Inhibition of the ubiquitin-proteasome system induces stress granule formation.

The inhibition of the ubiquitin-dependent proteasome system (UPS) via specific drugs is one type of approach used to combat cancer. Although it has been suggested that UPS inhibition prevents the rapid decay of AU-rich element (ARE)-containing messages, very little is known about the cellular mechanisms leading to this effect. Here we establish a link between the inhibition of UPS activity, the formation of cytoplasmic stress granules (SGs), and mRNA metabolism. The assembly of the SGs requires the phosphorylation of the translation initiation factor eIF2alpha by a mechanism involving the stress kinase GCN2. On prolonged UPS inhibition and despite the maintenance of eIF2alpha phosphorylation, SGs disassemble and translation recovers in an Hsp72 protein-dependent manner. The formation of these SGs coincides with the disassembly of processing bodies (PBs), known as mRNA decay entities. As soon as the SGs assemble, they recruit ARE-containing messages such as p21(cip1) mRNA, which are stabilized under these conditions. Hence, our findings suggest that SGs could be considered as one of the players that mediate the early response of the cell to proteasome inhibitors by interfering temporarily with mRNA decay pathways.

[1]  N. Sonenberg,et al.  Eukaryotic translation initiation factors and regulators. , 2003, Current opinion in structural biology.

[2]  Wei Li,et al.  RNA-Binding Proteins Tia-1 and Tiar Link the Phosphorylation of Eif-2α to the Assembly of Mammalian Stress Granules , 1999, The Journal of cell biology.

[3]  Stefan Hüttelmaier,et al.  ZBP1 regulates mRNA stability during cellular stress , 2006, The Journal of cell biology.

[4]  D. Nandi,et al.  The ubiquitin-proteasome system , 2006, Journal of Biosciences.

[5]  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.

[6]  J. Lykke-Andersen.  Identification of a Human Decapping Complex Associated with hUpf Proteins in Nonsense-Mediated Decay , 2002, Molecular and Cellular Biology.

[7]  N. Sonenberg,et al.  A role for the eIF4E-binding protein 4E-T in P-body formation and mRNA decay , 2005, The Journal of cell biology.

[8]  N. Steinmetz,et al.  Monitoring apoptosis in real time. , 2002, Cancer journal.

[9]  J. Lykke-Andersen.  Making structural sense of nonsense-mediated decay , 2004, Nature Structural &Molecular Biology.

[10]  S. Capaccioli,et al.  Post‐transcriptional regulation of gene expression by degradation of messenger RNAs , 2003, Journal of cellular physiology.

[11]  J. Lykke-Andersen,et al.  Multiple processing body factors and the ARE binding protein TTP activate mRNA decapping. , 2005, Molecular cell.

[12]  Myriam Gorospe,et al.  HuR Regulates p21 mRNA Stabilization by UV Light , 2000, Molecular and Cellular Biology.

[13]  D. Green,et al.  Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl , 1995, The Journal of experimental medicine.

[14]  R. Elliott,et al.  Cap-dependent and hepatitis C virus internal ribosome entry site-mediated translation are modulated by phosphorylation of eIF2alpha under oxidative stress. , 2006, The Journal of general virology.

[15]  D. Ron,et al.  Mammalian stress granules represent sites of accumulation of stalled translation initiation complexes. , 2003, American journal of physiology. Cell physiology.

[16]  Kenneth R Diller,et al.  Correlation of HSP70 expression and cell viability following thermal stimulation of bovine aortic endothelial cells. , 2005, Journal of biomechanical engineering.

[17]  P. Anderson,et al.  Stress granules: sites of mRNA triage that regulate mRNA stability and translatability. , 2002, Biochemical Society transactions.

[18]  Wei Li,et al.  Evidence that ternary complex (eIF2-GTP-tRNA(i)(Met))-deficient preinitiation complexes are core constituents of mammalian stress granules. , 2002, Molecular biology of the cell.

[19]  O. Pereira-smith,et al.  Competition of CUGBP1 and calreticulin for the regulation of p21 translation determines cell fate , 2004, The EMBO journal.

[20]  C. Takimoto,et al.  Heat shock protein and proteasome targeting agents. , 2002, Hematology/oncology clinics of North America.

[21]  P. Anderson,et al.  MK2‐induced tristetraprolin:14‐3‐3 complexes prevent stress granule association and ARE‐mRNA decay , 2004, The EMBO journal.

