Casein Kinase 2 Is Linked to Stress Granule Dynamics through Phosphorylation of the Stress Granule Nucleating Protein G3BP1

ABSTRACT Stress granules (SGs) are large macromolecular aggregates that contain translation initiation complexes and mRNAs. Stress granule formation coincides with translational repression, and stress granules actively signal to mediate cell fate decisions by signaling to the translation apparatus to (i) maintain translational repression, (ii) mount various transcriptional responses, including innate immunity, and (iii) repress apoptosis. Previous work showed that G3BP1 is phosphorylated at serine 149, which regulates G3BP1 oligomerization, stress granule assembly, and RNase activity intrinsic to G3BP1. However, the kinase that phosphorylates G3BP1 was not identified, leaving a key step in stress granule regulation uncharacterized. Here, using chemical inhibition, genetic depletion, and overexpression experiments, we show that casein kinase 2 (CK2) promotes stress granule dynamics. These results link CK2 activity with SG disassembly. We also show that casein kinase 2 phosphorylates G3BP1 at serine 149 in vitro and in cells. These data support a role for casein kinase 2 in regulation of protein synthesis by downregulating stress granule formation through G3BP1.

[1]  J. Tazi,et al.  Revisiting G3BP1 as a RasGAP Binding Protein: Sensitization of Tumor Cells to Chemotherapy by the RasGAP 317–326 Sequence Does Not Involve G3BP1 , 2011, PloS one.

[2]  R. Lloyd,et al.  Inhibition of cytoplasmic mRNA stress granule formation by a viral proteinase. , 2007, Cell host & microbe.

[3]  James S. Duncan,et al.  Protein kinase CK2 is a constitutively active enzyme that promotes cell survival: strategies to identify CK2 substrates and manipulate its activity in mammalian cells. , 2010, Methods in enzymology.

[4]  Michael B. Yaffe,et al.  Scansite 2.0: proteome-wide prediction of cell signaling interactions using short sequence motifs , 2003, Nucleic Acids Res..

[5]  Anne E Carpenter,et al.  CellProfiler: image analysis software for identifying and quantifying cell phenotypes , 2006, Genome Biology.

[6]  Sebastian A. Wagner,et al.  A Proteome-wide, Quantitative Survey of In Vivo Ubiquitylation Sites Reveals Widespread Regulatory Roles* , 2011, Molecular & Cellular Proteomics.

[7]  Roy Parker,et al.  Formation and Maturation of Phase-Separated Liquid Droplets by RNA-Binding Proteins. , 2015, Molecular cell.

[8]  M. Zamora,et al.  Translation of Eukaryotic Translation Initiation Factor 4GI (eIF4GI) Proceeds from Multiple mRNAs Containing a Novel Cap-dependent Internal Ribosome Entry Site (IRES) That Is Active during Poliovirus Infection* , 2005, Journal of Biological Chemistry.

[9]  Pavel Ivanov,et al.  Stress granules and cell signaling: more than just a passing phase? , 2013, Trends in biochemical sciences.

[10]  Jun O. Liu,et al.  Eukaryotic Initiation Factor 2α-independent Pathway of Stress Granule Induction by the Natural Product Pateamine A* , 2006, Journal of Biological Chemistry.

[11]  S. Moro,et al.  Tetrabromocinnamic Acid (TBCA) and Related Compounds Represent a New Class of Specific Protein Kinase CK2 Inhibitors , 2007, Chembiochem : a European journal of chemical biology.

[12]  Lucas C. Reineke,et al.  Diversion of stress granules and P-bodies during viral infection , 2013, Virology.

[13]  Masayasu Oie,et al.  Both G3BP1 and G3BP2 contribute to stress granule formation , 2013, Genes to cells : devoted to molecular & cellular mechanisms.

[14]  O. Issinger,et al.  The effect of polylysine on casein-kinase-2 activity is influenced by both the structure of the protein/peptide substrates and the subunit composition of the enzyme. , 1992, European journal of biochemistry.

