title: Receptor-recognized α₂-macroglobulin binds to cell surface-associated GRP78 and activates mTORC1 and mTORC2 signaling in prostate cancer cells.
abstract: OBJECTIVE
  Tetrameric α(2)-macroglobulin (α(2)M), a plasma panproteinase inhibitor, is activated upon interaction with a proteinase, and undergoes a major conformational change exposing a receptor recognition site in each of its subunits. Activated α(2)M (α(2)M*) binds to cancer cell surface GRP78 and triggers proliferative and antiapoptotic signaling. We have studied the role of α(2)M* in the regulation of mTORC1 and TORC2 signaling in the growth of human prostate cancer cells.
  
  
  METHODS
  Employing immunoprecipitation techniques and Western blotting as well as kinase assays, activation of the mTORC1 and mTORC2 complexes, as well as down stream targets were studied. RNAi was also employed to silence expression of Raptor, Rictor, or GRP78 in parallel studies.
  
  
  RESULTS
  Stimulation of cells with α(2)M* promotes phosphorylation of mTOR, TSC2, S6-Kinase, 4EBP, Akt(T308), and Akt(S473) in a concentration and time-dependent manner. Rheb, Raptor, and Rictor also increased. α(2)M* treatment of cells elevated mTORC1 kinase activity as determined by kinase assays of mTOR or Raptor immunoprecipitates. mTORC1 activity was sensitive to LY294002 and rapamycin or transfection of cells with GRP78 dsRNA. Down regulation of Raptor expression by RNAi significantly reduced α(2)M*-induced S6-Kinase phosphorylation at T389 and kinase activity in Raptor immunoprecipitates. α(2)M*-treated cells demonstrate about a twofold increase in mTORC2 kinase activity as determined by kinase assay of Akt(S473) phosphorylation and levels of p-Akt(S473) in mTOR and Rictor immunoprecipitates. mTORC2 activity was sensitive to LY294002 and transfection of cells with GRP78 dsRNA, but insensitive to rapamycin. Down regulation of Rictor expression by RNAi significantly reduces α(2)M*-induced phosphorylation of Akt(S473) phosphorylation in Rictor immunoprecipitates.
  
  
  CONCLUSIONS
  Binding of α(2)M* to prostate cancer cell surface GRP78 upregulates mTORC1 and mTORC2 activation and promotes protein synthesis in the prostate cancer cells.
authors.affiliation: Department of Pathology, Duke University Medical Center, Durham, North Carolina, United States of America.
authors.forename: Uma K
authors.initials: UK
authors.lastname: Misra
authors.forename: Salvatore V
authors.initials: SV
authors.lastname: Pizzo
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D000091342:Endoplasmic Reticulum Chaperone BiP; D020868:Gene Silencing; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000378:metabolism*; D008297:Male; D000076222:Mechanistic Target of Rapamycin Complex 1; D000076225:Mechanistic Target of Rapamycin Complex 2; D008954:Models, Biological; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D046912:Multiprotein Complexes / Q000378:metabolism*; D009479:Neuropeptides / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D000076226:Rapamycin-Insensitive Companion of mTOR Protein; D000076205:Ras Homolog Enriched in Brain Protein; D000076223:Regulatory-Associated Protein of mTOR; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C087000:EIF4EBP1 protein, human; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D046912:Multiprotein Complexes; D009479:Neuropeptides; D010750:Phosphoproteins; C490211:RHEB protein, human; C492571:RICTOR protein, human; C463659:RPTOR protein, human; D000076226:Rapamycin-Insensitive Companion of mTOR Protein; D000076205:Ras Homolog Enriched in Brain Protein; D000076223:Regulatory-Associated Protein of mTOR; C000624653:TSC2 protein, human; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D000511:alpha-Macroglobulins; D000076222:Mechanistic Target of Rapamycin Complex 1; D000076225:Mechanistic Target of Rapamycin Complex 2; D051057:Proto-Oncogene Proteins c-akt; D019893:Ribosomal Protein S6 Kinases; D058570:TOR Serine-Threonine Kinases; D020559:Monomeric GTP-Binding Proteins