Kinesin is a candidate for cross-bridging microtubules and intermediate filaments - Selective binding of kinesin to detyrosinated tobulin and vimentin
Kinesin is a candidate for cross-bridging microtubules and intermediate filaments - Selective binding of kinesin to detyrosinated tobulin and vimentin
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DOI：
10.1074/jbc.273.16.9797
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发表时间：
1998-04-17
影响因子：
4.8
通讯作者：
Gundersen, GG
中科院分区：
文献类型：
--
作者：
Liao, GJ;Gundersen, GG
我们先前表明，稳定的脱酪氨酸（Glu）微管起到在成纤维细胞中定位波形蛋白中间丝的作用（Gurland，G.，Gundersen，G. G.（1995）J. Cell Biol.131，1275-1290）。为了鉴定介导Glu微管-波形蛋白相互作用的候选蛋白，我们将微管与微管相互作用蛋白和饱和水平的Glu或酪氨酸化（Tyr）微管蛋白的抗体一起孵育。Glu微管蛋白的抗体比Tyr微管蛋白的抗体更有效地防止从成纤维细胞或脑提取物获得的驱动蛋白的微管结合。Scatchard图分析显示驱动蛋白头部与Glu微管的结合亲和力比与Tyr微管的结合亲和力高2.8倍。纯化的脑驱动蛋白与波形蛋白共沉积，但不与神经丝共沉积，表明驱动蛋白特异性地与波形蛋白结合，而没有辅助分子。驱动蛋白与波形蛋白的结合对ATP不敏感，驱动蛋白头部不能与波形蛋白结合。通过SDS-聚丙烯酰胺凝胶电泳，在波形蛋白/驱动蛋白颗粒中检测到类似于120 kDa的驱动蛋白重链和类似于64 kDa的轻链。轻链与一般驱动蛋白轻链抗体反应，但不与识别脑中驱动蛋白轻链的两种已知亚型的另外两种抗体反应，这表明参与与波形蛋白结合的驱动蛋白可能是特异性的。这些结果表明，驱动蛋白为基础的机制，波形蛋白与去酪氨酸微管的优先相互作用。
We showed previously that stable, detyrosinated (Glu) microtubules function to localize vimentin intermediate filaments in fibroblasts (Gurland, G., and Gundersen, G. G. (1995) J. Cell Biol. 131, 1275-1290), To identify candidate proteins that mediate the Glu microtubule-vimentin interaction, we incubated microtubules with microtubule-interacting proteins and saturating levels of antibodies to Glu or tyrosinated (Tyr) tubulin. Antibodies to Glu tubulin prevented the microtubule binding of kinesin obtained from fibroblast or brain extracts more effectively than antibodies to Tyr tubulin. Scatchard plot analysis showed that kinesin heads bound to Glu microtubules with an similar to 2.8-fold higher affinity than to Tyr microtubules, Purified brain kinesin cosedimented with vimentin, but not with neurofilaments, indicating that kinesin specifically associates with vimentin without accessory molecules. Kinesin binding to vimentin was not sensitive to ATP, and kinesin heads failed to bind to vimentin. By SDS-polyacrylamide gel electrophoresis, a kinesin heavy chain of similar to 120 kDa and a light chain of similar to 64 kDa were detected in vimentin/kinesin pellets. The light chain reacted with a general kinesin light chain antibody, but not with two other antibodies that recognize the two known isoforms of kinesin light chain in brain, suggesting that the kinesin involved in binding to vimentin may be a specific one. These results demonstrate a kinesin-based mechanism for the preferential interaction of vimentin with detyrosinated microtubules.