Identification of phosphorylated serine-15 and -82 residues of HSPB1 in 5-fluorouracil-resistant colorectal cancer cells by proteomics.
Identification of phosphorylated serine-15 and -82 residues of HSPB1 in 5-fluorouracil-resistant colorectal cancer cells by proteomics.
复制标题
通过蛋白质组学鉴定 5-氟尿嘧啶耐药结直肠癌细胞中 HSPB1 的磷酸化丝氨酸 15 和 -82 残基。
DOI：
10.1016/j.jprot.2011.09.023
复制
发表时间：
2012
影响因子：
3.3
通讯作者：
Tanigawa N.
中科院分区：
文献类型：
--
作者：
Sakai A;Otani M;Miyamoto A;Yoshida H;Furuya E;Tanigawa N.
为了鉴定与5-氟尿嘧啶（5-FU）耐药有关的蛋白质，对人结肠直肠癌（CRC）细胞系DLD-1及其5-FU耐药亚克隆DLD-1/5-FU之间的总蛋白质和磷酸化蛋白质进行了比较。利用双向电泳和MALDI-TOF/TOF技术，在DLD-1/5-FU耐药细胞中发现了17个表达上调和19个表达下调的蛋白质点。在DLD-1/5-FU细胞中，7种抗凋亡蛋白（HSPB 1、蛋白酶体亚基α-5、过渡型内质网ATP酶、14 -3-3 β、14 - 3-3 γ、14-3-3 σ和磷酸甘油酸激酶1）表达上调，4种促凋亡蛋白（cofilin-1、丙酮酸激酶M2、甘油醛-3-磷酸脱氢酶和核磷蛋白）表达下调。结果表明，DLD-1/5-FU细胞的获得性耐药性是由阻止药物诱导的凋亡引起的，特别是通过增强的HSPB 1及其磷酸化形式的组成型表达。DLD-1/5-FU细胞内源性HSPB 1的短干扰RNA敲低可恢复对5-FU的敏感性。MALDI-TOF/TOF和2-DE Western blot分析表明，HSPB 1的磷酸化残基主要为Ser-15和Ser-82（二磷酸化），其次为Ser-15、Ser-78和Ser-82（三磷酸化）。目前的研究结果表明，磷酸化HSPB 1可能在5-FU耐药中起重要作用。
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.