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 have now defined a signaling pathway by which ITSN activates Elk-1 independent of MEK and MAPK. In addition, we want to determine the importance of this signaling activity for ITSN function. Given the role of ITSN in the regulation of endocytosis, we are currently examining the possible function of ITSN in modulating RTK function through regulation of RTK endocytosis, recycling and degradation. We have recently demonstrated that an ITSN-binding protein called epsin is posttranslationally modified by the covalent attachment of ubiquitin suggesting that ITSN may also be linked to the process of ubiquitination. We have characterized the sequence requirements of epsin which direct the ubiquitination of the protein and have uncovered a novel role for a recently describe motif termed the ubiquitin-interacting motif (UIM) in promoting epsin ubiquitination. We are in the process of determining the specific enzymes responsible for epsin ubiquitination. In addition, we are testing whether the UIMs from other proteins may perform a similar function.
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