Phosphorylation of GSTP1 by EGFR Promotes Formation of the Inhibitory GSTP1-JNK Complex and Suppresses JNK Downstream Signaling and Apoptosis in Brain Tumor Cells [Molecular Bases of Disease]

October 1st, 2015 by

Under normal physiologic conditions, the GSTP1 protein exists intracellularly as a dimer in reversible equilibrium with its monomeric subunits. In the latter form, GSTP1 binds to the MAP kinase, JNK, and inhibits JNK downstream signaling. In tumor cells, which frequently are characterized by constitutively high GSTP1 expression, GSTP1 undergoes phosphorylation by EGFR at tyrosine residues 3, 7 and 198. Here, we report on the effect of this EGFR-dependent GSTP1 tyrosine phosphorylation on the interaction of GSTP1 with JNK, on the regulation of JNK downstream signaling by GSTP1 and on tumor cell survival. Using in vitro and in vivo growing human brain tumors, we show that tyrosine phosphorylation shifts the GSTP1 dimer-monomer equilibrium to the monomeric state and facilitates the formation of the GSTP1-JNK complex, in which JNK is functionally inhibited. Targeted mutagenesis and functional analysis demonstrated that the increased GSTP1 binding to JNK results from phosphorylation of the GSTP1 C-terminal Tyr198 by EGFR and is associated with a more than 2.5-fold decrease in JNK downstream signaling and a significant suppression of both spontaneous and drug-induced apoptosis in the tumor cells. The findings define a novel mechanism of regulatory control of JNK signaling that is mediated by the EGFR/GSTP1 crosstalk and provides a survival advantage for tumors with activated EGFR and high GSTP1 expression. The results lay the foundation for a novel strategy of dual EGFR/GSTP1 for treating EGFR+ve, GSTP1 expressing GBMs.
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Alteration/Deficiency in Activation 3 (ADA3) Protein, a Cell Cycle Regulator, Associates with Centromere through CENP-B and Regulates Chromosome Segregation [DNA and Chromosomes]

October 1st, 2015 by

Alteration/Deficiency in Activation 3 (ADA3) is a conserved component of several transcriptional co-activator and histone acetyl transferase (HAT) complexes. Recently, we generated Ada3 knockout mice and demonstrated that deletion of Ada3 leads to early embryonic lethality. Use of Ada3FL/FL mouse embryonic fibroblasts (MEFs) with deletion of Ada3 using adenovirus Cre showed a critical role of ADA3 in cell cycle progression through mitosis. Here, we demonstrate an association of ADA3 with high order repeat (HOR) region of the alpha-satellite region on human X chromosome centromeres that is consistent with its role in mitosis. Given the role of centromere proteins (CENPs) in mitosis, we next analyzed if ADA3 associates with centromere through CENPs. Both in vivo proximity ligation assay and immunofluorescence studies confirmed the association of ADA3 with CENP-B protein, a highly conserved centromeric protein which binds to the 17-bp DNA sequences on alpha-satellite DNA. Deletional analysis showed ADA3 directly associates with CENP-B through its N-terminus and a CENP-B binding deficient mutant of ADA3 was incompetent in cell proliferation rescue. Notably, knockdown of ADA3 decreased binding of CENP-B onto the centromeres, suggesting ADA3 is required for the loading of CENP-B on to the centromeres. Finally, we show that deletion of Ada3 from Ada3FL/FLMEFs exhibited various chromosome segregation defects. Taken together, we demonstrate a novel ADA3 interaction with CENP-B-centromere that may account for its previously known function in mitosis. This study together with its known function in maintaining genomic stability and its mis-localization in cancers, suggests an important role of ADA3 in mitosis.
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Syk is Recruited to Stress Granules and Promotes their Clearance through Autophagy [Cell Biology]