[22]  Shulin Li,et al.  T Cell Receptor (TCR) Mini-Gene mRNA Expression Regulated by Nonsense Codons: A Nuclear-associated Translation-like Mechanism , 1997, The Journal of experimental medicine.

[23]  Xiaodong Wang,et al.  Apaf-1, a Human Protein Homologous to C. elegans CED-4, Participates in Cytochrome c–Dependent Activation of Caspase-3 , 1997, Cell.

[24]  J. Calvet,et al.  The heat shock response in HeLa cells is accompanied by elevated expression of the c-fos proto-oncogene , 1987, Molecular and cellular biology.

[25]  P. Anderson,et al.  Importance of eIF2alpha phosphorylation and stress granule assembly in alphavirus translation regulation. , 2005, Molecular biology of the cell.

[26]  H. Wong,et al.  Proteasome inhibitors induce heat shock response and increase IL-6 expression in human intestinal epithelial cells. , 2002, American journal of physiology. Regulatory, integrative and comparative physiology.

[27]  A. Ciechanover,et al.  The ubiquitin system. , 1998, Annual review of biochemistry.

[28]  A. Hershko,et al.  The ubiquitin system for protein degradation and some of its roles in the control of the cell division cycle* , 2005, Cell Death and Differentiation.

[29]  R. Schneider,et al.  Ubiquitin-dependent mechanism regulates rapid turnover of AU-rich cytokine mRNAs , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[30]  J. Steitz,et al.  Protein ligands mediate the CRM1-dependent export of HuR in response to heat shock. , 2001, RNA.

[31]  É. Khandjian,et al.  Fragile X Mental Retardation protein determinants required for its association with polyribosomal mRNPs. , 2003, Human molecular genetics.

[32]  É. Khandjian,et al.  Trapping of messenger RNA by Fragile X Mental Retardation protein into cytoplasmic granules induces translation repression. , 2002, Human molecular genetics.

[33]  R. Cuesta,et al.  Chaperone hsp27 inhibits translation during heat shock by binding eIF4G and facilitating dissociation of cap-initiation complexes. , 2000, Genes & development.

[34]  E McEwen,et al.  Translational control is required for the unfolded protein response and in vivo glucose homeostasis. , 2001, Molecular cell.

[35]  U. Certa,et al.  Roles of AUF1 isoforms, HuR and BRF1 in ARE-dependent mRNA turnover studied by RNA interference. , 2004, Nucleic acids research.

[36]  D. Erdmann,et al.  Proteasome inhibitor induced gene expression profiles reveal overexpression of transcriptional regulators ATF3, GADD153 and MAD1 , 2000, Oncogene.

[37]  N. Sonenberg,et al.  Translational control in stress and apoptosis , 2005, Nature Reviews Molecular Cell Biology.

[38]  N. Dantuma,et al.  Fluorescent reporters for the ubiquitin–proteasome system , 2005 .

[39]  J. Bass,et al.  Thermally Induced Injury and Heat‐Shock Protein Expression in Cells and Tissues , 2005, Annals of the New York Academy of Sciences.

[40]  Kalle Gehring,et al.  Poly(A) binding protein (PABP) homeostasis is mediated by the stability of its inhibitor, Paip2 , 2006, The EMBO journal.

[41]  E. Pilipenko,et al.  20S proteasome differentially alters translation of different mRNAs via the cleavage of eIF4F and eIF3. , 2004, Molecular cell.

[42]  J. Lykke-Andersen,et al.  Recruitment and activation of mRNA decay enzymes by two ARE-mediated decay activation domains in the proteins TTP and BRF-1. , 2005, Genes & development.

[43]  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.

[44]  J. Nylandsted,et al.  Heat Shock Protein 70 Promotes Cell Survival by Inhibiting Lysosomal Membrane Permeabilization , 2004, The Journal of experimental medicine.

[45]  J. Schaper,et al.  Time course of the apoptotic cascade and effects of caspase inhibitors in adult rat ventricular cardiomyocytes. , 2001, Journal of molecular and cellular cardiology.

[46]  M. Sherman,et al.  Invited review: Interplay between molecular chaperones and signaling pathways in survival of heat shock. , 2002, Journal of applied physiology.

[47]  R. Vabulas,et al.  Protein Synthesis upon Acute Nutrient Restriction Relies on Proteasome Function , 2005, Science.

[48]  J. Tazi,et al.  mRNA degradation machines in eukaryotic cells. , 2002, Biochimie.

