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.

Eukaryotic cells have developed specific mechanisms to overcome environmental stress. Here we show that the Src homology 2/3 (SH2/SH3) domain-containing protein Nck-1 prevents the unfolded protein response normally induced by pharmacological endoplasmic reticulum (ER) stress agents. Overexpression of Nck-1 enhances protein translation, whereas it abrogates eukaryotic initiation factor 2␣ (eIF2␣) phosphorylation and inhibition of translation in response to tunicamycin or thapsigargin treatment. Nck-1 overexpression also attenuates induction of the ER chaperone, the immunoglobulin heavy chain-binding protein (BiP), and impairs cell survival in response to thapsigargin. We provided evidence that in these conditions, the effects of Nck on the unfolded protein response (UPR) involve its second SH3 domain and a calyculin A-sensitive phosphatase activity. In addition, we demonstrated that protein translation is reduced in mouse embryonic fibroblasts lacking both Nck isoforms and is enhanced in similar cells expressing high levels of Nck-1. In these various mouse embryonic fibroblasts, we also provided evidence that Nck modulates the activation of the ER resident eIF2␣ kinase PERK and consequently the phosphorylation of eIF2␣ on Ser-51 in response to stress. Our study establishes that Nck is required for optimal protein translation and demonstrates that, in addition to its adaptor function in mediating signaling from the plasma membrane, Nck also mediates signaling from the ER membrane compartment.

In mammals, Nck represented by two genes, is a 47-kDa SH2͞SH3 domain-containing protein lacking intrinsic enzymatic function. Here, we reported that the first and the third SH3 domains of Nck-1 interact with the C-terminal region of the ␤ subunit of the eukaryotic initiation factor 2 (eIF2␤). Binding of eIF2␤ was specific to the SH3 domains of Nck-1, and in vivo, the interaction Nck͞eIF2␤ was demonstrated by reciprocal coimmunoprecipitations. In addition, Nck was detected in a molecular complex with eIF2␤ in an enriched ribosomal fraction, whereas no other SH2͞SH3 domain-containing adapters were found. Cell fractionation studies demonstrated that the presence of Nck in purified ribosomal fractions was enhanced after insulin stimulation, suggesting that growth factors dynamically regulate translocation of Nck to ribosomes. In HEK293 cells, we observed that transient overexpression of Nck-1 significantly enhanced Cap-dependent and -independent protein translation. This effect of Nck-1 required the integrity of its first and third SH3 domains originally found to interact with eIF2␤. Finally, in vitro, Nck-1 also increased protein translation, revealing a direct role for Nck-1 in this process. Our study demonstrates that in addition to mediate receptor tyrosine kinase signaling, Nck-1 modulates protein translation potentially through its direct interaction with an intrinsic component of the protein translation machinery. T ranslation initiation is a complex process in which initiator tRNA (Met-tRNA i ) and the 40S and 60S ribosomal subunits of initiator tRNA (Met-tRNA i ) are assembled into 80S ribosomes at the initiation codon of mRNA by the coordinated action of the eukaryotic initiation factors (eIFs). Thus far, signaling-dependent events regulating eIFs have involved changes in their intrinsic activity or protein-interacting properties, resulting from their phosphorylation͞dephosphorylation and͞or sequestration into inactive complex. For example, eIF2 is a molecular complex of three subunits (␣, ␤, and ␥) responsible for one of the earliest steps in the initiation of protein synthesis (1). eIF2 forms a ternary complex with the Met-tRNA and GTP and, in collaboration with other initiation factors, binds the 40S ribosomal subunit to give rise to the preinitiation 43S complex (2). Inhibition of protein synthesis correlates with the phosphorylation of the eIF2␣ subunit (1) by the heme-regulated eIF2␣ kinase (HRI) (3), the IFN-inducible RNA-dependent protein kinase (PKR) (4), the serum starvation kinase (GCN2) (5), and the endoplasmic reticulum stress kinase (PERK) (6). Phosphorylation of eIF2␣ on Ser-51 by these eIF2␣ kinases inhibits the early steps of translation by blocking on eIF2 the exchange of GDP for GTP, a reaction under the control of the guanine exchange factor, eIF2B (7,8). Thus, growth factors may enhance initiation of protein translation by preventing activation of eIF2␣ kinases or by activating specific phosphatases to maintain low levels of eIF2␣ phosphorylation. In contrast to eIF2␣, the ␤ and ␥ subu...