October 1st, 2015 by

Syk is a cytoplasmic kinase that serves multiple functions within the immune system to couple receptors for antigens and antigen-antibody complexes to adaptive and innate immune responses. Recent studies have identified additional roles for the kinase in cancer cells where its expression can either promote or suppress tumor cell growth depending on the context. Proteomic analyses of Syk-binding proteins identified several interacting partners also found to be recruited to stress granules. We show here that the treatment of cells with inducers of stress granule formation leads to the recruitment of Syk to these protein-RNA complexes. This recruitment requires the phosphorylation of Syk on tyrosine and results in the phosphorylation of proteins at or near the stress granule. Grb7 is identified as a Syk-binding protein involved in the recruitment of Syk to the stress granule. This recruitment promotes the formation of autophagosomes and the clearance of stress granules from the cell once the stress is relieved, enhancing the ability of cells to survive the stress stimulus.

Concentration-Dependent Effects of Nuclear Lamins on Nuclear Size in Xenopus and Mammalian Cells [Developmental Biology]

October 1st, 2015 by Jevtić, P., Edens, L. J., Li, X., Nguyen, T., Chen, P., Levy, D. L.

A fundamental question in cell biology concerns the regulation of organelle size. While nuclear size is exquisitely controlled in different cell types, inappropriate nuclear enlargement is used to diagnose and stage cancer. Clarifying the functional significance of nuclear size necessitates an understanding of the mechanisms and proteins that control nuclear size. One structural component implicated in the regulation of nuclear morphology is the nuclear lamina, a meshwork of intermediate lamin filaments that lines the inner nuclear membrane. However, there has not been a systematic investigation of how the level and type of lamin expression influences nuclear size, in part due to difficulties in precisely controlling lamin expression levels in vivo. In this study, we circumvent this limitation by studying nuclei in Xenopus laevis egg and embryo extracts, open biochemical systems that allow for precise manipulation of lamin levels by the addition of recombinant proteins. We find that nuclear growth and size are sensitive to the levels of nuclear lamins, with low and high concentrations increasing and decreasing nuclear size, respectively. Interestingly, each type of lamin that we tested (lamins B1, B2, B3, and A) similarly affected nuclear size whether added alone or in combination, suggesting that total lamin concentration, and not lamin type, is more critical to determining nuclear size. Furthermore, we show that altering lamin levels in vivo, both in Xenopus embryos and mammalian tissue culture cells, also impacts nuclear size. These results have implications for normal development and carcinogenesis where both nuclear size and lamin expression levels change.

Poly(ADP-ribosyl)ation of FOXP3 Mediated by PARP-1 Regulates the Function of Regulatory T Cells [Cell Biology]

October 1st, 2015 by Luo, X., Nie, J., Wang, S., Chen, Z., Chen, W., Li, D., Hu, H., Li, B.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an ADP-ribosylating enzyme participating in diverse cellular functions. The roles of PARP-1 in the immune system, however, have not been well understood. Here we find that PARP-1 interacts with FOXP3 and induces its poly(ADP-ribosyl)ation. By using PARP-1 inhibitors, we show that reduced poly(ADP-ribosyl)ation of FOXP3 results in not only FOXP3 stabilization and increased FOXP3 downstream genes, but also enhanced suppressive function of regulatory T cells (Treg). Our results suggest that PARP-1 negatively regulates the suppressive function of Treg cells at the post-translational level via FOXP3 poly(ADP-ribosyl)ation. This finding has implications in developing PARP-1 inhibitors as potential agents for the prevention and treatment of autoimmune diseases.

Structural Basis for Clonal Diversity of the Public T Cell Response to a Dominant Human Cytomegalovirus Epitope [Protein Structure and Folding]