[49]  J. Steitz,et al.  Overexpression of HuR, a nuclear–cytoplasmic shuttling protein, increases the in vivo stability of ARE‐containing mRNAs , 1998, The EMBO journal.

[50]  S. Tenenbaum,et al.  NF-κB-Mediated MyoD Decay during Muscle Wasting Requires Nitric Oxide Synthase mRNA Stabilization, HuR Protein, and Nitric Oxide Release , 2005, Molecular and Cellular Biology.

[51]  R. Cuesta,et al.  Control of mRNA decay by heat shock-ubiquitin-proteasome pathway. , 1999, Science.

[52]  M. Scheffner,et al.  The Role of the Ubiquitin Ligase E6-AP in Human Papillomavirus E6-mediated Degradation of PDZ Domain-containing Proteins* , 2007, Journal of Biological Chemistry.

[53]  R. Orlowski,et al.  The proteasome and proteasome inhibitors in cancer therapy. , 2006, Annual review of pharmacology and toxicology.

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

[55]  R. Wek,et al.  Phosphorylation of the α-Subunit of the Eukaryotic Initiation Factor-2 (eIF2α) Reduces Protein Synthesis and Enhances Apoptosis in Response to Proteasome Inhibition* , 2005, Journal of Biological Chemistry.

[56]  Roy Parker,et al.  Targeting of Aberrant mRNAs to Cytoplasmic Processing Bodies , 2006, Cell.

[57]  T. Anthony,et al.  Coping with stress: eIF2 kinases and translational control. , 2006, Biochemical Society transactions.

[58]  G. Stoecklin,et al.  A novel mechanism of tumor suppression by destabilizing AU-rich growth factor mRNA , 2003, Oncogene.

[59]  C. Holding,et al.  Partitioning of IGFBP-5 actions in myogenesis: IGF-independent anti-apoptotic function , 2004, Journal of Cell Science.

[60]  Paola Briata,et al.  p38-dependent phosphorylation of the mRNA decay-promoting factor KSRP controls the stability of select myogenic transcripts. , 2005, Molecular cell.

[61]  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.

[62]  H. Beere.  `The stress of dying': the role of heat shock proteins in the regulation of apoptosis , 2004, Journal of Cell Science.

[63]  P. Anderson,et al.  Visibly stressed: the role of eIF2, TIA-1, and stress granules in protein translation , 2002, Cell stress & chaperones.

[64]  L. Paillard,et al.  AU-rich elements and associated factors: are there unifying principles? , 2006, Nucleic acids research.

[65]  Roy Parker,et al.  Movement of Eukaryotic mRNAs Between Polysomes and Cytoplasmic Processing Bodies , 2005, Science.

[66]  Jung-Chun Lin,et al.  Cell stress modulates the function of splicing regulatory protein RBM4 in translation control , 2007, Proceedings of the National Academy of Sciences.

[67]  W. Markesbery,et al.  Proteasome inhibition induces reversible impairments in protein synthesis , 2006, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[68]  M. Rudnicki,et al.  Caspase 3 Cleavage of the Ste20-Related Kinase SLK Releases and Activates an Apoptosis-Inducing Kinase Domain and an Actin-Disassembling Region , 2000, Molecular and Cellular Biology.

[69]  E. Bennett,et al.  Application and analysis of the GFPu family of ubiquitin-proteasome system reporters. , 2005, Methods in enzymology.

[70]  M. Sherman,et al.  Proteasome Inhibitors Activate Stress Kinases and Induce Hsp72 , 1998, The Journal of Biological Chemistry.

[71]  M. Glickman,et al.  Regulating the 26S proteasome. , 2002, Current topics in microbiology and immunology.

[72]  J. Adams.  The development of proteasome inhibitors as anticancer drugs. , 2004, Cancer cell.

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

[74]  J. Steitz,et al.  HuR binding to cytoplasmic mRNA is perturbed by heat shock. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[75]  David E. Golan,et al.  Dynamic Shuttling of Tia-1 Accompanies the Recruitment of mRNA to Mammalian Stress Granules , 2000, The Journal of cell biology.

[76]  Neal N. Iwakoshi,et al.  Proteasome inhibitors disrupt the unfolded protein response in myeloma cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[77]  S. de Jong,et al.  Proteasome inhibitor MG132 sensitizes HPV‐positive human cervical cancer cells to rhTRAIL‐induced apoptosis , 2006, International journal of cancer.