title: Identification of phosphorylated serine-15 and -82 residues of HSPB1 in 5-fluorouracil-resistant colorectal cancer cells by proteomics.
abstract: To identify the proteins involved in 5-fluorouracil (5-FU) resistance, a comparison of the total and phosphorylated proteins between the human colorectal cancer (CRC) cell line DLD-1 and its 5-FU-resistant subclone DLD-1/5-FU was performed. Using 2-DE and MALDI-TOF/TOF-based proteomics, 17 up-regulated and 19 down-regulated protein spots were identified in the 5-FU-resistant DLD-1/5-FU cells compared with the parent cell lines. In DLD-1/5-FU cells, 7 anti-apoptotic proteins (HSPB1, proteasome subunit α-5, transitional endoplasmic reticulum ATPase, 14-3-3 β, 14-3-3 γ, 14-3-3 σ, and phosphoglycerate kinase 1) were up-regulated and 4 proapoptotic proteins (cofilin-1, pyruvate kinase M2, glyceraldehyde-3-phosphate dehydrogenase, and nucleophosmin) were down-regulated. The results show that the acquired drug resistance of DLD-1/5-FU cells is caused by the prevention of drug-induced apoptosis, in particular through the enhanced constitutive expression of HSPB1 and its phosphorylated form. Short interfering RNA knockdown of endogenous HSPB1 in DLD-1/5-FU cells restored the sensitivity to 5-FU. Furthermore, MALDI-TOF/TOF and 2-DE Western blot analysis identified the phosphorylated residues of HSPB1 as Ser-15 and Ser-82 in the main (diphosphorylated) form and Ser-15, Ser-78, and Ser-82 in the minor (triphosphorylated) form. The current findings indicate that phosphorylated HSPB1 may play an important role in 5-FU resistance.
authors: Department of Chemistry, 2-7 Daigaku-machi, Takatsuki City, Osaka 569-8686, Japan. asakai@art.osaka-med.ac.jp
authors: Akiko
authors: A
authors: Sakai
authors: Mieko
authors: M
authors: Otani
authors: Akiko
authors: A
authors: Miyamoto
authors: Hideji
authors: H
authors: Yoshida
authors: Eisuke
authors: E
authors: Furuya
authors: Nobuhiko
authors: N
authors: Tanigawa
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D019008:Drug Resistance, Neoplasm; D055785:Gene Knockdown Techniques; D055551:HSP27 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006360:Heat-Shock Proteins; D006801:Humans; D018832:Molecular Chaperones; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D040901:Proteomics; D012694:Serine / Q000235:genetics / Q000378:metabolism; D014500:Uracil Nucleotides / Q000494:pharmacology*
chemical_list: D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D014500:Uracil Nucleotides; D012694:Serine; C016462:5-fluorouridine 5'-phosphate