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GST (B-14) antibody

RRID:AB_627677

Antibody ID

AB_627677

Target Antigen

GST (B-14) c elegans/worm, schistosoma japonicum

Proper Citation

(Santa Cruz Biotechnology Cat# sc-138, RRID:AB_627677)

Clonality

monoclonal antibody

Comments

validation status unknown check with seller; recommendations: Immunoprecipitation; Immunofluorescence; WB, IP; Western Blot

Host Organism

mouse

Vendor

Santa Cruz Biotechnology

GRASP55 Senses Glucose Deprivation through O-GlcNAcylation to Promote Autophagosome-Lysosome Fusion.

  • Zhang X
  • Dev. Cell
  • 2018 Apr 23

Literature context:


Abstract:

The Golgi apparatus is the central hub for protein trafficking and glycosylation in the secretory pathway. However, how the Golgi responds to glucose deprivation is so far unknown. Here, we report that GRASP55, the Golgi stacking protein located in medial- and trans-Golgi cisternae, is O-GlcNAcylated by the O-GlcNAc transferase OGT under growth conditions. Glucose deprivation reduces GRASP55 O-GlcNAcylation. De-O-GlcNAcylated GRASP55 forms puncta outside of the Golgi area, which co-localize with autophagosomes and late endosomes/lysosomes. GRASP55 depletion reduces autophagic flux and results in autophagosome accumulation, while expression of an O-GlcNAcylation-deficient mutant of GRASP55 accelerates autophagic flux. Biochemically, GRASP55 interacts with LC3-II on the autophagosomes and LAMP2 on late endosomes/lysosomes and functions as a bridge between LC3-II and LAMP2 for autophagosome and lysosome fusion; this function is negatively regulated by GRASP55 O-GlcNAcylation. Therefore, GRASP55 senses glucose levels through O-GlcNAcylation and acts as a tether to facilitate autophagosome maturation.

Funding information:
  • NINDS NIH HHS - NS43987(United States)

EGFR-Phosphorylated Platelet Isoform of Phosphofructokinase 1 Promotes PI3K Activation.

  • Lee JH
  • Mol. Cell
  • 2018 Apr 19

Literature context:


Abstract:

EGFR activates phosphatidylinositide 3-kinase (PI3K), but the mechanism underlying this activation is not completely understood. We demonstrated here that EGFR activation resulted in lysine acetyltransferase 5 (KAT5)-mediated K395 acetylation of the platelet isoform of phosphofructokinase 1 (PFKP) and subsequent translocation of PFKP to the plasma membrane, where the PFKP was phosphorylated at Y64 by EGFR. Phosphorylated PFKP binds to the N-terminal SH2 domain of p85α, which is distinct from binding of Gab1 to the C-terminal SH2 domain of p85α, and recruited p85α to the plasma membrane resulting in PI3K activation. PI3K-dependent AKT activation results in enhanced phosphofructokinase 2 (PFK2) phosphorylation and production of fructose-2,6-bisphosphate, which in turn promotes PFK1 activation. PFKP Y64 phosphorylation-enhanced PI3K/AKT-dependent PFK1 activation and GLUT1 expression promoted the Warburg effect, tumor cell proliferation, and brain tumorigenesis. These findings underscore the instrumental role of PFKP in PI3K activation and enhanced glycolysis through PI3K/AKT-dependent positive-feedback regulation.

Funding information:
  • NCI NIH HHS - T32 CA121938(United States)

Nuclear Receptor Nur77 Facilitates Melanoma Cell Survival under Metabolic Stress by Protecting Fatty Acid Oxidation.

  • Li XX
  • Mol. Cell
  • 2018 Feb 1

Literature context:


Abstract:

Fatty acid oxidation (FAO) is crucial for cells to overcome metabolic stress by providing ATP and NADPH. However, the mechanism by which FAO is regulated in tumors remains elusive. Here we show that Nur77 is required for the metabolic adaptation of melanoma cells by protecting FAO. Glucose deprivation activates ERK2 to phosphorylate and induce Nur77 translocation to the mitochondria, where Nur77 binds to TPβ, a rate-limiting enzyme in FAO. Although TPβ activity is normally inhibited by oxidation under glucose deprivation, the Nur77-TPβ association results in Nur77 self-sacrifice to protect TPβ from oxidation. FAO is therefore able to maintain NADPH and ATP levels and prevent ROS increase and cell death. The Nur77-TPβ interaction further promotes melanoma metastasis by facilitating circulating melanoma cell survival. This study demonstrates a novel regulatory function of Nur77 with linkage of the FAO-NADPH-ROS pathway during metabolic stress, suggesting Nur77 as a potential therapeutic target in melanoma.

