Premont Richard T, Perry Stephen J, Schmalzigaug Robert, Roseman J Tyler, Xing Yanghui, Claing Audrey
Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Cell Signal. 2004 Sep;16(9):1001-11. doi: 10.1016/j.cellsig.2004.02.002.
GIT proteins are GTPase-activating proteins (GAPs) for ADP-ribosylation factor (ARF) small GTP-binding proteins, and interact with the PIX family of Rac1/Cdc42 guanine nucleotide exchange factors. GIT and PIX transiently localize p21-activated protein kinases (PAKs) to remodeling focal adhesions through binding to paxillin. To understand the role of these interactions, the association of GIT and PIX proteins was examined in detail. Two separable binding interactions link GIT and PIX proteins, GIT and PIX proteins each dimerize and a beta-PIX fragment containing the GIT-binding region failed to inhibit the association of the GIT and PIX proteins. Endogenous GIT and PIX co-fractionate at a very high molecular size. Purified 6xHis-tagged beta-PIX from Sf9 cells co-expressing untagged GIT1 yields recombinant GIT1/beta-PIX complexes that have equal amounts of beta-PIX and GIT1 and co-fractionate at the same large size as native GIT/PIX complexes. Thus, GIT and PIX proteins are tightly associated as a multimeric nexus capable of linking together important signaling molecules, including PAKs.
GIT蛋白是ADP核糖基化因子（ARF）小GTP结合蛋白的GTP酶激活蛋白（GAP），并与Rac1/Cdc42鸟嘌呤核苷酸交换因子的PIX家族相互作用。GIT和PIX通过与桩蛋白结合，将p21激活的蛋白激酶（PAK）短暂定位于重塑粘着斑。为了了解这些相互作用的作用，对GIT和PIX蛋白的关联进行了详细研究。两种可分离的结合相互作用将GIT和PIX蛋白联系起来，GIT和PIX蛋白各自二聚化，并且包含GIT结合区域的β-PIX片段未能抑制GIT和PIX蛋白的关联。内源性GIT和PIX在非常大的分子大小下共同分级分离。从共表达无标签GIT1的Sf9细胞中纯化的6xHis标记的β-PIX产生重组GIT1/β-PIX复合物，其具有等量的β-PIX和GIT1，并且在与天然GIT/PIX复合物相同的大尺寸下共同分级分离。因此，GIT和PIX蛋白紧密结合形成一个多聚体连接点，能够将包括PAK在内的重要信号分子连接在一起。
