Detyrosination of tubulin regulates the interaction of intermediate filaments with microtubules in vivo via a kinesin-dependent mechanism
Detyrosination of tubulin regulates the interaction of intermediate filaments with microtubules in vivo via a kinesin-dependent mechanism
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DOI：
10.1091/mbc.10.4.1105
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发表时间：
1999-04-01
影响因子：
3.3
通讯作者：
Gundersen, GG
中科院分区：
文献类型：
--
作者：
Kreitzer, G;Liao, GJ;Gundersen, GG
微管蛋白的翻译后修饰形式在细胞中稳定的微管（MTs）亚群中积累，但它们本身并不参与产生微管的稳定性。我们先前表明，稳定的、去酪氨酸化（Glu）的微管在成纤维细胞中起到定位波形蛋白中间丝（IFs）的作用。为了确定微管蛋白去酪氨酸化还是微管稳定性是中间丝优先与Glu微管结合的关键因素，我们将不可聚合的Glu微管蛋白微量注射到细胞中。如果去酪氨酸化是关键的，那么可溶性Glu微管蛋白应该是中间丝 - 微管相互作用的竞争性抑制剂。在微量注射之前，通过用碘乙酰胺（IAA）处理使Glu微管蛋白变得不可聚合且不可酪氨酸化。微量注射的IAA - Glu微管蛋白破坏了中间丝与微管的相互作用，通过中间丝向核周位置塌陷来检测，并且对细胞中Glu或酪氨酸化微管的阵列没有可检测到的影响。相反，无论是IAA - 酪氨酸化微管蛋白还是未处理的可组装成微管的Glu微管蛋白，在微量注射时都不会导致中间丝塌陷。通过微量注射α - 微管蛋白的片段，对Glu微管蛋白上负责干扰Glu微管 - 中间丝相互作用的表位进行了定位。Glu微管蛋白的14 - kDa C末端片段（α - C Glu）诱导中间丝塌陷，而α - 微管蛋白的36 - kDa N末端片段没有改变中间丝阵列。该表位所需的不仅仅是C末端的去酪氨酸化位点，因为模拟Glu微管蛋白C末端的短肽（7聚体）没有破坏中间丝的分布。我们先前表明驱动蛋白可能介导Glu微管和中间丝的相互作用。在这项研究中我们发现，在体外驱动蛋白与微管的结合被破坏体内中间丝 - Glu微管相互作用的相同试剂（即IAA - Glu微管蛋白和α - C Glu）所抑制。这些结果首次表明微管蛋白去酪氨酸化作为一种信号，通过一种可能涉及驱动蛋白的机制将中间丝招募到微管上。
Posttranslationally modified forms of tubulin accumulate in the subset of stabilized microtubules (MTs) in cells but are not themselves involved in generating MT stability. We showed previously that stabilized, detyrosinated (Glu) MTs function to localize vimentin intermediate filaments (IFs) in fibroblasts. To determine whether tubulin detyrosination or MT stability is the critical element in the preferential association of Ifs with Glu MTs, we microinjected nonpolymerizable Glu tubulin into cells. If detyrosination is critical, then soluble Glu tubulin should be a competitive inhibitor of the IF-MT interaction. Before microinjection, Glu tubulin was rendered nonpolymerizable and nontyrosinatable by treatment with iodoacetamide (IAA). Microinjected IAA-Glu tubulin disrupted the interaction of Ifs with MTs, as assayed by the collapse of Ifs to a perinuclear location, and had no detectable effect on the array of Glu or tyrosinated MTs in cells. Conversely, neither IAA-tyrosinated tubulin nor untreated Glu tubulin, which assembled into MTs, caused collapse of Ifs when microinjected. The epitope on Glu tubulin responsible for interfering with the Glu MT-IF interaction was mapped by microinjecting tubulin fragments of alpha-tubulin. The 14-kDa C-terminal fragment of Glu tubulin (alpha-C Glu) induced IF collapse, whereas the 36-kDa N-terminal fragment of alpha-tubulin did not alter the IF array. The epitope required more than the detyrosination site at the C terminus, because a short peptide (a 7-mer) mimicking the C terminus of Glu tubulin did not disrupt the IF distribution. We previously showed that kinesin may mediate the interaction of Glu MTs and IFs. In this study we found that kinesin binding to MTs in vitro was inhibited by the same reagents (i.e., IAA-Glu tubulin and alpha-C Glu) that disrupted the IF-Glu MT interaction in vivo. These results demonstrate for the first time that tubulin detyrosination functions as a signal for the recruitment of Ifs to MTs via a mechanism that is likely to involve kinesin.