The attenuation of protein synthesis via the phosphorylation of eIF2α is a major stress response of all eukaryotic cells. The growth-arrest- and DNA-damage-induced transcript 34 (GADD34) bound to the serine/threonine protein phosphatase 1 (PP1) is the necessary eIF2α phosphatase complex that returns mammalian cells to normal protein synthesis following stress. The molecular basis by which GADD34 recruits PP1 and its substrate eIF2α are not fully understood, hindering our understanding of the remarkable selectivity of the GADD34:PP1 phosphatase for eIF2α. Here, we report detailed structural and functional analyses of the GADD34:PP1 holoenzyme and its recruitment of eIF2α. The data highlight independent interactions of PP1 and eIF2α with GADD34, demonstrating that GADD34 functions as a scaffold both in vitro and in cells. This work greatly enhances our molecular understanding of a major cellular eIF2α phosphatase and establishes the foundation for future translational work.
Since the discovery of the prototypical Sprouty (Spry) protein in Drosophila, there has been an effort to determine how these novel modulators of the Ras/MAP-kinase pathway function. A clue to their mechanism of action comes from the several highly conserved sequences within all the currently known Spry isoforms: an approximately 110-residue cysteine-rich sequence in the C-terminal half that directs Spry proteins to a concentration of signaling proteins at the plasma membrane; a small motif surrounding a tyrosine residue (Y55 in human Spry2) that is responsible for interaction with other proteins. In cultured mammalian cells, hSpry2 inhibits epidermal growth factor receptor (EGFR) endocytosis and subsequently sustains the activation of MAP kinase but negatively regulates the same pathway following stimulation of fibroblast growth factor receptors (FGFRs). Current evidence indicates that Cbl is a key protein that interacts directly with Spry2 following activation of receptor tyrosine kinases (RTKs). It appears to be the ability of Cbl to interact as an E3 ubiquitin ligase on specific target proteins and as a docking protein in other contexts that dictates the differential effects Spry2 has on the Ras/MAP-kinase pathway following EGFR and FGFR activation.
Abstract Comprehensive understanding of aberrant splicing in gastric cancer is lacking. We RNA-sequenced 19 gastric tumor–normal pairs and identified 118 high-confidence tumor-associated (TA) alternative splicing events (ASEs) based on high-coverage sequencing and stringent filtering, and also identified 8 differentially expressed splicing factors (SFs). The TA ASEs occurred in genes primarily involved in cytoskeletal organization. We constructed a correlative network between TA ASE splicing ratios and SF expression, replicated it in independent gastric cancer data from The Cancer Genome Atlas and experimentally validated it by knockdown of the nodal SFs (PTBP1, ESRP2 and MBNL1). Each SF knockdown drove splicing alterations in several corresponding TA ASEs and led to alterations in cellular migration consistent with the role of TA ASEs in cytoskeletal organization. We have therefore established a robust network of dysregulated splicing associated with tumor invasion in gastric cancer. Our work is a resource for identifying oncogenic splice forms, SFs and splicing-generated tumor antigens as biomarkers and therapeutic targets.
