The glomerular filtration barrier in the kidney is formed in part by a specialized intercellular junction known as the slit diaphragm, which connects adjacent actin-based foot processes of kidney epithelial cells (podocytes). Mutations affecting a number of slit diaphragm proteins, including nephrin (encoded by NPHS1), lead to renal disease owing to disruption of the filtration barrier and rearrangement of the actin cytoskeleton, although the molecular basis for this is unclear. Here we show that nephrin selectively binds the Src homology 2 (SH2)/SH3 domain-containing Nck adaptor proteins, which in turn control the podocyte cytoskeleton in vivo. The cytoplasmic tail of nephrin has multiple YDxV sites that form preferred binding motifs for the Nck SH2 domain once phosphorylated by Src-family kinases. We show that this Nck-nephrin interaction is required for nephrin-dependent actin reorganization. Selective deletion of Nck from podocytes of transgenic mice results in defects in the formation of foot processes and in congenital nephrotic syndrome. Together, these findings identify a physiological signalling pathway in which nephrin is linked through phosphotyrosine-based interactions to Nck adaptors, and thus to the underlying actin cytoskeleton in podocytes. Simple and widely expressed SH2/SH3 adaptor proteins can therefore direct the formation of a specialized cellular morphology in vivo.

The SH2 domain-containing SHP-1 tyrosine phosphatase has been shown to negatively regulate a broad spectrum of growth factor-and cytokine-driven mitogenic signaling pathways. Included among these is the cascade of intracellular events evoked by stem cell factor binding to c-Kit, a tyrosine kinase receptor which associates with and is dephosphorylated by SHP-1. Using a series of glutathione S-transferase ( The pivotal role of the SH2 domain-containing SHP-1 (PTP1C, HCP, or SHPTP1) tyrosine phosphatase in the regulation of hemopoietic cell growth and development is now well recognized (1). In contrast to the structurally similar, ubiquitously expressed SHP-2 (Syp or PTP1D) tyrosine phosphatase and its Drosophila csw homolog (10, 37), SHP-1 appears to exert primarily inhibitory effects on the signaling cascades in which it participates (34). SHP-1 has been shown, for example, to suppress the growth-promoting properties of the activated interleukin 3 (IL-3), erythropoietin, and colony-stimulating factor 1 receptors, an effect mediated either directly by receptor dephosphorylation or indirectly by dephosphorylation of receptor-associated protein tyrosine kinases (PTKs) (4,19,55,56). SHP-1 has also been implicated in downregulation of the signaling pathways evoked by engagement of the B-and Tlymphocyte antigen receptors (5, 32, 33), antigen receptor comodulators such as CD22, Fc␥RIIB, and CD5, and cytosolic signaling molecules such as Vav and Grb2/Sos1 which are involved in Ras activation (6,8,20). The capacity of SHP-1 to negatively modulate this broad spectrum of signaling effectors is consistent with the enormous overexpansion of multiple hemopoietic cell populations manifested by motheaten (me) and viable motheaten (me v ) mice, animals now known to be homozygous for loss-of-function mutations in the SHP-1 gene (21,46). The presence of two SH2 domains in SHP-1, as well as the possibility for altering its C-terminal SH2 domain by alternative splicing of a 39-amino-acid segment (46), provides a structural explanation for the diverse range of molecular interactions in which this phosphotyrosine phosphatase (PTP) appears to participate. Thus, while the precise structural basis for and physiologic effects of SHP-1 interactions with the molecules with which it has been shown to associate require further investigation, the current data concerning SHP-1 functions identify this PTP as a critical player in the modulation of hemopoietic cell growth and function.In addition to the regulation of cell proliferation, SHP-1 has also been implicated in the control of signaling cascades coupling growth factor receptors to hemopoietic cell differentiation. This role for SHP-1 has become particularly well appreciated in the context of data derived from studies of SHP-1 interactions with the transmembrane PTK receptor encoded by the c-Kit proto-oncogene. The latter receptor subserves pivotal functions in promoting the development, survival, and proliferation of hemopoietic stem cells, neural crest-derived cells, and germ cells, a role...

