actin regulation.     

GRB2 links signaling to actin assembly by enhancing interaction of neural Wiskott-Aldrich syndrome protein (N-WASp) with actin-related protein (ARP2/3) complex

Proteins of the Wiskott-Aldrich Syndrome protein (WASp) family connect signaling pathways to the actin polymerization-driven cell motility. The ubiquitous homolog of WASp, N-WASp, is a multidomain protein that interacts with the Arp2/3 complex and G-actin via its C-terminal WA domain to stimulate actin polymerization. The activity of N-WASp is enhanced by the binding of effectors like Cdc42-guanosine 5'-3-O-(thio)triphosphate, phosphatidylinositol bisphosphate, or the Shigella IcsA protein. Here we show that the SH3-SH2-SH3 adaptor Grb2 is another activator of N-WASp that stimulates actin polymerization by increasing the amount of N-WASp. Arp2/3 complex. The concentration dependence of N-WASp activity, sedimentation velocity and cross-linking experiments together suggest that N-WASp is subject to self-association, and Grb2 enhances N-WASp activity by binding preferentially to its active monomeric form. Use of peptide inhibitors, mutated Grb2, and isolated SH3 domains demonstrate that the effect of Grb2 is mediated by the interaction of its C-terminal SH3 domain with the proline-rich region of N-WASp. Cdc42 and Grb2 bind simultaneously to N-WASp and enhance actin polymerization synergistically. Grb2 shortens the delay preceding the onset of Escherichia coli (IcsA) actin-based reconstituted movement. These results suggest that Grb2 may activate Arp2/3 complex-mediated actin polymerization downstream from the receptor tyrosine kinase signaling pathway.     

Direct Regulation of Nephrin Tyrosine Phosphorylation by Nck Adaptor Proteins

The transmembrane protein nephrin is a key component of the kidney slit diaphragm that contributes to the morphology of podocyte foot processes through signaling to the underlying actin cytoskeleton. We have recently reported that tyrosine phosphorylation of the cytoplasmic tail of nephrin facilitates recruitment of Nck SH2/SH3 adaptor proteins and subsequent actin remodeling and that phosphorylation of the Nck binding sites on nephrin is decreased during podocyte injury. We now demonstrate that Nck directly modulates nephrin phosphorylation through formation of a signaling complex with the Src family kinase Fyn. The ability of Nck to enhance nephrin phosphorylation is compromised in the presence of a Src family kinase inhibitor and when the SH3 domains of Nck are mutated. Furthermore, induced loss of Nck expression in podocytes in vivo is associated with a rapid reduction in nephrin tyrosine phosphorylation. Our results suggest that Nck may facilitate dynamic signaling events at the slit diaphragm by promoting Fyn-dependent phosphorylation of nephrin, which may be important in the regulation of foot process morphology and response to podocyte injury.     

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

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, CD3ε, 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.     

Tyrosine-phosphorylated SOCS3 interacts with the Nck and Crk-L adapter proteins and regulates Nck activation

Suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine and growth factor signal transduction. Although the affect of SOCS proteins on the Jak-STAT pathway has been well characterized, their role in the regulation of other signaling modules is not well understood. In the present study, we demonstrate that SOCS3 physically interacts with the SH2/SH3-containing adapter proteins Nck and Crk-L, which are known to couple activated receptors to multiple downstream signaling pathways and the actin cytoskeleton. Our data show that the SOCS3/Nck and SOCS3/Crk-L interactions depend on tyrosine phosphorylation of SOCS3 Tyr(221) within the conserved SOCS box motif and intact SH2 domains of Nck and Crk-L. Furthermore, SOCS3 Tyr(221) forms a YXXP motif, which is a consensus binding site for the Nck and Crk-L SH2 domains. Expression of SOCS3 in NIH3T3 cells induces constitutive recruitment of a Nck-GFP fusion protein to the plasma membrane and constitutive tyrosine phosphorylation of endogenous Nck. Our findings suggest that SOCS3 regulates multiple cytokine and growth factor-activated signaling pathways by acting as a recruitment factor for adapter proteins.