[15]  J. Tazi,et al.  Control of Fetal Growth and Neonatal Survival by the RasGAP-Associated Endoribonuclease G3BP , 2005, Molecular and Cellular Biology.

[16]  Lucas C. Reineke,et al.  The Stress Granule Protein G3BP1 Recruits Protein Kinase R To Promote Multiple Innate Immune Antiviral Responses , 2014, Journal of Virology.

[17]  D. Fabbro,et al.  Biochemical and three-dimensional-structural study of the specific inhibition of protein kinase CK2 by [5-oxo-5,6-dihydroindolo-(1,2-a)quinazolin-7-yl]acetic acid (IQA). , 2003, The Biochemical journal.

[18]  L. Cesaro,et al.  Mass spectrometry analysis of a protein kinase CK2beta subunit interactome isolated from mouse brain by affinity chromatography. , 2008, Journal of proteome research.

[19]  J. Pelletier,et al.  Inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation. , 2006, Molecular biology of the cell.

[20]  C. Brangwynne,et al.  Getting RNA and Protein in Phase , 2012, Cell.

[21]  Lucas C. Reineke,et al.  Arginine Demethylation of G3BP1 Promotes Stress Granule Assembly* , 2016, The Journal of Biological Chemistry.

[22]  Randal J. Kaufman,et al.  Heme-regulated Inhibitor Kinase-mediated Phosphorylation of Eukaryotic Translation Initiation Factor 2 Inhibits Translation, Induces Stress Granule Formation, and Mediates Survival upon Arsenite Exposure* , 2005, Journal of Biological Chemistry.

[23]  D. Weil,et al.  Translationally repressed mRNA transiently cycles through stress granules during stress. , 2008, Molecular biology of the cell.

[24]  M. Lederer,et al.  Stress granules are dispensable for mRNA stabilization during cellular stress , 2014, Nucleic acids research.

[25]  L. Pinna,et al.  Structural determinants of protein kinase CK2 regulation by autoinhibitory polymerization. , 2012, ACS chemical biology.

[26]  F. V. van Kuppeveld,et al.  MDA5 Localizes to Stress Granules, but This Localization Is Not Required for the Induction of Type I Interferon , 2013, Journal of Virology.

[27]  Emmanuel Ampofo,et al.  Functional interaction of protein kinase CK2 and activating transcription factor 4 (ATF4), a key player in the cellular stress response. , 2013, Biochimica et biophysica acta.

[28]  P. Anderson,et al.  Mammalian stress granules and processing bodies. , 2007, Methods in enzymology.

[29]  A. Ortega,et al.  Human G3BP1 interacts with β-F1-ATPase mRNA and inhibits its translation , 2010, Journal of Cell Science.

[30]  Lucas C. Reineke,et al.  Large G3BP-induced granules trigger eIF2α phosphorylation , 2012, Molecular biology of the cell.

[31]  P. Silver,et al.  Metazoan stress granule assembly is mediated by P-eIF2alpha-dependent and -independent mechanisms. , 2009, RNA.

[32]  U. Landegren,et al.  Direct observation of individual endogenous protein complexes in situ by proximity ligation , 2006, Nature Methods.

[33]  Timothy D. Craggs,et al.  Phase Transition of a Disordered Nuage Protein Generates Environmentally Responsive Membraneless Organelles , 2015, Molecular cell.

[34]  L. Pinna,et al.  Protein Kinase CK2α′ Is Induced by Serum as a Delayed Early Gene and Cooperates with Ha-ras in Fibroblast Transformation* , 1998, The Journal of Biological Chemistry.

[35]  P. Ivanov,et al.  Hydrogen peroxide induces stress granule formation independent of eIF2α phosphorylation. , 2012, Biochemical and biophysical research communications.

[36]  P. Anderson,et al.  Stress granule assembly is mediated by prion-like aggregation of TIA-1. , 2004, Molecular biology of the cell.

[37]  Masahiro Morita,et al.  mTORC1 and CK2 coordinate ternary and eIF4F complex assembly , 2016, Nature Communications.

