The catalytic activity of the Src homology 2 (SH2) domain-containing tyrosine phosphatase, SHP-2, is required for virtually all of its signaling effects. Elucidating the molecular mechanisms of SHP-2 signaling, therefore, rests upon the identification of its target substrates. In this report, we have used SHP-2 substratetrapping mutants to identify the major vault protein (MVP) as a putative SHP-2 substrate. MVP is the predominant component of vaults that are cytoplasmic ribonucleoprotein complexes of unknown function. We show that MVP is dephosphorylated by SHP-2 in vitro and it forms an enzyme-substrate complex with SHP-2 in vivo. In response to epidermal growth factor (EGF), SHP-2 associates via its SH2 domains with tyrosyl-phosphorylated MVP. MVP also interacts with the activated form of the extracellular-regulated kinases (Erks) in response to EGF and a constitutive complex between tyrosyl-phosphorylated MVP, SHP-2, and the Erks was detected in MCF-7 breast cancer cells. Using MVP-deficient fibroblasts, we demonstrate that MVP cooperates with Ras for optimal EGF-induced Elk-1 activation and is required for cell survival. We propose that MVP functions as a novel scaffold protein for both SHP-2 and Erk. The regulation of MVP tyrosyl phosphorylation by SHP-2 may play an important role in cell survival signaling.Tyrosyl phosphorylation is a dynamic and reversible posttranslational modification catalyzed by the opposing actions of PTKs 1 and PTPs. The coordinated actions of PTKs and PTPs are crucial for the regulation of a multitude of physiological processes such as cell growth, differentiation, migration, adhesion, immune response, cell survival, and apoptosis. Although there is overwhelming evidence to demonstrate a critical role for PTPs in the regulation of cellular physiology, a detailed molecular understanding of the actions of many of these enzymes remains to be determined. In large part, the lack of a precise molecular basis for how PTPs function has stemmed from the difficult task of identifying their substrates. The SH2 domain-containing PTP, SHP-2, is an ubiquitously expressed PTP that has two SH2 domains at the NH 2 terminus, a PTP domain, and a COOH-terminal tail (1-3). Much evidence derived from genetic studies demonstrates that SHP-2 plays a positive role in transducing signals from receptor PTKs (4). Further evidence in mammalian systems for a positive signaling role for SHP-2 has been obtained from mice containing a targeted deletion within exon 3 of murine SHP-2 that deletes the NH 2 terminus SH2 domain (5). SHP-2 exon 3-deleted mice exhibit embryonic lethality (5). Fibroblasts derived from them are defective for the activation of the MAPKs such as the Erks in response to a number of polypeptide growth factors (5-8). These data, as well as those derived from others (9, 10), place SHP-2 upstream of the Erks, and in some cases upstream of either Ras (11), phosphatidylinositol 3-kinase (8, 12, 13) or the Src family kinases (6,14,15).The ability of SHP-2 to transduce downstream signals in such a d...

The cAMP-response element-binding protein (CREB)-binding protein and p300 are two highly conserved transcriptional coactivators and histone acetyltransferases that integrate signals from diverse signal transduction pathways in the nucleus and also link chromatin remodeling with transcription. In this report, we have examined the role of p300 in the control of the G 1 phase of the cell cycle in nontransformed immortalized human breast epithelial cells (MCF10A) and fibroblasts (MSU) by using adenovirus vectors expressing p300-specific antisense sequences. Quiescent MCF10A and MSU cells expressing p300-specific antisense sequences synthesized p300 at much reduced levels and exited G1 phase without serum stimulation. These cells also showed an increase in cyclin A and cyclin A-and E-associated kinase activities characteristic of S phase induction. Further analysis of the p300-depleted quiescent MCF10A cells revealed a 5-fold induction of c-MYC and a 2-fold induction of c-JUN. A direct target of c-MYC, CAD, which is requiredfor DNA synthesis, was also found to be up-regulated, indicating that up-regulation of c-MYC functionally contributed to DNA synthesis. Furthermore, S phase induction in p300-depleted cells was reversed when antisense c-MYC was expressed in these cells, indicating that up-regulation of c-MYC may directly contribute to S phase induction. Adenovirus E1A also induced DNA synthesis and increased the levels of c-MYC and c-JUN in serum-starved MCF10A cells in a p300-dependent manner. Our results suggest an important role of p300 in cell cycle regulation at G1 and raise the possibility that p300 may negatively regulate early response genes, including c-MYC and c-JUN, thereby preventing DNA synthesis in quiescent cells.

