Joe Patrick A, Banerjee Asok, Ludueña Richard F
Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.
Cell Motil Cytoskeleton. 2008 Jun;65(6):476-86. doi: 10.1002/cm.20274.
Tubulin is the target for some very powerful anti-mitotic and anti-tumor drugs. The betaIII tubulin isotype is found in very few normal tissues, but is often found in tumors, where it has been implicated in resistance to anti-tumor drugs. The betaIII isotype occurs in fish, amphibians, birds and mammals and its unique features are highly conserved in evolution. One of these features is the replacement of cys239 by ser239. Cys239 is unusual in being highly sensitive to oxidation; in fact, oxidation of this residue inhibits microtubule assembly. The betaIII isotype also has a very unusual cys124, where other beta isotypes have ser/ala124. The striking conservation in betaIII of vertebrates strongly suggests that cys124 and ser239 play functional roles. We have prepared the C124S and S239C mutants of betaIII and tested their effects on the functional properties of tubulin. We have found that both the betaIII C124S and betaIII S239C mutants bind colchicine less well than does wild-type alphabetaIII, and also make transfected HeLa cells more resistant to colchicine. However, the double mutant, betaIII C124S/S239C, binds colchicine still less well than do either of the single mutants, but in contrast to the former, the double mutant increases the cells' sensitivity to colchicine. Our results indicate that the roles that these residues play in colchicine binding and microtubule integrity are far more complex than previously imagined and that the specific residues at which betaIII differs from the other isotypes act collectively to keep betaIII in a functional conformation.
微管蛋白是一些非常强效的抗有丝分裂和抗肿瘤药物的作用靶点。βIII微管蛋白亚型在极少的正常组织中存在，但在肿瘤中却经常被发现，并且它与肿瘤对抗肿瘤药物的耐药性有关。βIII亚型存在于鱼类、两栖类、鸟类和哺乳动物中，其独特特征在进化过程中高度保守。其中一个特征是第239位的半胱氨酸被丝氨酸取代。半胱氨酸239不同寻常之处在于它对氧化高度敏感；事实上，该残基的氧化会抑制微管组装。βIII亚型还具有一个非常特殊的半胱氨酸124，而其他β亚型在此位置是丝氨酸/丙氨酸124。脊椎动物βIII中这种显著的保守性强烈表明半胱氨酸124和丝氨酸239发挥着功能作用。我们制备了βIII的C124S和S239C突变体，并测试了它们对微管蛋白功能特性的影响。我们发现βIII C124S和βIII S239C突变体与秋水仙碱的结合能力均不如野生型αβIII，并且使转染的HeLa细胞对秋水仙碱更具抗性。然而，双突变体βIII C124S/S239C与秋水仙碱的结合能力比任何一个单突变体都更差，但与前者不同的是，双突变体增加了细胞对秋水仙碱的敏感性。我们的结果表明，这些残基在秋水仙碱结合和微管完整性方面所起的作用远比之前想象的要复杂得多，并且βIII与其他亚型不同的特定残基共同作用以使βIII保持功能构象。
