Wars2 is a determinant of angiogenesis.

Coronary flow (CF) measured ex vivo is largely determined by capillary density that reflects angiogenic vessel formation in the heart in vivo. Here we exploit this relationship and show that CF in the rat is influenced by a locus on rat chromosome 2 that is also associated with cardiac capillary density. Mitochondrial tryptophanyl-tRNA synthetase (Wars2), encoding an L53F protein variant within the ATP-binding motif, is prioritized as the candidate at the locus by integrating genomic data sets. WARS2(L53F) has low enzyme activity and inhibition of WARS2 in endothelial cells reduces angiogenesis. In the zebrafish, inhibition of wars2 results in trunk vessel deficiencies, disordered endocardial-myocardial contact and impaired heart function. Inhibition of Wars2 in the rat causes cardiac angiogenesis defects and diminished cardiac capillary density. Our data demonstrate a pro-angiogenic function for Wars2 both within and outside the heart that may have translational relevance given the association of WARS2 with common human diseases

Phosphorylation at tyrosine 262 promotes GADD34 protein turnover.

In mammalian cells, metabolic and environmental stress increases the phosphorylation of the eukaryotic translational initiation factor, eIF2α, and attenuates global protein synthesis. Subsequent transcriptional activation of GADD34 assembles an eIF2α phosphatase that feeds back to restore mRNA translation. Active proteasomal degradation of GADD34 protein then reestablishes the sensitivity of cells to subsequent bouts of stress. Mass spectrometry established GADD34 phosphorylation on multiple serines, threonines, and tyrosines. Phosphorylation at tyrosine 262 enhanced the rate of the GADD34 protein turnover. Substrate-trapping studies identified TC-PTP (PTPN2) as a potential GADD34 phosphatase, recognizing phosphotyrosine 262. Reduced GADD34 protein levels in TC-PTP-null MEFs following ER stress emphasized the importance of TC-PTP in determining the cellular levels of GADD34 protein. The susceptibility of TC-PTP-null MEFs to ER stress-induced apoptosis was significantly ameliorated by ectopic expression of GADD34. The data suggested that GADD34 phosphorylation on tyrosine 262 modulates endoplasmic reticulum stress signaling and cell fate

The Cysteine-Rich Sprouty Translocation Domain Targets Mitogen-Activated Protein Kinase Inhibitory Proteins to Phosphatidylinositol 4,5-Bisphosphate in Plasma Membranes

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 Cδ, 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 protein-tagged 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

Structural and Functional Analysis of the GADD34:PP1 eIF2α Phosphatase

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

Chronic oxidative stress promotes GADD34-mediated phosphorylation of the TAR DNA-binding protein TDP-43, a modification linked to neurodegeneration

Oxidative and endoplasmic reticulum (ER) stresses are hallmarks of the pathophysiology of ALS and other neurodegenerative diseases. In these stresses, different kinases phosphorylate eukaryotic initiation factor eIF2α, enabling the translation of stress response genes; among these is GADD34, the protein product of which recruits the α-isoform of protein phosphatase 1 catalytic subunit (PP1α) and eIF2α to assemble a phosphatase complex catalyzing eIF2α dephosphorylation and resumption of protein synthesis. Aberrations in this pathway underlie the aforementioned disorders. Previous observations indicating that GADD34 is induced by arsenite, a thiol-directed oxidative stressor, in the absence of eIF2α phosphorylation suggest other roles for GADD34. Here, we report that arsenite-induced oxidative stress differs from thapsigargin- or tunicamycin-induced ER stress in promoting GADD34 transcription and the preferential translation of its mRNA in the absence of eIF2α phosphorylation. Arsenite also stabilized GADD34 protein, slowing its degradation. In response to oxidative stress, but not ER stress, GADD34 recruited TDP-43, and enhanced cytoplasmic distribution and cysteine modifications of TDP-43 promoted its binding to GADD34. Arsenite also recruited a TDP-43 kinase, casein kinase-1ϵ (CK1ϵ), to GADD34. Concomitant with TDP-43 aggregation and proteolysis after prolonged arsenite exposure, GADD34-bound CK1ϵ catalyzed TDP-43 phosphorylations at serines 409/410, which were diminished or absent in GADD34−/− cells. Our findings highlight that the phosphatase regulator, GADD34, also functions as a kinase scaffold in response to chronic oxidative stress and recruits CK1ϵ and oxidized TDP-43 to facilitate its phosphorylation, as seen in TDP-43 proteinopathies.ASTAR (Agency for Sci., Tech. and Research, S’pore)MOH (Min. of Health, S’pore)Published versio

