Department of Oncology and Biochemistry, London Regional Cancer Program, University of Western Ontario and London Health Sciences Center, London, Ontario, Canada.
J Biol Chem. 2010 Aug 20;285(34):26461-74. doi: 10.1074/jbc.M110.121491. Epub 2010 Jun 17.
An oncogenic form of RHAMM (receptor for hyaluronan-mediated motility, mouse, amino acids 163-794 termed RHAMM(Delta163)) is a cell surface hyaluronan receptor and mitotic spindle protein that is highly expressed in aggressive human cancers. Its regulation of mitotic spindle integrity is thought to contribute to tumor progression, but the molecular mechanisms underlying this function have not previously been defined. Here, we report that intracellular RHAMM(Delta163) modifies the stability of interphase and mitotic spindle microtubules through ERK1/2 activity. RHAMM(-/-) mouse embryonic fibroblasts exhibit strongly acetylated interphase microtubules, multi-pole mitotic spindles, aberrant chromosome segregation, and inappropriate cytokinesis during mitosis. These defects are rescued by either expression of RHAMM or mutant active MEK1. Mutational analyses show that RHAMM(Delta163) binds to alpha- and beta-tubulin protein via a carboxyl-terminal leucine zipper, but in vitro analyses indicate this interaction does not directly contribute to tubulin polymerization/stability. Co-immunoprecipitation and pulldown assays reveal complexes of RHAMM(Delta163), ERK1/2-MEK1, and alpha- and beta-tubulin and demonstrate direct binding of RHAMM(Delta163) to ERK1 via a D-site motif. In vitro kinase analyses, expression of mutant RHAMM(Delta163) defective in ERK1 binding in mouse embryonic fibroblasts, and blocking MEK1 activity collectively confirm that the effect of RHAMM(Delta163) on interphase and mitotic spindle microtubules is mediated by ERK1/2 activity. Our results suggest a model wherein intracellular RHAMM(Delta163) functions as an adaptor protein to control microtubule polymerization during interphase and mitosis as a result of localizing ERK1/2-MEK1 complexes to their tubulin-associated substrates.
一种致癌形式的 RHAMM（透明质酸介导的运动受体，鼠，氨基酸 163-794 称为 RHAMM(Delta163)) 是一种细胞表面透明质酸受体和有丝分裂纺锤体蛋白，在侵袭性人类癌症中高度表达。它对有丝分裂纺锤体完整性的调节被认为有助于肿瘤的进展，但以前尚未定义其功能的分子机制。在这里，我们报告细胞内 RHAMM(Delta163) 通过 ERK1/2 活性改变了间期和有丝分裂纺锤体微管的稳定性。RHAMM(-/-) 小鼠胚胎成纤维细胞表现出强烈的乙酰化间期微管、多极有丝分裂纺锤体、异常染色体分离和有丝分裂期间不适当的胞质分裂。这些缺陷可以通过表达 RHAMM 或突变的活性 MEK1 来挽救。突变分析表明，RHAMM(Delta163) 通过羧基末端亮氨酸拉链与α-和β-微管蛋白结合，但体外分析表明这种相互作用不会直接导致微管聚合/稳定性。共免疫沉淀和下拉实验显示 RHAMM(Delta163)、ERK1/2-MEK1 和α-和β-微管蛋白的复合物，并证明 RHAMM(Delta163) 通过 D 位基序直接与 ERK1 结合。体外激酶分析、在小鼠胚胎成纤维细胞中表达不能与 ERK1 结合的突变 RHAMM(Delta163) 以及阻断 MEK1 活性共同证实，RHAMM(Delta163) 对间期和有丝分裂纺锤体微管的影响是由 ERK1/2 活性介导的。我们的结果表明，细胞内 RHAMM(Delta163) 作为一种衔接蛋白，通过将 ERK1/2-MEK1 复合物定位到其微管相关底物上来控制间期和有丝分裂期间的微管聚合。
