Binding of pp170 to microtubules is regulated by phosphorylation.
Binding of pp170 to microtubules is regulated by phosphorylation.
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pp170 与微管的结合受磷酸化调节。
DOI：
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发表时间：
1991
影响因子：
4.8
通讯作者：
T. Kreis
中科院分区：
文献类型：
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作者：
J. Rickard;T. Kreis
我们已经使用单克隆抗体亲和柱从HeLa细胞中纯化分子量为170，000的蛋白质（命名为pp 170），我们先前鉴定该蛋白质为核苷酸敏感性微管结合蛋白（Rickard，J.E.，和Kreis，T. E.等（1990）J. Cell Biol.110，1623-1633）。我们在这里表明，亲和纯化的pp 170直接结合到紫杉醇聚合的微管蛋白。这种关联不受MgATP的直接影响。然而，MgATP的加入可以抑制pp 170在HeLa细胞微管结合蛋白存在下与微管的结合。MgATP的这种作用与微管相关激酶对pp 170的磷酸化有关。马铃薯酸性磷酸酶使pp 170去磷酸化并恢复pp 170与微管结合的能力。此外，在高速上清液提取物中，PP 170与微管的结合被磷酸酶抑制剂冈田酸抑制，这与PP 170磷酸化对微管结合的抑制作用一致。在体内，pp 170在丝氨酸残基上被磷酸化，磷酸基团的半衰期约为2小时。微管与诺考达唑解聚废除纳入32 P的蛋白质，显然是通过增加其去磷酸化的速率。用紫杉醇稳定微管可使32 P掺入pp 170的速率降低约50%，但对磷酸盐损失没有显著影响。这些数据表明，pp 170是一种微管结合蛋白，并且微管相互作用被pp 170的磷酸化抑制。pp 170的体内磷酸化状态的微管活性药物的敏感性表明，这种翻译后修饰可能是一个重要的调节器的相互作用的pp 170与微管在细胞中。
We have used a monoclonal antibody affinity column to purify from HeLa cells a protein of molecular weight 170,000 (designated pp170) which we previously identified as a nucleotide-sensitive microtubule-binding protein (Rickard, J. E., and Kreis, T. E. (1990) J. Cell Biol. 110, 1623-1633). We show here that the affinity-purified pp170 binds directly to taxol-polymerized tubulin. This association is not affected directly by MgATP. Addition of MgATP can, however, inhibit binding of pp170 to microtubules in the presence of microtubule-binding proteins from HeLa cells. This effect of MgATP correlates with phosphorylation of pp170 by a microtubule-associated kinase. Potato acid phosphatase dephosphorylates the pp170 and restores the ability of pp170 to bind to microtubules. Furthermore, binding of pp170 to microtubules in a high speed supernatant extract is inhibited by the phosphatase inhibitor okadaic acid, consistent with an inhibitory effect of pp170 phosphorylation on microtubule binding. In vivo, pp170 is phosphorylated on serine residues, with a half-life for the phosphate groups of approximately 2 h. Depolymerization of microtubules with nocodazole abolishes incorporation of 32P into the protein, apparently by increasing the rate of its dephosphorylation. Stabilization of microtubules with taxol reduces the rate of 32P incorporation into pp170 by approximately 50%, but has no significant effect on phosphate loss. These data establish that pp170 is a microtubule-binding protein, and that the microtubule interaction is inhibited by phosphorylation of pp170. The sensitivity of the in vivo phosphorylation state of pp170 to microtubule-active drugs suggests that this posttranslational modification may be an important regulator of the interaction of pp170 with microtubules in cells.