Oxidative stress promotes SIRT1 recruitment to the GADD34/PP1 alpha complex to activate its deacetylase function

Phosphorylation of the eukaryotic translation initiation factor, eIF2 alpha, by stress-activated protein kinases and dephosphorylation by the growth arrest and DNA damage-inducible protein (GADD34)-containing phosphatase is a central node in the integrated stress response. Mass spectrometry demonstrated GADD34 acetylation at multiple lysines. Substituting K-315 and K-322 with alanines or glutamines did not impair GADD34's ability to recruit protein phosphatase 1 alpha (PP1 alpha) or eIF2 alpha, suggesting that GADD34 acetylation did not modulate eIF2 alpha phosphatase activity. Arsenite (Ars)-induced oxidative stress increased cellular GADD34 levels and enhanced Sirtuin 1 (SIRT1) recruitment to assemble a cytoplasmic complex containing GADD34, PP1 alpha, eIF2 alpha and SIRT1. Induction of GADD34 in WT MEFs paralleled the dephosphorylation of eIF2 alpha (phosphoserine-51) and SIRT1 (phosphoserine-47). By comparison, eIF2 alpha and SIRT1 were persistently phosphorylated in Ars-treated GADD34 -/- MEFs. Expressing WT GADD34, but not a mutant unable to bind PP1 alpha in GADD34 -/- MEFs restored both eIF2 alpha and SIRT1 dephosphorylation. SIRT1 dephosphorylation increased its deacetylase activity, measured in vitro and in cells. Loss of function of GADD34 or SIRT1 enhanced cellular p-eIF2 alpha levels and attenuated cell death following Ars exposure. These results highlighted a novel role for the GADD34/PP1 alpha complex in coordinating the dephosphorylation and reactivation of eIF2 alpha and SIRT1 to determine cell fate following oxidative stress

Direct Association of Sprouty-related Protein with an EVH1 Domain (SPRED) 1 or SPRED2 with DYRK1A Modifies Substrate/Kinase Interactions*

The mammalian SPRED (Sprouty-related protein with an EVH1 domain) proteins include a family of three members, SPRED1–3. Currently, little is known about their biochemistry. The best described, SPRED1, has been shown to inhibit the Ras/ERK pathway downstream of Ras. All three SPREDs have a cysteine-rich domain (CRD) that has high homology to the CRD of the Sprouty family of proteins, several of which are also Ras/ERK inhibitors. In the belief that binding partners would clarify SPRED function, we assayed for their associated proteins. Here, we describe the direct and endogenous interaction of SPRED1 and SPRED2 with the novel kinase, DYRK1A. DYRK1A has become the subject of recent research focus as it plays a central role in Caenorhabditis elegans oocyte maturation and egg activation, and there is strong evidence that it could be involved in Down syndrome in humans. Both SPRED1 and SPRED2 inhibit the ability of DYRK1A to phosphorylate its substrates, Tau and STAT3. This inhibition occurs via an interaction of the CRD of the SPREDs with the kinase domain of DYRK1A. DYRK1A substrates must bind to the kinase to enable phosphorylation, and SPRED proteins compete for the same binding site to modify this process. Our accumulated evidence indicates that the SPRED proteins are likely physiological modifiers of DYRK1A

Structure of a novel phosphotyrosine-binding domain in Hakai that targets E-cadherin

10.1038/emboj.2011.496EMBO Journal3151308-1319EMJO