In response to stress, the endoplasmic reticulum (ER) signaling machinery triggers the inhibition of protein synthesis and up-regulation of genes whose products are involved in protein folding, cell cycle exit, and/or apoptosis. We demonstrate that the misfolding agents azetidine-2-carboxylic acid (Azc) and tunicamycin initiate signaling from the ER, resulting in the activation of Jun-N-terminal kinase, p44 MAPK /extracellular signal-regulated kinase-1 (ERK-1), and p38 MAPK through IRE1␣-dependent mechanisms. To characterize the ER proximal signaling events involved, immuno-isolated ER membranes from rat fibroblasts treated with ER stress inducers were used to reconstitute the activation of the stress-activated protein kinase/mitogen-activate protein kinase (MAPK) pathways in vitro. This allowed us to demonstrate a role for the SH2/SH3 domain containing adaptor Nck in ERK-1 activation after Azc treatment. We also show both in vitro and in vivo that under basal conditions ER-associated Nck represses ERK-1 activation and that upon ER stress this pool of Nck dissociates from the ER membrane to allow ERK-1 activation. Moreover, under the same conditions, Nck-null cells elicit a stronger ERK-1 activation in response to Azc stress, thus, correlating with an enhanced survival phenotype. These data delineate a novel mechanism for the regulation of ER stress signaling to the MAPK pathway and demonstrate a critical role for Nck in ER stress and cell survival.

Netrins are a family of secreted proteins that guide the migration of cells and axonal growth cones during development. DCC (deleted in colorectal cancer) is a receptor for netrin-1 implicated in mediating these responses. Here, we show that DCC interacts constitutively with the SH3/SH2 adaptor Nck in commissural neurons. This interaction is direct and requires the SH3 but not SH2 domains of Nck-1. Moreover, both DCC and Nck-1 associate with the actin cytoskeleton, and this association is mediated by DCC. A dominant negative Nck-1 inhibits the ability of DCC to induce neurite outgrowth in N1E-115 cells and to activate Rac1 in fibroblasts in response to netrin-1. These studies provide evidence for an important role of mammalian Nck-1 in a novel signaling pathway from an extracellular guidance cue to changes in the actin-based cytoskeleton responsible for axonal guidance.In the developing nervous system, newborn neurons extend their axons to their targets in response to attractive and repulsive cues. The neuronal growth cone, located at the tip of the growing axon, is a highly motile structure that can be viewed as a sophisticated signal transduction device capable of recognizing extracellular guidance cues and translating them into directed neurite outgrowth (1, 2). Over the past years, a combination of cellular and genetic studies has led to the identification of several proteins playing a critical role in guiding axons along their pathways, including members of the ephrin, semaphorin, netrin, slit, reelin, and nerve growth factor families of proteins (3). Several classes of transmembrane proteins have also been identified and characterized as their respective binding receptors (4). For instance, two classes of receptors, DCC and the UNC-5 proteins, have been described for the netrins. Netrins are bifunctional molecules attracting and repelling different classes of axons. The DCC family of receptors mediates growth cone attraction by netrins (5-8), whereas the UNC-5 proteins are required for the repulsive effect of the netrins (9 -11). However, the pathway is more complicated since UNC-5-mediated repulsion requires the function of DCC proteins in some cases (12)(13)(14).We recently determined that the small GTPases Rac1, Cdc42, and RhoA play a key role in the cytosolic signaling events induced by the netrin-1 receptor DCC (15, 16). Several putative Src homology 3 (SH3) 1 binding motifs, PXXP (17), are found in the cytoplasmic tail of DCC, suggesting a possible interaction with an SH3-containing adaptor molecule to mediate netrin-1 signaling to Rho GTPases. A role for the fly protein Dock in axon guidance has been well characterized during Drosophila eye and nervous system development (18 -21). Nck, the mammalian homolog of Dock, is a ubiquitously expressed protein composed of a single SH2 and three SH3 domains and is represented by two genes (22,23).In this paper, we provide biochemical and functional evidence for the involvement of Nck in mediating the netrin-1 receptor DCC signaling. We demonstrate that DCC as...

Ternary complex (TC) and eIF4F complex assembly are the two major rate-limiting steps in translation initiation regulated by eIF2α phosphorylation and the mTOR/4E-BP pathway, respectively. How TC and eIF4F assembly are coordinated, however, remains largely unknown. We show that mTOR suppresses translation of mRNAs activated under short-term stress wherein TC recycling is attenuated by eIF2α phosphorylation. During acute nutrient or growth factor stimulation, mTORC1 induces eIF2β phosphorylation and recruitment of NCK1 to eIF2, decreases eIF2α phosphorylation and bolsters TC recycling. Accordingly, eIF2β mediates the effect of mTORC1 on protein synthesis and proliferation. In addition, we demonstrate a formerly undocumented role for CK2 in regulation of translation initiation, whereby CK2 stimulates phosphorylation of eIF2β and simultaneously bolsters eIF4F complex assembly via the mTORC1/4E-BP pathway. These findings imply a previously unrecognized mode of translation regulation, whereby mTORC1 and CK2 coordinate TC and eIF4F complex assembly to stimulate cell proliferation.