The mammalian protein synthesizing system is highly organized in vivo, and its substrate, tRNA, is channeled throughout the translation process. However, the cellular components responsible for this organization are not known. To examine this question a series of studies was carried out using intact and permeabilized Chinese hamster ovary cells. We show that cold shock dramatically reduces the protein synthetic capacity of these cells by as much as 95%. The loss of activity can be reversed by a short recovery period under conditions that allow energy metabolism to occur; transcription and translation during the recovery period are not needed. While individual components of the translation apparatus are not inactivated by the cold shock, the supramolecular organization of the system appears to be altered and F-actin levels are found to decrease. Resumption of protein synthesis during the recovery period coincides closely with the restoration of F-actin to normal levels. Moreover, disruption of actin filaments, but not microtubules, also leads to a major reduction in translation. These data support the conclusion that the cellular microfilament network plays an important role in the structure and function of the translation system and that perturbations of this network can have profound effects on protein synthesis.Considerable evidence has now accumulated showing that the mammalian protein synthetic machinery is highly organized in vivo, both structurally and functionally (1-3). For example, recent work with a permeabilized Chinese hamster ovary cell (CHO) 1 system, that closely mimics the living cell, demonstrated that a channeled tRNA cycle functions during the translation process (3), i.e. aminoacyl-tRNA and tRNA, the intermediates in the process, are directly transferred from the aminoacyl-tRNA synthetases, to the elongation factor, to the ribosome, and back to the synthetases, without dissociation into the cellular fluid. Such studies suggest that the various parts of the translation apparatus must associate as tRNA is transferred from one component to the next. In fact, since only small amounts of RNA and protein leak out of permeabilized cells under conditions in which large, exogenously-added macromolecules can enter, it is likely that the components of the translation machinery are permanently organized into a supramolecular structure (2). Additional evidence for an organized translation machinery comes from the isolation of a multienzyme aminoacyl-tRNA synthetase complex and other assemblies which contain multiple translation components (4 -8). Interactions among various individual components of the translation system also have been demonstrated in vitro, supporting the idea that similar associations occur in vivo (9 -12). Thus, elucidating the components responsible for this supramolecular organization and how they influence protein synthesis is essential for a complete understanding of the translation process in vivo.Several pieces of information suggest that the cytoskeletal elements of the cell serv...

We recently reported that the transcriptional coactivator and histone acetyltransferase p300 plays an important role in the G 1 phase of the cell cycle by negatively regulating c-myc and thereby preventing premature G 1 exit (Kolli, et al. Here, we show that antisense-mediated depletion of CBP in serum-deprived human cells leads to induction of c-myc and that such cells emerge from quiescence without growth factors at a rate comparable with that of p300-depleted cells. The CBP-depleted cells contained significantly reduced levels of the cyclin-dependent kinase inhibitor p21 and low levels of p107 and p130 (but not pRb) phosphorylation, suggesting that these factors, along with elevated levels of c-Myc, contribute to induction of DNA synthesis. Antisense c-Myc reversed the phosphorylation of p107 and p130 and the induction of S phase in CBP-depleted cells, indicating that up-regulation of c-myc is directly responsible for the induction of S phase. Furthermore, the serum-stimulated p300/CBP-depleted cells did not traverse beyond S phase, and a significant number of these cells died of apoptosis, which was not related to p53 levels. These cells also contained significantly higher levels of c-Myc compared with normal cells. When c-myc expression was blocked by antisense cMyc, the apoptosis of the serum-stimulated CBPdepleted cells was reversed, indicating that high levels of c-Myc contribute to apoptosis. Thus, despite their high degree of structural and functional similarities, normal levels of both p300 and CBP are essential for keeping c-myc in a repressed state in G 1 and thereby preventing inappropriate entry of cells into S phase. In addition, both these proteins also provide important functions in coordinated cell cycle progression. p300 and the CBP 1 are two large highly homologous and conserved transcriptional adapter proteins containing histone acetyltransferase activity that link transcription with chroma-

Amino acids are required for the activation of the mammalian target of rapamycin complex 1 (mTORC1), which plays a critical role in cell growth, proliferation, and metabolism. The branched-chain amino acid leucine is an essential nutrient that stimulates mTORC1 to promote protein synthesis by activating p70 S6 kinase 1 (S6K1). Here we show that the protein tyrosine phosphatase SHP-2 is required for leucine-induced activation of S6K1 in skeletal myoblasts. In response to leucine, S6K1 activation is inhibited in myoblasts either lacking SHP-2 expression or overexpressing a catalytically inactive mutant of SHP-2. Activation of S6K1 by leucine requires the mobilization of intracellular calcium (Ca 2؉ ), which we show is mediated by SHP-2 in an inositol-1,4,5-trisphosphate-dependent manner. Ectopic Ca 2؉ mobilization rescued the S6K1 activation defect in SHP-2-deficient myoblasts. SHP-2 was identified to act upstream of phospholipase C ␤4, linking it to the generation of nutrient-induced Ca 2؉ release and S6K1 phosphorylation. Consistent with these results, SHP-2-deficient myoblasts exhibited impaired leucine sensing, leading to defective autophagy and reduced myoblast size. These data define a new role for SHP-2 as a nutrient-sensing regulator in skeletal myoblasts that is required for the activation of S6K1.