Funding information:
  • NIAMS NIH HHS - R01 AR052785(United States)

Subdomain 2, Not the Transmembrane Domain, Determines the Dimerization Partner of Growth Hormone Receptor and Prolactin Receptor.

  • Liu Y
  • Endocrinology
  • 2017 Oct 1

Literature context:


Abstract:

Growth hormone receptor (GHR) and prolactin (PRL) receptor (PRLR) are homologous transmembrane class I cytokine receptors. In humans, GH interacts with GHR homodimers or PRLR homodimers and PRL interacts with only PRLR homodimers to promote signaling. In human breast cancer cells endogenously expressing both receptors, GHR and PRLR specifically coimmunoprecipitate. We previously devised a split luciferase complementation assay to study GHR and PRLR assemblages. In this technique, firefly luciferase is split into two fragments (N- and C-terminal fragments of the luciferase), each without enzyme activity and tethered to the tails of two receptors. The fragments restore luciferase activity when brought close to each other by the receptors. Real-time ligand-induced complementation changes reflect the arrangement of receptors and indicate that GHR/PRLR is arranged as a heteromultimer comprised of GHR-GHR homodimers and PRLR-PRLR homodimers. We now dissect determinants for GHR and PRLR homodimerization versus heteroassociation. GHR and PRLR have extracellular domains comprised of the ligand-binding N-terminal subdomain 1 and a membrane-proximal subdomain 2 (S2), which fosters receptor-receptor contact. Based on previous studies of S2 versus the transmembrane domain (TMD) in GHR dimerization, we constructed GHR(PRLRS2), GHR(PRLRS2-TMD), and GHR(PRLRTMD), replacing GHR's S2 alone, S2 plus TMD, and TMD alone with PRLR's counterpart. We tested by complementation the ability of these chimeras and GHR or PRLR to homodimerize or heteroassociate. Comparing various combinations, we found GHR(PRLRS2) and GHR(PRLRS2-TMD) behaved as PRLR, whereas GHR(PRLRTMD) behaved as GHR regarding their dimerization partners. We conclude that S2 of GHR and PRLR, rather than their TMDs, determines their dimerization partner.

The Elongation Factor Spt6 Maintains ESC Pluripotency by Controlling Super-Enhancers and Counteracting Polycomb Proteins.

  • Wang AH
  • Mol. Cell
  • 2017 Oct 19

Literature context:


Abstract:

Spt6 coordinates nucleosome dis- and re-assembly, transcriptional elongation, and mRNA processing. Here, we report that depleting Spt6 in embryonic stem cells (ESCs) reduced expression of pluripotency factors, increased expression of cell-lineage-affiliated developmental regulators, and induced cell morphological and biochemical changes indicative of ESC differentiation. Selective downregulation of pluripotency factors upon Spt6 depletion may be mechanistically explained by its enrichment at ESC super-enhancers, where Spt6 controls histone H3K27 acetylation and methylation and super-enhancer RNA transcription. In ESCs, Spt6 interacted with the PRC2 core subunit Suz12 and prevented H3K27me3 accumulation at ESC super-enhancers and associated promoters. Biochemical as well as functional experiments revealed that Spt6 could compete for binding of the PRC2 methyltransferase Ezh2 to Suz12 and reduce PRC2 chromatin engagement. Thus, in addition to serving as a histone chaperone and transcription elongation factor, Spt6 counteracts repression by opposing H3K27me3 deposition at critical genomic regulatory regions.

Funding information:
  • Intramural NIH HHS - ZIA AR041126-17()

Collaboration between Distinct Rab Small GTPase Trafficking Circuits Mediates Bacterial Clearance from the Bladder Epithelium.