<div>Abstract<p>The Sprouty proteins are increasingly being recognized to be deregulated in various types of cancers. This deregulation is often associated with aberrant signaling of receptor tyrosine kinases and its downstream effectors, leading to the mitogen-activated protein kinase (MAPK) signaling pathway. In human hepatocellular carcinoma, where the MAPK activity is enhanced via multiple hepatocarcinogenic factors, we observed a consistent reduced expression of the sprouty 2 (<i>Spry2</i>) transcript and protein in malignant hepatocytes compared with normal or cirrhotic hepatocytes. The expression pattern of <i>Spry2</i> in hepatocellular carcinoma resembles that of several potential tumor markers of hepatocellular carcinoma and also that of several angiogenic factors and growth factor receptors. In contrast to previous studies of <i>Spry2</i> down-regulation in other cancers, we have ruled out loss of heterozygosity or the methylation of promoter sites, two common mechanisms responsible for the silencing of genes with tumor suppressor properties. Functionally, we show that Spry2 inhibits both extracellular signal-regulated kinase signaling as well as proliferation in hepatocellular carcinoma cell lines, whereas knocking down <i>Spry2</i> levels in NIH3T3 cells causes mild transformation. Our study clearly indicates a role for <i>Spry2</i> in hepatocellular carcinoma, and an understanding of the regulatory controls of its expression could provide new means of regulating the angiogenic switch in this hypervascular tumor, thereby potentially controlling tumor growth. (Cancer Res 2006; 66(4): 2048-58)</p></div>
<div>Abstract<p>Sprouty (Spry) proteins were found to be endogenous inhibitors of the Ras/mitogen-activated protein kinase pathway that play an important role in the remodeling of branching tissues. We investigated <i>Spry</i> expression levels in various cancers and found that <i>Spry1</i> and <i>Spry2</i> were down-regulated consistently in breast cancers. Such prevalent patterns of down-regulation may herald the later application of these isoforms as tumor markers that are breast cancer specific and more profound than currently characterized markers. <i>Spry1</i> and <i>2</i> were expressed specifically in the luminal epithelial cells of breast ducts, with higher expression during stages of tissue remodeling when the epithelial ducts are forming and branching. These findings suggest that Sprys might be involved as a modeling counterbalance and surveillance against inappropriate epithelial expansion. The abrogation of endogenous Spry activity in MCF-7 cells by the overexpression of a previously characterized dominant-negative mutant of Spry, hSpry2<sup>Y55F</sup> resulted in enhanced cell proliferation <i>in vitro</i>. The hSpry2<sup>Y55F</sup> stably expressing cells also formed larger and greater number of colonies in the soft-agar assay. An <i>in vivo</i> nude mice assay showed a dramatic increase in the tumorigenic potential of hSpry2<sup>Y55F</sup> stable cells. The consistent down-regulation of <i>Spry1</i> and <i>2</i> in breast cancer and the experimental evidence using a dominant-negative hSpry2<sup>Y55F</sup> indicate that Spry proteins may actively maintain tissue integrity that runs amok when their expression is decreased below normal threshold levels. This alludes to a previously unrecognized role for Sprys in cancer development.</p></div>
Sprouty (Spry) proteins have been revealed as inhibitors of the Ras/mitogen-activated protein kinase (MAPK) cascade, a pathway crucial for developmental processes initiated by activation of various receptor tyrosine kinases.In COS-1 and Swiss 3T3 cells, all Spry isoforms translocate to the plasma membrane, notably ruffles, following activation.Here we show that microinjection of active Rac induced the translocation of Spry isoforms, indicating that the target of the Spry translocation domain (SpryTD) is downstream of active Rac.Targeted disruption of actin polymerization revealed that the SpryTD target appeared upstream of cytoskeletal rearrangements.Accumulated evidence indicated that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] is the likely SpryTD target.Human Spry2TD (hSpry2TD) binds to PtdIns(4,5)P 2 in vesicle-binding assays.hSpry2TD colocalizes with the pleckstrin homology domain of phospholipase C␦, which binds PtdIns(4,5)P 2 .The plasma membrane localization of hSpry2TD was abolished in ionomycin-treated MDCK cells or when PtdIns(4,5)P 2 was specifically dephosphorylated by overexpression of an engineered, green fluorescent proteintagged inositol 5-phosphatase.Similarly, Spred, a novel Ras/MAPK inhibitor recently found to contain the conserved cysteine-rich SpryTD, also translocated to peripheral membranes and bound to PtdIns(4,5)P 2 .Alignment of the Spry and Spred proteins led us to identify a translocation-defective point mutant, hSpry2 D252.Targeting of hSpry2 to PtdIns(4,5)P 2 was shown to be essential for the down-regulation of Ras/MAPK signaling.
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