October 1st, 2015 by

Cytomegalovirus (CMV) is a ubiquitous and persistent human pathogen that is kept in check by CD8+ cytotoxic T lymphocytes (CTLs). Individuals expressing the major histocompatibility complex (MHC) class I molecule HLA-A2 produce CTLs bearing T cell receptors (TCRs) that recognize the immunodominant CMV epitope NLVPMVATV (NLV). The NLV-specific T cell repertoire is characterized by a high prevalence of TCRs that are frequently observed in multiple unrelated individuals. These public TCRs feature identical, or nearly identical, complementarity-determining region 3α (CDR3α) and/or CDR3β sequences. The TCRs may express public CDR3α motifs alone, public CDR3β motifs alone, or dual public CDR3αβ motifs. In addition, the same public CDR3α motif may pair with different CDR3β motifs (and the reverse), giving rise to highly diverse NLV-specific TCR repertoires. To investigate the structural underpinnings of this clonal diversity, we determined crystal structures of two public TCRs (C7 and C25) in complex with NLV-HLA-A2. These TCRs utilize completely different CDR3α and CDR3β motifs that, in addition, can associate with multiple variable α (Vα) and Vβ regions in NLV-specific T cell repertoires. The C7-NLV-HLA-A2 and C25-NLV-HLA-A2 complexes exhibit divergent TCR footprints on peptide-MHC, such that C25 is more focused on the central portion of the NLV peptide than is C7. These structures, combined with molecular modeling, show how the public CDR3α motif of C25 may associate with different Vα regions, and how the public CDR3α motif of C7 may pair with different CDR3β motifs. This interchangeability of TCR V regions and CDR3 motifs permits multiple structural solutions to binding an identical peptide-MHC ligand, and thereby the generation of a clonally diverse public T cell response to CMV.
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Essential role of the EF-hand domain in targeting sperm phospholipase C{zeta} to membrane PIP2 [Developmental Biology]

October 1st, 2015 by

Sperm-specific PLC-zeta is widely considered to be the physiological stimulus that triggers intracellular Ca2+ oscillations and egg activation during mammalian fertilization. Although PLC-zeta is structurally similar to PLC-delta1, it lacks a PH domain and it remains unclear how PLC-zeta targets its PIP2 membrane substrate. Recently, the PLC-delta1 EF-hand domain was shown to bind to anionic phospholipids through a number of cationic residues, suggesting a potential mechanism for how PLCs might interact with their target membranes. Those critical cationic EF-hand residues in PLC-delta1 are notably conserved in PLC-zeta. We investigated the potential role of these conserved cationic residues in PLC-zeta by generating a series of mutants that sequentially neutralized three positively-charged residues (K49, K53 and R57) within the mouse PLC-zeta EF-hand domain. Microinjection of the PLC-zeta EF-hand mutants into mouse eggs enabled their Ca2+ oscillation-inducing activities to be compared with wild-type PLC-zeta. Further, the mutant proteins were purified and the in vitro PIP2 hydrolysis and binding properties were monitored. Our analysis suggests that PLC-zeta binds significantly to PIP2 but not to phosphatidic acid or phosphatidylserine, and that sequential reduction of the net positive charge within the 1st EF-hand domain of PLC-zeta significantly alters in vivo Ca2+ oscillation-inducing activity and in vitro interaction with PIP2 without affecting its Ca2+ sensitivity. Our findings are consistent with theoretical predictions provided by a mathematical model that links oocyte Ca2+ frequency and the binding ability of different PLC-zeta mutants to PIP2. Moreover, a PLC-zeta mutant with mutations in the cationic residues within the 1st EF hand domain and the XY linker region dramatically reduces the binding of PLC-zeta to PIP2, leading to complete abolishment of its Ca2+ oscillation-inducing activity.

Molecular mechanisms and kinetic effects of FXYD1 and phosphomimetic mutants on purified human Na,K-ATPase [Membrane Biology]