  • Miao Y
  • Cell Host Microbe
  • 2017 Sep 13

Literature context:


Abstract:

Rab small GTPases control membrane trafficking through effectors that recruit downstream mediators such as motor proteins. Subcellular trafficking typically involves multiple Rabs, with each specific step mediated by a distinct Rab protein. We describe a collaboration between two distinct Rab-protein-orchestrated trafficking circuits in bladder epithelial cells (BECs) that expels intracellular uropathogenic Escherichia coli (UPEC) from their intracellular niche. RAB11a and RAB27b and their trafficking circuitry are simultaneously involved in UPEC expulsion. While RAB11a recruits its effector RAB11FIP3 and cytoskeletal motor Dynein, RAB27b mobilizes the effector MyRIP and motor Myosin VIIa to mediate bacterial expulsion. This collaboration is coordinated by deposition of the exocyst complex on bacteria-containing vesicles, an event triggered by the innate receptor Toll-like receptor 4. Both RAB11a and RAB27b are recruited and activated by the exocyst complex components SEC6/SEC15. Thus, the cell autonomous defense system can mobilize and coalesce multiple subcellular trafficking circuitries to combat infections.

Nucleus-Translocated ACSS2 Promotes Gene Transcription for Lysosomal Biogenesis and Autophagy.

  • Li X
  • Mol. Cell
  • 2017 Jun 1

Literature context:


Abstract:

Overcoming metabolic stress is a critical step in tumor growth. Acetyl coenzyme A (acetyl-CoA) generated from glucose and acetate uptake is important for histone acetylation and gene expression. However, how acetyl-CoA is produced under nutritional stress is unclear. We demonstrate here that glucose deprivation results in AMP-activated protein kinase (AMPK)-mediated acetyl-CoA synthetase 2 (ACSS2) phosphorylation at S659, which exposed the nuclear localization signal of ACSS2 for importin α5 binding and nuclear translocation. In the nucleus, ACSS2 binds to transcription factor EB and translocates to lysosomal and autophagy gene promoter regions, where ACSS2 incorporates acetate generated from histone acetylation turnover to locally produce acetyl-CoA for histone H3 acetylation in these regions and promote lysosomal biogenesis, autophagy, cell survival, and brain tumorigenesis. In addition, ACSS2 S659 phosphorylation positively correlates with AMPK activity in glioma specimens and grades of glioma malignancy. These results underscore the significance of nuclear ACSS2-mediated histone acetylation in maintaining cell homeostasis and tumor development.

Funding information:
  • NCI NIH HHS - R01 CA109035()
  • NCI NIH HHS - R01 CA169603()
  • NCI NIH HHS - R01 CA204996()
  • NINDS NIH HHS - R01 NS094615()

Mechanisms of Ubiquitin-Nucleosome Recognition and Regulation of 53BP1 Chromatin Recruitment by RNF168/169 and RAD18.

  • Hu Q
  • Mol. Cell
  • 2017 May 18

Literature context:


Abstract:

The protein 53BP1 plays a central regulatory role in DNA double-strand break repair. 53BP1 relocates to chromatin by recognizing RNF168-mediated mono-ubiquitylation of histone H2A Lys15 in the nucleosome core particle dimethylated at histone H4 Lys20 (NCP-ubme). 53BP1 relocation is terminated by ubiquitin ligases RNF169 and RAD18 via unknown mechanisms. Using nuclear magnetic resonance (NMR) spectroscopy and biochemistry, we show that RNF169 bridges ubiquitin and histone surfaces, stabilizing a pre-existing ubiquitin orientation in NCP-ubme to form a high-affinity complex. This conformational selection mechanism contrasts with the low-affinity binding mode of 53BP1, and it ensures 53BP1 displacement by RNF169 from NCP-ubme. We also show that RAD18 binds tightly to NCP-ubme through a ubiquitin-binding domain that contacts ubiquitin and nucleosome surfaces accessed by 53BP1. Our work uncovers diverse ubiquitin recognition mechanisms in the nucleosome, explaining how RNF168, RNF169, and RAD18 regulate 53BP1 chromatin recruitment and how specificity can be achieved in the recognition of a ubiquitin-modified substrate.

Funding information:
  • NCI NIH HHS - R01 CA132878()
  • NIGMS NIH HHS - R01 GM116829()

Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing.

  • Moura M
  • Elife
  • 2017 May 2

Literature context:


Abstract:

Faithfull genome partitioning during cell division relies on the Spindle Assembly Checkpoint (SAC), a conserved signaling pathway that delays anaphase onset until all chromosomes are attached to spindle microtubules. Mps1 kinase is an upstream SAC regulator that promotes the assembly of an anaphase inhibitor through a sequential multi-target phosphorylation cascade. Thus, the SAC is highly responsive to Mps1, whose activity peaks in early mitosis as a result of its T-loop autophosphorylation. However, the mechanism controlling Mps1 inactivation once kinetochores attach to microtubules and the SAC is satisfied remains unknown. Here we show in vitro and in Drosophila that Protein Phosphatase 1 (PP1) inactivates Mps1 by dephosphorylating its T-loop. PP1-mediated dephosphorylation of Mps1 occurs at kinetochores and in the cytosol, and inactivation of both pools of Mps1 during metaphase is essential to ensure prompt and efficient SAC silencing. Overall, our findings uncover a mechanism of SAC inactivation required for timely mitotic exit.

