Faucher C, Capdevielle J, Canal I, Ferrara P, Mazarguil H, McGuire W L, Darbon J M
Institut National de la Santé et de la Recherche Médicale, Unité 133, Faculté de Médecine, Toulouse, France.
J Biol Chem. 1993 Jul 15;268(20):15168-73.
We have previously reported the presence of a 28-kDa protein in human mammary adenocarcinoma MCF-7 cells, whose phosphorylation by phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and permeant diacylglycerol 1,2-dioctanoyl-sn-glycerol was correlated to growth arrest induced by the protein kinase C (PKC) activators. We now investigate the possible identity of this protein with the estrogen-regulated "24-kDa" protein shown as related to the mammalian heat shock protein 27 (Fuqua, S. A. W., Blum-Salingaros, M., and McGuire, W. L. (1989) Cancer Res 49, 4126-4129). 32P-Labeled 28-kDa protein from TPA-treated MCF-7 cells was immunoprecipitated with a 24-kDa-specific monoclonal antibody. Immunoblots from cell extracts fractionated by two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis demonstrated that TPA induced the conversion of a 28-kDa isoform "a" (pI 6.7) to a more acidic isoform "b" (pI 6.2). Two-dimensional gel analysis of [3H]leucine-labeled MCF-7 cell extracts demonstrated that conversely to TPA, which induced only phosphorylation of 28-kDa protein, heat shock induced both synthesis (increase of isoform a) and phosphorylation (conversion of isoforms a to b) of the protein. 32P labeling of MCF-7 cells allowed demonstration of the presence of an extra phosphoisoform "c" (pI 5.9) upon TPA as well as heat shock treatment. When cells were pretreated with the bisindolylmaleimide GF109203X, a selective inhibitor of PKC, the heat shock-induced phosphorylation was unchanged, while the TPA effect was almost abolished, suggesting that the heat shock-activated protein kinase was very likely different from PKC. However, peptide mapping of the 28-kDa phosphoprotein suggested identical sites of phosphorylation upon TPA and heat shock stimulation. Partial amino acid sequencing of the 28-kDa protein revealed identity with both the 24-kDa protein and the mammalian HSP27. The fact that estrogens and PKC, respectively, regulate expression and phosphorylation of this 24/28-kDa protein strongly argues for its key role in MCF-7 cell proliferation and differentiation.
我们之前报道过在人乳腺腺癌MCF-7细胞中存在一种28 kDa的蛋白质，其被佛波酯12-O-十四酰佛波醇-13-乙酸酯（TPA）和渗透性二酰甘油1,2-二辛酰-sn-甘油磷酸化与蛋白激酶C（PKC）激活剂诱导的生长停滞相关。我们现在研究这种蛋白质是否可能与雌激素调节的“24 kDa”蛋白质相同，该蛋白质显示与哺乳动物热休克蛋白27相关（富夸，S. A. W.，布卢姆-萨林加罗斯，M.，以及麦圭尔，W. L.（1989年）《癌症研究》49，4126 - 4129）。用24 kDa特异性单克隆抗体对来自TPA处理的MCF-7细胞的32P标记的28 kDa蛋白质进行免疫沉淀。通过二维等电聚焦/SDS-聚丙烯酰胺凝胶电泳分离细胞提取物的免疫印迹表明，TPA诱导28 kDa同工型“a”（pI 6.7）转变为酸性更强的同工型“b”（pI 6.2）。对[3H]亮氨酸标记的MCF-7细胞提取物进行二维凝胶分析表明，与仅诱导28 kDa蛋白质磷酸化的TPA相反，热休克诱导该蛋白质的合成（同工型a增加）和磷酸化（同工型a转变为b）。对MCF-7细胞进行32P标记显示，在TPA以及热休克处理后存在一种额外的磷酸同工型“c”（pI 5.9）。当细胞用双吲哚马来酰亚胺GF109203X（一种PKC的选择性抑制剂）预处理时，热休克诱导的磷酸化不变，而TPA的作用几乎被消除，这表明热休克激活的蛋白激酶很可能与PKC不同。然而，对28 kDa磷蛋白的肽图谱分析表明，在TPA和热休克刺激下磷酸化位点相同。对28 kDa蛋白质的部分氨基酸测序显示其与24 kDa蛋白质和哺乳动物HSP27相同。雌激素和PKC分别调节这种24/28 kDa蛋白质的表达和磷酸化这一事实有力地证明了其在MCF-7细胞增殖和分化中的关键作用。