October 1st, 2015 by

Phospholemman (FXYD1) is a single trans-membrane protein regulator of Na,K-ATPase, expressed strongly in heart, skeletal muscle and brain, and phosphorylated by protein kinases A and C at S68 and S63, respectively. Binding of FXYD1 reduces Na,K-ATPase activity and phosphorylation at S68 or S63 relieves the inhibition. Despite the accumulated information on physiological effects, whole cell studies provide only limited information on molecular mechanisms. As a complementary approach, we utilized purified human Na,K-ATPase (α1β1 and α2β1) reconstituted with FXYD1 or mutants, S63E, S68E and S63E,S68E that mimic phosphorylation at S63 and S68. Compared to Control α1β1, FXYD1 reduces Vmax and turn-over rate and raises K0.5Na. The phosphomimetic mutants reverse these effects and reduce K0.5Na below Control K0.5Na. Effects on α2β1 are similar but smaller. Experiments in proteoliposomes reconstituted with α1β1 show analogous effects of FXYD1 on K0.5Na, which are abolished by phosphomimetic mutants and also by increasing mole fractions of DOPS in the proteoliposomes. Stopped-flow experiments using the dye RH421 show that FXYD1 slows the conformational transition E2(2K)ATP → E1(3Na)ATP but does not affect 3NaE1P → E2P3Na. This regulatory effect is explained simply by molecular modeling, which indicates that a cytoplasmic helix (residues 60-70) docks between the αN and αP domains in the E2 conformation but docking is weaker in E1 (also for phosphomimetic mutants). Taken together with previous work showing that FXYD1 also raises binding affinity for the Na selective site III, these results provide a rather comprehensive picture of the regulatory mechanism of FXYD1 that complements the physiological studies.

ACTH Responses Require Actions of the Melanocortin-2 Receptor Accessory Protein on the Extracellular Surface of the Plasma Membrane [Membrane Biology]

September 30th, 2015 by Malik, S., Dolan, T. M., Maben, Z. J., Hinkle, P. M.

The melanocortin-2 (MC2) receptor is a G protein-coupled receptor that mediates responses to ACTH. The MC2 receptor acts in concert with an accessory protein (MRAP) that is absolutely required for ACTH binding and signaling. MRAP has a single transmembrane domain and forms a highly unusual anti-parallel homodimer that is stably associated with MC2 receptors at the plasma membrane. Despite the physiological importance of the interaction between the MC2 receptor and MRAP, there is little understanding of how the accessory protein works. The dual topology of MRAP has made it impossible to determine whether highly conserved and necessary regions of MRAP are required on the intracellular or extracellular face of the plasma membrane. The strategy used here was to fix the orientation of two anti-parallel MRAP molecules and then introduce inactivating mutations on one side of the membrane or the other. This was achieved by engineering proteins containing tandem copies of MRAP fused to the amino-terminus of the MC2 receptor. The data firmly establish that only the Nout copy of MRAP, oriented with critical segments on the extracellular side of the membrane, is essential. The transmembrane domain of MRAP is also required in only the Nout orientation. Finally, activity of MRAP-MRAP-MC2-receptor fusion proteins with inactivating mutations in either MRAP or the receptor is rescued by co-expression of free wild-type MRAP or free wild-type receptor. These results show that the basic MRAP/MRAP/receptor signaling unit forms higher order complexes and that these multimers signal.
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Homocitrullination is a Novel Histone H1 Epigenetic Mark Dependent on Aryl Hydrocarbon Receptor Recruitment of Carbamoyl Phosphate Synthase 1 [Gene Regulation]

September 30th, 2015 by Joshi, A. D., Mustafa, M. G., Lichti, C. F., Elferink, C. J.

The Aryl hydrocarbon Receptor (AhR), a regulator of xenobiotic toxicity, is a member of the eukaryotic Per-Arnt-Sim domain protein family of transcription factors. Recent evidence identified a novel AhR DNA recognition sequence called the non-consensus xenobiotic response element (NC-XRE). AhR binding to the NC-XRE in response to activation by the canonical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin, resulted in concomitant recruitment of Carbamoyl Phosphate Synthase 1 (CPS1) to the NC-XRE. Studies presented here demonstrate that CPS1 is a bona fide nuclear protein involved in homocitrullination (hcit), including a key lysine residue on histone H1 (H1K34hcit). H1K34hcit represents a hitherto unknown epigenetic mark implicated in enhanced gene expression of the peptidylarginine deiminase 2 gene, itself a chromatin modifying protein. Collectively, our data suggest that AhR activation promotes CPS1 recruitment to DNA enhancer sites in the genome resulting in a specific enzyme-independent post-translational modification (PTM) of the linker histone H1 protein (H1K34hcit), pivotal in altering local chromatin structure and transcriptional activation.
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