The proapoptotic protein Bad is a key player in cell survival decisions, and is regulated post-translationally by several signaling networks. We expressed Bad in mouse embryonic fibroblasts to sensitize them to apoptosis, and tested cell lines derived from knock-out mice to establish the significance of the interaction between the adaptor protein Grb10 and the Raf-1 protein kinase in anti-apoptotic signaling pathways targeting Bad. When compared with wild-type cells, both Grb10 and Raf-1-deficient cells exhibit greatly enhanced sensitivity to apoptosis in response to Bad expression. Structure-function analysis demonstrates that, in this cellular model, the SH2, proline-rich, and pleckstrin homology domains of Grb10, as well as its Akt phosphorylation site and consequent binding by 14-3-3, are all necessary for its anti-apoptotic functions. As for Raf-1, its kinase activity, its ability to be phosphorylated by Src on Tyr-340/341 and the binding of its Ras-associated domain to the Grb10 SH2 domain are all necessary to promote cell survival. Silencing the expression of either Grb10 or Raf-1 by small interfering RNAs as well as mutagenesis of specific serine residues on Bad, coupled with signaling inhibitor studies, all indicate that Raf-1 and Grb10 are required for the ability of both the phosphatidylinositol 3-kinase/Akt and MAP kinase pathways to modulate the phosphorylation and inactivation of Bad. Because total Raf-1, ERK, and Akt kinase activities are not impaired in the absence of Grb10, we propose that this adapter protein creates a subpopulation of Raf-1 with specific anti-apoptotic activity.The best known models for receptor-mediated anti-apoptotic cascades include interleukin-3 signaling in hematopoietic cells and insulin-like growth factor 1 (IGF-1) 3 signaling in fibroblasts. To date, several distinct anti-apoptotic signaling pathways have been characterized (1, 2) including activation of the Akt kinase following the production of phosphatidylinositol 3,4-biphosphate by phosphoinositol 3-kinase (PI3K) (3, 4). A second anti-apoptotic pathway is the mitogenic MAPK cascade that is characterized by the sequential activation of the Raf, MEK, ERK, and p90 RSK kinases (5, 6). Finally, Peruzzi et al. (6) have identified an additional IGF-1R-dependent pathway that requires the mitochondrial relocalization of a complex containing a 14-3-3 protein, the E3 ubiquitin ligase Nedd4, and the Raf-1 kinase (6). One of the end points of these signaling pathways is the serine phosphorylation and inactivation of the Bcl-2 protein family member Bad (7). This phosphorylation event is followed by the binding of Bad to 14-3-3 proteins and its sequestration in the cytoplasm, thus preventing it from inactivating the anti-apoptotic proteins Bcl-2 and Bcl-x L on the mitochondrial surface (8). Several serine residues have been identified as sites for Bad-inactivating phosphorylation; Akt promotes cell survival by phosphorylating serine 136 (9, 10), serine 112 is the substrate for the MAPK downstream signaling effector p90 RSK (5, 11...

The e ects of Fibroblast Growth Factor-2 (FGF-2) on breast cancer cell DNA synthesis are controversial. To elucidate the mechanisms by which FGF-2 stimulates or inhibits DNA synthesis, we analysed FGF-2 signaling pathways in breast cancer MCF-7 and MCF-7 cells overexpressing Ha-Ras (MCF-7ras). We found that FGF-2-induction of DNA synthesis correlates with Ras transient activation, FRS-2 tyrosine phosphorylation and low level of expression of p66 Shc . In addition, Nckassociated proteins are highly tyrosine phosphorylated and JNK reaches a higher level of activation when FGF-2 triggers DNA synthesis. Interestingly upon FGF-2 treatment, JNK activation and DNA synthesis are dependent on Rac-1 activity. These results con®rm that in MCF-7 cells, induction of DNA synthesis by FGF-2 requires a transient activation of the Ras/MAPK cascade and demonstrates for the ®rst time that intact Rac-1 and Nck signaling networks are required.