Intersectin Adaptor Protein In Regulation Of Endocytosis

我们小组感兴趣的是了解交叉素(ITSN)接头蛋白在调节信号转导通路中的作用，特别是受体酪氨酸激酶(RTK)。ITSN是一个不断增长的适配器蛋白家族的成员，它拥有保守的Eps15同源(EH)结构域以及额外的蛋白质识别基序。含EH的蛋白质在调节依赖于笼蛋白的内吞作用中起着不可或缺的作用。ITSN由两个NH4末端的EH结构域、一个卷曲线圈(CC)区域和5个串联的Src同源3(SH3)结构域组成。此外，ITSN还有一个更大的剪接变体，称为ITSN-L，它具有编码DBL同源(DH)结构域、Pleckstrin同源(PH)结构域和C2结构域的COOH-末端延伸。DH结构域是Ras类GTP酶的Rho亚家族的鸟嘌呤核苷酸交换因子(GEF)，包括Rho、Rac和Cdc42。这些结构域与直接与脂质和膜相互作用的PH结构域协同工作。因此，ITSN-L可能在神经系统内调节Rho家族的激活。C2结构域结合磷脂膜、蛋白质或可溶性肌醇多磷酸，既依赖于钙离子，也不依赖于钙离子。虽然各种实验都表明ITSN参与了内吞作用的调节，但我们现在已经证明了ITSN激活了信号转导通路。过去十年的研究表明，内吞作用和有丝分裂信号之间存在联系。因此，我们的发现表明，ITSN可能是这两个细胞过程之间的一个可能的分子联系。我们目前的重点是了解ITSN激活信号通路的机制。ITSN的靶标之一是ELK-1转录因子。虽然ELK-1经典地被认为是RAS-MAPK通路的靶标，但我们已经证明ITSN以MEK和MAPK非依赖性的方式激活ELK-1。因此，ITSN似乎刺激了一条与经典的RAS-MAPK途径不同的途径，以激活ELK-1。我们最近发表的工作表明，ITSN刺激了ITSN激活ELK-1所必需的JNK依赖的途径。此外，我们还发现，ITSN还激活了一个独立的RAS通路，该通路不会导致JNK或ERK MAPK通路的激活。因此，我们将注意力集中在确定ITSN激活的RAS池在细胞中的作用上。我们还继续描述了泛素相互作用基序(UIM)的生化功能。我们最近发表了来自许多但不是所有蛋白质的UIM既能结合多泛素链又能促进蛋白质泛素化的研究结果。我们发现UIMs以一种方向依赖的方式促进泛素化，这一发现表明UIMs也可能通过分子内和分子间相互作用来调节蛋白质的功能。

Our group is interested in understanding the role of the Intersectin (ITSN) adaptor protein in regulating signal transduction cascades in general and receptor tyrosine kinases (RTKs) in particular. ITSN is a member of a growing family of adaptor proteins that possess conserved Eps15 homology (EH) domains as well as additional protein recognition motifs. EH-containing proteins play an integral role in regulating clathrin-dependent endocytosis. ITSN consists of two NH4-terminal EH domains, a coiled-coil (CC) region and 5 tandem Src homology 3 (SH3) domains. In addition, there is a larger splice variant of ITSN, termed ITSN-L, which possess a COOH-terminal extension encoding a Dbl homology (DH) domain, a Pleckstrin homology (PH) domain and a C2 domain . DH domains function as guanine nucleotide exchange factors (GEFs) for the Rho subfamily of Ras-like GTPases which include Rho, Rac and Cdc42. These domains function in concert with PH domains which direct interaction with lipids and membrane. Thus, ITSN-L may serve to regulate Rho family activation within the nervous system. C2 domains bind phospholipid membranes, proteins or soluble inositol polyphosphates using both Ca+2-dependent and -independent mechanisms. Although a variety of experiments have implicated ITSN in the regulation of endocytosis, we have now demonstrated that ITSN activates signal transduction pathways. Research over the past decade has suggested a link between endocytosis and mitogenic signaling. Thus, our findings suggest that ITSN may be one possible molecular link between these two cellular processes. Our current focus is on understanding the mechanism by which ITSN activates signaling pathways. One of the targets of ITSN is the Elk-1 transcription factor. Although Elk-1 is classically known as a target of the Ras-MAPK pathway, we have demonstrated that ITSN activates Elk-1 in a MEK and MAPK-independent manner. Thus, ITSN appears to stimulate a pathway distinct from the classic Ras-MAPK pathway in order to activate Elk-1. We recently published our work demonstrating that ITSN stimulates a JNK-dependent pathway necessary for Elk-1 activation by ITSN. In addition, we discovered that ITSN also activates an independent Ras pathway which does not lead to activation of the JNK or ERK MAPK pathways. Thus we have focused our attention on determining the role of this pool of ITSN-activated Ras in the cell. We have also continued our characterization of the biochemical function of ubiquitin-interacting motifs (UIMs). We recently published that UIMs from many but not all proteins are capable of both binding polyubiquitin chains and promoting ubiquitylation of proteins. We discovered that UIMs function in an orientation-dependent manner to promote ubiquitylation, a finding which suggested that UIMs may also regulate protein function through intra and inter molecular interactions.