Regulation of Microtubule Dynamic Instability in Vitro by Differentially Phosphorylated Stathmin
Regulation of Microtubule Dynamic Instability in Vitro by Differentially Phosphorylated Stathmin
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DOI：
10.1074/jbc.m900343200
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发表时间：
2009-06-05
影响因子：
4.8
通讯作者：
Wilson, Leslie
中科院分区：
文献类型：
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作者：
Manna, Tapas;Thrower, Douglas A.;Wilson, Leslie
Stathmin是微管聚合和动力学的重要调节因子。当去磷酸化时，它以两种方式破坏微管的稳定性，通过将可溶性微管蛋白隔离到组装不能组装的T2S复合体中来减少微管聚合物的质量(每个Stathmin分子两个α：β微管蛋白二聚体)，以及通过直接与微管结合来增加从生长到正负端缩短的开关频率(灾难频率)。Stathmin在其四个丝氨酸残基(Ser(16)、Ser(25)、Ser(38)和Ser(63))中的一个或多个上的磷酸化降低了其微管破坏稳定的活性。然而，Stathmin的单个丝氨酸残基的磷酸化对微管动态不稳定性的影响还没有得到系统的研究。在此，我们分析了Stathmin在Ser(16)或Ser(63)处单磷酸化，以及在Ser(25)和Ser(38)处双磷酸化对其调节体外稳态微管动态不稳定性能力的影响。Ser(16)或Ser(63)处的磷酸化大大降低或取消了stathmin与可溶性微管蛋白结合和隔离的能力，以及它通过直接与微管结合而充当灾难因子的能力。相反，Ser(25)和Ser(38)的双磷酸化并不影响stathmin与微管或微管的结合，也不影响其促进灾难的活性。我们的结果表明，Stathmin对动态不稳定性的影响被Ser(16)和Ser(63)的磷酸化强烈但不同地减弱，并支持Ser(16)或Ser(63)特异的激酶选择性靶向为调节微管功能提供补充机制的假说。
Stathmin is an important regulator of microtubule polymerization and dynamics. When unphosphorylated it destabilizes microtubules in two ways, by reducing the microtubule polymer mass through sequestration of soluble tubulin into an assembly-incompetent T2S complex (two alpha:beta tubulin dimers per molecule of stathmin), and by increasing the switching frequency (catastrophe frequency) from growth to shortening at plus and minus ends by binding directly to the microtubules. Phosphorylation of stathmin on one or more of its four serine residues (Ser(16), Ser(25), Ser(38), and Ser(63)) reduces its microtubule-destabilizing activity. However, the effects of phosphorylation of the individual serine residues of stathmin on microtubule dynamic instability have not been investigated systematically. Here we analyzed the effects of stathmin singly phosphorylated at Ser(16) or Ser(63), and doubly phosphorylated at Ser(25) and Ser(38), on its ability to modulate microtubule dynamic instability at steady-state in vitro. Phosphorylation at either Ser(16) or Ser(63) strongly reduced or abolished the ability of stathmin to bind to and sequester soluble tubulin and its ability to act as a catastrophe factor by directly binding to the microtubules. In contrast, double phosphorylation of Ser(25) and Ser(38) did not affect the binding of stathmin to tubulin or microtubules or its catastrophe-promoting activity. Our results indicate that the effects of stathmin on dynamic instability are strongly but differently attenuated by phosphorylation at Ser(16) and Ser(63) and support the hypothesis that selective targeting by Ser(16)-specific or Ser(63)-specific kinases provides complimentary mechanisms for regulating microtubule function.