Nguyen L T, Beauregard G, Tessier S, Allard P, Atfi A, Durocher Y, Chapdelaine A, Potier M, Chevalier S
Department of Biochemistry, University of Montreal and Research Center, Maisonneuve-Rosemont Hospital, Montreal, Canada.
Biochem Cell Biol. 1996;74(1):75-85. doi: 10.1139/o96-008.
Because protein tyrosine kinases play a crucial role in the regulation of cell division and carcinogenesis, we have herein measured such enzyme activities (specific activity and subcellular distribution) and compared their characteristics with respect to hydrodynamic properties and radiation inactivation sizes as well as renaturation after electrophoresis in denaturing conditions in canine prostatic epithelial cells either in a resting (freshly isolated) or in a dividing (cultured cells) state. In quiescent cells, most protein tyrosine kinase activity was expressed by soluble proteins with a Stokes' radius (Rs) of 3.05 nm, a sedimentation coefficient (S20,w) of 4.0 S, and a molecular mass of 50 kDa. By contrast, in dividing cells (three days in primary culture), the specific activity was higher and the enzyme was mainly membrane bound. The use of a detergent (Triton X-100) allowed the extraction of most of that enzyme; its partial specific volume, S20,w and Rs were then 0.883 cm3/g, 4.0 S, and 5.6 nm, respectively, hence yielding a molecular mass of 215 kDa, which decreased to 125-145 kDa when corrected for detergent binding. Probing these chromatography-peak fractions, 50 kDa from cytosol of resting cells and 215 kDa from membrane extracts of dividing cells, with a phosphotyrosine antibody following their incubation with ATP and electrophoresis in denaturing conditions revealed the presence of a common 50-kDa phosphotyrosylated protein along with three other bands (130, 75, and 40 kDa) in the high-Mr peak of enzyme. However, the radiation inactivation size for protein tyrosine kinases expressed in both resting and dividing cells were similar, 47.2 +/- 8.7 and 44.5 +/- 6.1 kDa, respectively. Furthermore, by renaturation after electrophoresis in denaturing conditions, major protein tyrosine kinase polypeptides of 50 kDa were identified in both cell populations. Taken together, these results indicate that, in dividing prostatic epithelial cells, membrane-bound protein tyrosine kinases of low molecular weight with properties similar to those of monomeric soluble forms present in quiescent cells are part of high-molecular weight complexes. This activation process may be critical for hormone-independent proliferation of prostatic epithelial cells.
由于蛋白质酪氨酸激酶在细胞分裂和致癌作用的调控中起着关键作用，我们在此测量了此类酶的活性（比活性和亚细胞分布），并比较了它们在水动力学性质、辐射失活大小以及在变性条件下电泳后复性方面的特征，这些研究对象是处于静止（新鲜分离）或分裂（培养细胞）状态的犬前列腺上皮细胞。在静止细胞中，大多数蛋白质酪氨酸激酶活性由 Stokes 半径（Rs）为 3.05 nm、沉降系数（S20,w）为 4.0 S 且分子量为 50 kDa 的可溶性蛋白质所表现。相比之下，在分裂细胞（原代培养三天）中，比活性更高，且该酶主要与膜结合。使用去污剂（Triton X-100）可提取出大部分该酶；其偏比容、S20,w 和 Rs 分别为 0.883 cm3/g、4.0 S 和 5.6 nm，由此得出分子量为 215 kDa，校正去污剂结合后降至 125 - 145 kDa。在用 ATP 孵育并在变性条件下电泳后，用磷酸酪氨酸抗体探测这些色谱峰馏分（静止细胞胞质溶胶中的 50 kDa 和分裂细胞膜提取物中的 215 kDa），结果显示在酶的高分子量峰中存在一种共同的 50 kDa 磷酸酪氨酸化蛋白以及其他三条带（130、75 和 40 kDa）。然而，静止细胞和分裂细胞中表达的蛋白质酪氨酸激酶的辐射失活大小相似，分别为 47.2±8.7 kDa 和 44.5±6.1 kDa。此外，通过在变性条件下电泳后的复性，在两个细胞群体中均鉴定出了主要的 50 kDa 蛋白质酪氨酸激酶多肽。综上所述，这些结果表明，在分裂的前列腺上皮细胞中，具有与静止细胞中存在的单体可溶性形式相似性质的低分子量膜结合蛋白质酪氨酸激酶是高分子量复合物的一部分。这一激活过程可能对前列腺上皮细胞的激素非依赖性增殖至关重要。
