O'Bryan Lab Research
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.
Figure 1. Cellular signaling. Shown is an idealized pathway involving activation of a transmembrane tyrosine kinase receptor. Binding of ligand results
in activation of the intrinsic kinase activity of the receptor leading to receptor autophosphorylation as well as phosphorylation of substrates. Activated receptors are recruited into
clathrin-coated pits followed by internalization into intracellular vesicles that serve as a platform for compartmentalized signaling. Receptors are then sorted for either degradation in
the lysosome or recycled back to the plasma membrane. Thus, endocytosis is both a negative and positive regulator of cellular signaling. Current challenges in signal transduction are to
determine how the various signals are integrated within a cell to produce a given response or phenotype. GF, growth factor; Ub, ubiquitin; AP2, adaptor protein 2; DAG, diacylglycerol; TF,
transcription factor; Dyn, dynamin; , clathrin triskelion.
Figure. 2. ITSN coordinately regulates multiple biochemical pathways.The modular structure of ITSN allows for the interaction with multiple pathways in the cell. As a
result, loss of ITSN expression or alternatively, overexpression, as is found in Down Syndrome, will impact multiple cellular pathways leading to alteration in cell growth and survival
(1 & 2), endocytosis (3) and receptor regulation (4).
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:
- 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
- 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.
- 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.
- 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.