The ability of cells to respond to extracellular signals is critical for the normal homeostasis of an organism. The binding of peptide growth factors to transmembrane receptors such receptor tyrosine kinases (RTKs) results in the activation of numerous biochemical pathways that regulate the processes of cell growth, differentiation, development and apoptosis (Figure. 1). Disruption of these pathways underlies the pathogenesis of many diseases in humans, including cancer and neurodegeneration. My particular research interests lie in understanding a class of signal transduction molecules referred to as adaptor or scaffolding proteins. These molecules consist predominantly of discrete protein:protein interaction domains and their primary role is to regulate the temporal and spatial assembly of complexes that modulate the action of receptors such as RTKs. The research in my laboratory is currently focused on one such scaffold called intersectin (ITSN). ITSN is a highly conserved, multi-domain protein consisting of two NH2-terminal Eps 15 homology (EH) domains, a central coiled-coil (CC) and five COOH-terminal Src homology 3 (SH3) domains. In addition, there is a larger isoform termed ITSN-L that possesses a COOH--terminal extension encoding a guanine nucleotide exchange factor (GEF) domain for Rho family of GTPases. As the name implies, ITSN is involved in the regulation of numerous biochemical pathways and thus stands at a nexus in the regulation of cell function (Figure. 2). I am particularly interested in understanding the mechanism by which ITSN interfaces with RTKs given its ability to cooperate with these receptors in the activation of cellular signaling pathways and in the oncogenic transformation of cells.

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Work from a number of laboratories indicates that ITSN regulates clathrin-dependent endocytosis through the recruitment of proteins important for clathrin-coated pit assembly. Although endocytosis is classically thought of as a mechanism for attenuating cellular signaling, emerging evidence indicate that components of the endocytic machinery play a positive role in activating cellular signaling pathways. Indeed, my laboratory discovered that ITSN activates cellular signaling pathways in addition to its role in endocytosis. These findings provide direct evidence that endocytosis both activates as well as attenuates cellular signaling. The work in my laboratory is focused on several aspects of ITSN function:

1. Determining the mechanism by which ITSN stimulates mitogenic signaling pathways. We demonstrated that the various modular domains of ITSN are involved in activating different signaling pathways. Given these findings we are in the process of characterizing binding partners for ITSN and determining their role in ITSN function. One such partner is Sos1, a guanine nucleotide exchange factor that stimulates the formation of active, GTP-bound Ras, one of the most frequently mutated genes in human cancers. We discovered that ITSN regulates Ras activation on intracellular vesicles. Interestingly, this pool of Ras does not activate the typical downstream Ras effectors. Thus, I am interested in determining the role of this pool of ITSN-activated Ras.


2. Determining the role of ITSN in ubiquitination. My laboratory also discovered that ITSN interacts with the ubiquitination machinery. We have recently found that ITSN associates with a number of E3 ubiquitin ligases which are responsible for the covalent attachment of ubiquitin to cellular proteins. One of these ITSN-associated ligases, Cbl, is a major regulator of RTKs thus providing an additional link between ITSN and RTK pathways. We are currently exploring the role of ITSN in Cbl-mediated regulation of RTKs as well as the importance of the additional ITSN-associated E3 ligases that we have identified.


3. Determining the role of ITSN in development and disease. We have begun to explore the importance of ITSN during development through the use of transgenic and knockout animal models. Intriguingly, ITSN is localized to human chromosome 21 in the Down Syndrome Critical Region and is overexpressed in Down Syndrome patients as well as in a mouse model for Down Syndrome. These findings suggest that ITSN overexpression contributes in part to the sequelae associated with Down Syndrome. The development of ITSN transgenic animals as well as ITSN null mice will help to address this possibility.


4. Determining the importance UIM-directed ubiquitination. In a related project, we discovered that the ITSN-binding protein epsin was modified by ubiquitin conjugation. Our studies revealed that a short motif present in epsin, as well as other proteins, termed the ubiquitin-interacting motif (UIM), was responsible for this modification. This finding represented the first demonstration of a conserved sequence that was sufficient for promoting the ubiquitination of a protein. However, the UIMs are themselves not the site of ubiquitin attachment suggesting that the UIMs recruit the cellular machinery to epsin thereby leading to ubiquitination of the protein. In addition, UIMs bind to ubiquitin and ubiquitinated proteins suggesting that these motifs may regulate a network of protein:protein interactions and modifications akin to tyrosine phosphorylation and SH2 domains. Finally, UIM-directed ubiquitination does not lead to degradation of the modified protein thus raising the question as to the function of UIM-directed ubiquitination.