UCSF Helen Diller Family, Comprehensive Cancer Center, 2340 Sutter Street, San Francisco, CA 94115, USA.
Mol Cell Biol. 2010 May;30(9):2264-79. doi: 10.1128/MCB.01450-08. Epub 2010 Feb 16.
The neurofibromatosis type 1 (NF1) gene encodes the GTPase-activating protein (GAP) neurofibromin, which negatively regulates Ras activity. The yeast Saccharomyces cerevisiae has two neurofibromin homologs, Ira1 and Ira2. To understand how these proteins are regulated, we utilized an unbiased proteomics approach to identify Ira2 and neurofibromin binding partners. We demonstrate that the Gpb1/Krh2 protein binds and negatively regulates Ira2 by promoting its ubiquitin-dependent proteolysis. We extended our findings to show that in mammalian cells, the ETEA/UBXD8 protein directly interacts with and negatively regulates neurofibromin. ETEA contains both UBA and UBX domains. Overexpression of ETEA downregulates neurofibromin in human cells. Purified ETEA, but not a mutant of ETEA that lacks the UBX domain, ubiquitinates the neurofibromin GAP-related domain in vitro. Silencing of ETEA expression increases neurofibromin levels and downregulates Ras activity. These findings provide evidence for conserved ubiquitination pathways regulating the RasGAP proteins Ira2 (in yeast) and neurofibromin (in humans).
神经纤维瘤病 1 型 (NF1) 基因编码 GTP 酶激活蛋白 (GAP) 神经纤维瘤，它负调控 Ras 活性。酵母酿酒酵母有两种神经纤维瘤同源物，Ira1 和 Ira2。为了了解这些蛋白质是如何被调控的，我们利用无偏蛋白质组学方法来鉴定 Ira2 和神经纤维瘤的结合伙伴。我们证明 Gpb1/Krh2 蛋白通过促进其泛素依赖性蛋白水解来结合并负调控 Ira2。我们的研究结果进一步表明，在哺乳动物细胞中，ETEA/UBXD8 蛋白直接与神经纤维瘤相互作用并负调控其活性。ETEA 包含 UBA 和 UBX 结构域。ETEA 的过表达可下调人细胞中的神经纤维瘤。体外纯化的 ETEA，但不含有 UBX 结构域的 ETEA 突变体，可使神经纤维瘤 GAP 相关结构域泛素化。ETEA 表达的沉默可增加神经纤维瘤的水平并下调 Ras 活性。这些发现为调节 RasGAP 蛋白 Ira2（在酵母中）和神经纤维瘤（在人类中）的保守泛素化途径提供了证据。
