DNA repair protein O6-alkylguanine-DNA alkyltransferase is phosphorylated by two distinct and novel protein kinases in human brain tumour cells
DNA repair protein O6-alkylguanine-DNA alkyltransferase is phosphorylated by two distinct and novel protein kinases in human brain tumour cells
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DOI：
10.1042/0264-6021:3510393
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发表时间：
2000-10-15
影响因子：
4.1
通讯作者：
Srivenugopal, KS
中科院分区：
文献类型：
--
作者：
Mullapudi, SRS;Ali-Osman, F;Srivenugopal, KS
我们最近表明，人O-n-烷基鸟嘌呤-DNA烷基转移酶（AGT）是一种磷蛋白，是改善癌症化疗的重要靶标，磷酸化抑制其活性[Srivenugopal，Mullapudi，Shou，Hazra和Ali-Osman（2000）Cancer Res.60，282-287]。在本研究中，我们对人髓母细胞瘤细胞系HBT 228中磷酸化AGT的细胞激酶进行了表征。粗细胞提取物使用Mg ~（2+）比Mn ~（2+）更有效地磷酸化人重组AGT（rAGT）蛋白。[gamma-P-32]ATP和[gamma-P-32]GTP都是磷酸盐供体，前者的效率是前者的两倍。已知激活蛋白激酶A、蛋白激酶C和钙调蛋白依赖性激酶的特定组分不刺激rAGT的磷酸化。磷酸氨基酸分析后，在体外与ATP或GTP反应表明，AGT被修改在相同的氨基酸（丝氨酸，苏氨酸和酪氨酸）在完整的HBT 228细胞。尽管这些特性中的一些指向酪蛋白激酶II作为候选酶，但酪蛋白激酶II的已知抑制剂和激活剂不影响rAGT磷酸化。分馏的细胞提取物上的聚（谷氨酸/酪氨酸）-琼脂糖凝胶导致的AGT激酶，修饰的酪氨酸残基的吸附和排斥的一部分，磷酸化AGT的丝氨酸和苏氨酸残基。SDS/PAGE和非变性PAGE后的凝胶内激酶测定揭示了HBT 228细胞中存在75和130 kDa的两种AGT激酶。部分纯化的酪氨酸激酶，确定为130 kDa的酶，由相同的测定，强烈抑制tyrphostin 25，但不是由genestein。酪氨酸激酶利用ATP或GTP磷酸化AGT蛋白，该反应抑制AGT的DNA修复活性。还提供了激酶可能与细胞中AGT物理相关的证据。这些结果表明，两种新的细胞蛋白激酶，酪氨酸激酶和丝氨酸/苏氨酸激酶，都能够使用GTP作为供体，磷酸化AGT蛋白，并影响其功能。这些新的激酶可能作为加强AGT在人类肿瘤中的生化调节的潜在靶点。
We showed recently that human O-n-alkylguanine-DNA alkyltransferase (AGT), an important target for improving cancer chemotherapy, is a phosphoprotein and that phosphorylation inhibits its activity [Srivenugopal, Mullapudi, Shou, Hazra and Ali-Osman (2000) Cancer Res. 60, 282-287]. In the present study we characterized the cellular kinases that phosphorylate AGT in the human medulloblastoma cell line HBT228, Crude cell extracts used Mg2+ more efficiently than Mn2+ for phosphorylating human recombinant AGT (rAGT) protein. Both [gamma-P-32]ATP and [gamma-P-32]GTP served as phosphate donors, with the former being twice as efficient. Specific components known to activate protein kinase A, protein kinase C and calmodulin-dependent kinases did not stimulate the phosphorylation of rAGT. Phospho aminoacid analysis after reaction in vitro with ATP or GTP showed that AGT was modified at the same amino acids (serine, threonine and tyrosine) as in intact HBT228 cells. Although some of these properties pointed to casein kinase II as a candidate enzyme, known inhibitors and activators of casein kinase II did not affect rAGT phosphorylation. Fractionation of the cell extracts on poly(Glu/Tyr)-Sepharose resulted in the adsorption of an AGT kinase that modified the tyrosine residues and the exclusion of a fraction that phosphorylated AGT on serine and threonine residues. In-gel kinase assays after SDS/PAGE and non-denaturing PAGE revealed the presence of two AGT kinases of 75 and 130 kDa in HBT228 cells. The partly purified tyrosine kinase, identified as the 130 kDa enzyme by the same assays, was strongly inhibited by tyrphostin 25 but not by genestein. The tyrosine kinase used ATP or GTP to phosphoryl ate the AGT protein; this reaction inhibited the DNA repair activity of AGT. Evidence that the kinases might physically associate with AGT in cells was also provided. These results demonstrate that two novel cellular protein kinases, a tyrosine kinase and a serine/threonine kinase, both capable of using GTP as a donor, phosphorylate the AGT protein and affect its function. The new kinases might serve as potential targets for strengthening the biochemical modulation of AGT in human tumours.