Celastrol-Induced Nur77 Interaction with TRAF2 Alleviates Inflammation by Promoting Mitochondrial Ubiquitination and Autophagy.

  • Hu M
  • Mol. Cell
  • 2017 Apr 6

Literature context:


Abstract:

Mitochondria play an integral role in cell death, autophagy, immunity, and inflammation. We previously showed that Nur77, an orphan nuclear receptor, induces apoptosis by targeting mitochondria. Here, we report that celastrol, a potent anti-inflammatory pentacyclic triterpene, binds Nur77 to inhibit inflammation and induce autophagy in a Nur77-dependent manner. Celastrol promotes Nur77 translocation from the nucleus to mitochondria, where it interacts with tumor necrosis factor receptor-associated factor 2 (TRAF2), a scaffold protein and E3 ubiquitin ligase important for inflammatory signaling. The interaction is mediated by an LxxLL motif in TRAF2 and results not only in the inhibition of TRAF2 ubiquitination but also in Lys63-linked Nur77 ubiquitination. Under inflammatory conditions, ubiquitinated Nur77 resides at mitochondria, rendering them sensitive to autophagy, an event involving Nur77 interaction with p62/SQSTM1. Together, our results identify Nur77 as a critical intracellular target for celastrol and unravel a mechanism of Nur77-dependent clearance of inflamed mitochondria to alleviate inflammation.

Funding information:
  • NIDA NIH HHS - 1P30 DA035756-01(United States)

IRBIT controls apoptosis by interacting with the Bcl-2 homolog, Bcl2l10, and by promoting ER-mitochondria contact.

  • Bonneau B
  • Elife
  • 2016 Dec 20

Literature context:


Abstract:

IRBIT is a molecule that interacts with the inositol 1,4,5-trisphosphate (IP3)-binding pocket of the IP3 receptor (IP3R), whereas the antiapoptotic protein, Bcl2l10, binds to another part of the IP3-binding domain. Here we show that Bcl2l10 and IRBIT interact and exert an additive inhibition of IP3R in the physiological state. Moreover, we found that these proteins associate in a complex in mitochondria-associated membranes (MAMs) and that their interplay is involved in apoptosis regulation. MAMs are a hotspot for Ca2+ transfer between endoplasmic reticulum (ER) and mitochondria, and massive Ca2+ release through IP3R in mitochondria induces cell death. We found that upon apoptotic stress, IRBIT is dephosphorylated, becoming an inhibitor of Bcl2l10. Moreover, IRBIT promotes ER mitochondria contact. Our results suggest that by inhibiting Bcl2l10 activity and promoting contact between ER and mitochondria, IRBIT facilitates massive Ca2+ transfer to mitochondria and promotes apoptosis. This work then describes IRBIT as a new regulator of cell death.

Funding information:
  • Howard Hughes Medical Institute - R01 NS036715(United States)
  • NCATS NIH HHS - UL1 TR001105(United States)

The novel SH3 domain protein Dlish/CG10933 mediates fat signaling in Drosophila by binding and regulating Dachs.

  • Zhang Y
  • Elife
  • 2016 Oct 3

Literature context:


Abstract:

Much of the Hippo and planar cell polarity (PCP) signaling mediated by the Drosophila protocadherin Fat depends on its ability to change the subcellular localization, levels and activity of the unconventional myosin Dachs. To better understand this process, we have performed a structure-function analysis of Dachs, and used this to identify a novel and important mediator of Fat and Dachs activities, a Dachs-binding SH3 protein we have named Dlish. We found that Dlish is regulated by Fat and Dachs, that Dlish also binds Fat and the Dachs regulator Approximated, and that Dlish is required for Dachs localization, levels and activity in both wild type and fat mutant tissue. Our evidence supports dual roles for Dlish. Dlish tethers Dachs to the subapical cell cortex, an effect partly mediated by the palmitoyltransferase Approximated under the control of Fat. Conversely, Dlish promotes the Fat-mediated degradation of Dachs.

Funding information:
  • NICHD NIH HHS - T32 HD007516(United States)