Abstract
Apratoxin A is a potent cytotoxic marine natural product that rapidly inhibits signal transducer and activator of transcription (STAT) 3 phosphorylation by an undefined mechanism. We have used biochemical and proteomics approaches to illuminate upstream molecular events. Apratoxin A inhibits Janus kinase (JAK)/STAT signaling through rapid down-regulation of interleukin 6 signal transducer (gp130). Apratoxin A also depletes cancer cells of several cancer-associated receptor tyrosine kinases by preventing their N-glycosylation, leading to their rapid proteasomal degradation. A proteomics approach revealed that several proteins in the endoplasmic reticulum, the site of N-glycoprotein synthesis, are down-regulated upon apratoxin A exposure. Using in vitro cell free systems, we demonstrated that apratoxin A prevents cotranslational translocation of proteins destined for the secretory pathway. This process is reversible in living cells. Our study indicates that apratoxins are new tools to study the secretory pathway and raises the possibility that inhibition of cotranslational translocation may be exploited for anticancer drug development.
Footnotes
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This work was financially supported by the James and Esther King Biomedical Research Program, Florida Department of Health [Grant 06-NIR07] (H.L.). Initial seed funding was provided through a Junior Investigator Award (pilot grant) from the University of Florida Shands Cancer Center, American Cancer Society Institutional Research Grant [Grant ACS-IRG-01-188-01] (H.L.).
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ABBREVIATIONS: BIP, 70-kDa heat shock protein 5; CALR, calreticulin; c-MET, met proto-oncogene (hepatocyte growth factor receptor); DTT, dithiothreitol; ER, endoplasmic reticulum; FGFR, fibroblast growth factor receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GI50, concentration at which the cell growth is inhibited by 50%; gp130, interleukin 6 signal transducer; HER-2, ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2); HPLC, high-performance liquid chromatography; HYOU1, hypoxia up-regulated 1; IGF1R-β, insulin-like growth factor 1 receptor β; IL, interleukin; iTRAQ, isobaric tag for relative and absolute quantitation; JAK, Janus kinase; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PDGFR-β, platelet-derived growth factor receptor, β polypeptide; PDI, protein disulfide isomerase; PNGase F, peptide N-glycosidase F; RPN1, ribophorin 1; RTK, receptor tyrosine kinase; siRNA, small interfering RNA; SPARC, secreted protein, acidic, cysteine-rich (osteonectin); SRP, signal recognition particle; STAT, signal transducer and activator of transcription; sulfo-NHS-SS-Biotin, sulfosuccinimidyl 2-(biotinamido)-ethyl-1,3-dithiopropionate; VEGFR, vascular endothelial growth factor receptor.
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material. - Accepted April 29, 2009.
- Received March 9, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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