Intersectin Adaptor Protein In Regulation Of Endocytosis

我们的研究小组有兴趣了解Intersectin（ITSN）衔接蛋白在调节信号转导级联反应中的作用，特别是受体酪氨酸激酶（RTK）。ITSN是一个不断增长的衔接蛋白家族的成员，该家族具有保守的Eps 15同源（EH）结构域以及额外的蛋白识别基序。含EH的蛋白质在调节网格蛋白依赖性内吞作用中发挥不可或缺的作用。ITSN由两个NH 4末端EH结构域、一个卷曲螺旋（CC）区和5个串联的Src同源3（SH 3）结构域组成。此外，存在ITSN的较大剪接变体，称为ITSN-L，其具有编码Db 1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结构域作为Ras样GTP酶的Rho亚家族的鸟嘌呤核苷酸交换因子（GEF）起作用，所述Ras样GTP酶包括Rho、Rac和Cdc 42。这些结构域与PH结构域一起发挥作用，PH结构域直接与脂质和膜相互作用。因此，ITSN-L可以用于调节神经系统内的Rho家族激活。C2结构域结合磷脂膜，蛋白质或可溶性肌醇多磷酸使用Ca+2依赖性和非依赖性机制。虽然各种各样的实验表明ITSN参与了内吞作用的调节，但我们现在已经证明ITSN激活了信号转导途径。过去十年的研究表明，内吞作用和促有丝分裂信号之间存在联系。因此，我们的研究结果表明，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的靶点之一是Elk-1转录因子。虽然Elk-1是经典的Ras-MAPK通路的目标，我们已经证明，ITSN激活Elk-1在MEK和MAPK的独立方式。因此，ITSN似乎刺激不同于经典Ras-MAPK途径的途径以激活Elk-1。我们最近发表了我们的工作，证明ITSN刺激JNK依赖性途径，这是ITSN激活Elk-1所必需的。此外，我们发现ITSN还激活一个独立的Ras通路，该通路不导致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在细胞中的作用上。我们还继续我们的生化功能的泛素相互作用的图案（UIMs）的表征。我们最近发表了来自许多但不是所有蛋白质的UIM能够结合多聚泛素链并促进蛋白质的泛素化。我们发现，UIMs功能的方向依赖性的方式，以促进泛素化，这一发现表明，UIMs也可能通过内部和分子间的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.