Phosphorylation blocks the activity of tubulin polymerization-promoting protein (TPPP) -: Identification of sites targeted by different kinases
Phosphorylation blocks the activity of tubulin polymerization-promoting protein (TPPP) -: Identification of sites targeted by different kinases
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DOI：
10.1074/jbc.m703466200
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发表时间：
2007-10-05
影响因子：
4.8
通讯作者：
Ovadi, Judit
中科院分区：
文献类型：
--
作者：
Hlavanda, Emma;Klement, Eva;Ovadi, Judit
微管蛋白聚合促进蛋白（TPPP）是一种未折叠的脑特异性蛋白质，在体内外与微管蛋白/微管系统相互作用，并在人类病理性脑包涵体中富集。在这里，我们表明，TPPP诱导微管蛋白自组装成完整的经常捆绑微管，和特定位点的磷酸化明显影响TPPP的功能。体外磷酸化的野生型和截短形式（三角洲3- 43 TPPP）的人重组TPPP的激酶参与脑特异性过程。对于全长蛋白质，ERK 2、细胞周期蛋白依赖性激酶5（Cdk 5）和cAMP依赖性蛋白激酶（PKA）的化学计量分别为2.9 +/- 0.3、2.2 +/- 0.3和0.9 +/- 0.1 mol P/mol蛋白质，当N-末端尾缺失时，所有三种激酶均检测到0.4-0.5 mol P/mol蛋白质。通过质谱鉴定Cdk 5的磷酸化位点Thr（14）、Ser（18）、Ser（160）; ERK 2的磷酸化位点Ser（18）、Ser（160）和PKA的磷酸化位点Ser（32）。这些位点与生物信息学预测一致。从牛脑中分离的TPPP的三个N-末端位点也被发现在体内被磷酸化。亲和结合实验为TPPP和ERK 2之间的直接相互作用提供了证据。TPPP的磷酸化ERK 2或Cdk 5，但不是由PKA，扰乱TPPP和微管蛋白之间的相互作用诱导的结构改变，而不影响结合亲和力（Kd = 2.5-2.7 μ M）或化学计量（1摩尔TPPP/摩尔微管蛋白）的复合物。ERK 2或Cdk 5的磷酸化导致TPPP的微管组装活性丧失。我们的体外和体内数据的组合表明，ERK 2可以通过其未折叠的N-末端尾中的Thr（14）和/或Ser（18）的磷酸化来调节TPPP活性。
Tubulin polymerization- promoting protein (TPPP), an unfolded brain-specific protein interacts with the tubulin/microtubule system in vitro and in vivo, and is enriched in human pathological brain inclusions. Here we show that TPPP induces tubulin self-assembly into intact frequently bundled microtubules, and that the phosphorylation of specific sites distinctly affects the function of TPPP. In vitro phosphorylation of wild type and the truncated form (Delta 3-43TPPP) of human recombinant TPPP was performed by kinases involved in brain-specific processes. A stoichiometry of 2.9 +/- 0.3, 2.2 +/- 0.3, and 0.9 +/- 0.1 mol P/mol protein with ERK2, cyclin-dependent kinase 5 (Cdk5), and cAMP-dependent protein kinase (PKA), respectively, was revealed for the full-length protein, and 0.4-0.5 mol P/mol protein was detected with all three kinases when the N-terminal tail was deleted. The phosphorylation sites Thr(14), Ser(18), Ser(160) for Cdk5; Ser(18), Ser(160) for ERK2, and Ser(32) for PKA were identified by mass spectrometry. These sites were consistent with the bioinformatic predictions. The three N-terminal sites were also found to be phosphorylated in vivo in TPPP isolated from bovine brain. Affinity binding experiments provided evidence for the direct interaction between TPPP and ERK2. The phosphorylation of TPPP by ERK2 or Cdk5, but not by PKA, perturbed the structural alterations induced by the interaction between TPPP and tubulin without affecting the binding affinity (K-d = 2.5-2.7 mu M) or the stoichiometry (1 mol TPPP/mol tubulin) of the complex. The phosphorylation by ERK2 or Cdk5 resulted in the loss of microtubule-assembling activity of TPPP. The combination of our in vitro and in vivo data suggests that ERK2 can regulate TPPP activity via the phosphorylation of Thr(14) and/or Ser(18) in its unfolded N-terminal tail.