[38]  P. Anderson,et al.  Stress granules: the Tao of RNA triage. , 2008, Trends in biochemical sciences.

[39]  K. Chébli,et al.  A Novel Phosphorylation-Dependent RNase Activity of GAP-SH3 Binding Protein: a Potential Link between Signal Transduction and RNA Stability , 1998, Molecular and Cellular Biology.

[40]  K. Chébli,et al.  The RasGAP-associated endoribonuclease G3BP assembles stress granules , 2003, The Journal of cell biology.

[41]  C. Malbon,et al.  Arginine methylation of G3BP1 in response to Wnt3a regulates β-catenin mRNA , 2011, Journal of Cell Science.

[42]  Lucas C. Reineke,et al.  mRNA Decapping Enzyme 1a (Dcp1a)-induced Translational Arrest through Protein Kinase R (PKR) Activation Requires the N-terminal Enabled Vasodilator-stimulated Protein Homology 1 (EVH1) Domain* , 2013, The Journal of Biological Chemistry.

[43]  E. P. Kennedy,et al.  The enzymatic phosphorylation of proteins. , 1954, The Journal of biological chemistry.

[44]  O. Issinger,et al.  Stress-induced Activation of Protein Kinase CK2 by Direct Interaction with p38 Mitogen-activated Protein Kinase* , 2000, The Journal of Biological Chemistry.

[45]  Paul S. Russo,et al.  Phase Transitions in the Assembly of MultiValent Signaling Proteins , 2016 .

[46]  Wen Jiang,et al.  EMAN2: an extensible image processing suite for electron microscopy. , 2007, Journal of structural biology.

[47]  D. Litchfield,et al.  The Multiple Personalities of the Regulatory Subunit of Protein Kinase CK2: CK2 Dependent and CK2 Independent Roles Reveal a Secret Identity for CK2β , 2005, International journal of biological sciences.

[48]  K. Taniuchi,et al.  The N-Terminal Domain of G3BP Enhances Cell Motility and Invasion by Posttranscriptional Regulation of BART , 2011, Molecular Cancer Research.

[49]  H. Zou,et al.  Determination of CK2 specificity and substrates by proteome-derived peptide libraries. , 2013, Journal of proteome research.

[50]  M. Mann,et al.  Lysine Acetylation Targets Protein Complexes and Co-Regulates Major Cellular Functions , 2009, Science.

[51]  E. Krebs,et al.  CK2 phosphorylation of eukaryotic translation initiation factor 5 potentiates cell cycle progression , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[52]  Jimin Pei,et al.  Cell-free Formation of RNA Granules: Low Complexity Sequence Domains Form Dynamic Fibers within Hydrogels , 2012, Cell.

[53]  Christer Larsson,et al.  Regulation of PMP22 mRNA by G3BP1 affects cell proliferation in breast cancer cells , 2013, Molecular Cancer.

[54]  Arjun Bhutkar,et al.  Poly(ADP-ribose) regulates stress responses and microRNA activity in the cytoplasm. , 2011, Molecular cell.

[55]  L. Pinna,et al.  One‐thousand‐and‐one substrates of protein kinase CK2? , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[56]  U. Landegren,et al.  Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay. , 2008, Methods.

[57]  J. Tazi,et al.  RasGAP-Associated Endoribonuclease G3BP: Selective RNA Degradation and Phosphorylation-Dependent Localization , 2001, Molecular and Cellular Biology.

[58]  Jerry Pelletier,et al.  Uncoupling stress granule assembly and translation initiation inhibition. , 2009, Molecular biology of the cell.

[59]  M. Mann,et al.  Identifying and quantifying in vivo methylation sites by heavy methyl SILAC , 2004, Nature Methods.

[60]  Randal J. Kaufman,et al.  Stress granules and processing bodies are dynamically linked sites of mRNP remodeling , 2005, The Journal of cell biology.

[61]  Lucas C. Reineke,et al.  Stress Granules Regulate Double-Stranded RNA-Dependent Protein Kinase Activation through a Complex Containing G3BP1 and Caprin1 , 2015, mBio.