The C-terminal domains of human neurofibromin and its budding yeast homologs Ira1 and Ira2 regulate the metaphase to anaphase transition

The human tumor suppressor neurofibromin contains a cysteine and serine-rich domain/Ras-GTPase activating protein domain (CSRD/RasGAP) and a C-terminal domain (CTD). Domain studies of neurofibromin suggest it has other functions in addition to being a RasGAP, but the mechanisms underlying its tumor suppressor activity are not well understood. The budding yeast <i>Saccharomyces cerevisiae</i> is a good model system for studying neurofibromin function because it possesses Ira1 and Ira2, which are homologous to human neurofibromin in both sequence and function. We found that overexpression of CTD or a neurofibromin CTD-homologous domain (CHD) of Ira1/2 in budding yeast delayed degradation of the securin protein Pds1, whereas overexpression of CSRD/RasGAP did not affect Pds1 degradation. We also found that when CTD or CHD was overexpressed, the number of cells in metaphase was higher than in the control. These results demonstrate that CTD and CHD function in the metaphase to anaphase transition. In addition, Δ<i>ira1</i>Δ<i>ira2</i> cells bypassed mitotic arrest in response to spindle damage, indicating that Ira1 and Ira2 may be involved in the spindle assembly checkpoint (SAC). However, Δ<i>ira1</i>Δ<i>ira2</i>Δ<i>mad2</i> cells are more sensitive to spindle damage than Δ<i>mad2</i> or Δ<i>ira1</i>Δ<i>ira2</i> cells are, suggesting that Ira1/2 and Mad2 function in different pathways. Overexpression of CTD but not CSRD/RasGAP partially rescued the hypersensitivity of Δ<i>ira1</i>Δ<i>ira2</i>Δ<i>mad2</i> cells to microtubule-destabilizing drugs, indicating a role for CTD in the SAC pathway. Taken together, independently of RasGAP activity, the C-terminal domains of neurofibromin, Ira1, and Ira2 regulate the metaphase to anaphase transition in a Mad2-independent fashion.

人类肿瘤抑制蛋白神经纤维蛋白（neurofibromin）包含一个富含半胱氨酸与丝氨酸结构域/Ras鸟苷三磷酸酶激活蛋白结构域（CSRD/RasGAP）以及一个羧基末端结构域（CTD）。针对神经纤维蛋白的结构域研究提示，其除具备RasGAP功能外还存在其他生物学功能，但目前其肿瘤抑制活性的分子机制尚未被充分阐明。出芽酵母酿酒酵母（Saccharomyces cerevisiae）是研究神经纤维蛋白功能的优良模型系统，因其拥有在序列与功能上均与人类神经纤维蛋白同源的Ira1与Ira2蛋白。本研究发现，在出芽酵母中过表达CTD或Ira1/2的神经纤维蛋白羧基末端结构域同源结构域（CHD），会延迟分离酶抑制蛋白Pds1的降解；而过表达CSRD/RasGAP则不会对Pds1的降解产生影响。此外我们还观察到，当CTD或CHD过表达时，处于细胞分裂中期的细胞数量显著高于对照组。上述实验结果证明，CTD与CHD参与调控细胞周期中期向后期的转换过程。进一步研究发现，ira1Δira2双缺失突变体细胞可绕过纺锤体损伤诱导的有丝分裂阻滞，提示Ira1与Ira2可能参与纺锤体组装检验点（SAC）的调控。然而，ira1Δira2Δmad2三缺失突变体细胞对纺锤体损伤的敏感性高于mad2单缺失突变体或ira1Δira2双缺失突变体，这表明Ira1/2与Mad2通过不同的信号通路发挥功能。过表达CTD而非CSRD/RasGAP，可部分回补ira1Δira2Δmad2三缺失突变体细胞对微管解聚药物的超敏感性，这表明CTD在纺锤体组装检验点通路中发挥调控作用。综上，不依赖于RasGAP活性的前提下，神经纤维蛋白、Ira1与Ira2的羧基末端结构域以不依赖Mad2的方式调控细胞周期中期向后期的转换过程。