John O'Bryan

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. 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 1 (ITSN1) [1,2]. ITSN1 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 ITSN1-L that possesses a COOH-terminal extension encoding a guanine nucleotide exchange factor (GEF) domain for Rho family of GTPases. As the name implies, ITSN1 is involved in the regulation of numerous biochemical pathways and thus stands at a nexus in the regulation of cell function. I am particularly interested is understanding the mechanism by which ITSN1 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.

CURRENT PROJECTS

ITSN1 regulates clathrin-dependent endocytosis through the recruitment of proteins important for clathrin-coated pit assembly. Although endocytosis is classically viewed as a mechanism for attenuating cellular signaling, the endocytic machinery also participates in the activation of cellular signaling pathways. Indeed, my laboratory discovered that ITSN1 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 ITSN1 function:

1. Determining the mechanisms by which ITSN1 regulates mitogenic signaling pathways. ITSN1 associates with a variety of cellular signaling pathways through its multiple modular domains. Two areas of particular interest center on the regulation of E3 ubiquitin ligases and Ras GTPases.

a) Regulation of E3 ubiquitin ligases. We have defined a role for ITSN1 in the activation of the Cbl E3 ubiqutin ligase [3-5]. ITSN1 binds Cbl and enhances its ubiquitlylation of RTKs. We have defined additional components in this

regulation including Spry2 and the Shp2 protein tyrosine phosphatase. Furthermore, we have identified additional E3 ligases as targets of ITSN1 and are interested in defining the importance of ITSN1 in regulation of these

additional enzymes.

b) Regulation of Ras GTPase. We discovered that ITSN1 stimulates the compartmentalized activation of Ras on intracellular vesicles [6]. Surprisingly, however, this pool of ITSN1-activated Ras does not activate typical Ras

effector pathways such as the ERK-MAPK pathway. However, we have defined a novel PI3K isoform, namely PI3KC2beta, as the target of this ITSN1-Ras pathways [7,8]. Interestingly, our work has led to the discovery of a novel

function for nucleotide-free Ras (nfRas) in the inhibition of PI3KC2beta activity [7]. Our working model is that interaction of nfRas with PI3KC2beta mutually inhibits the function of both proteins and that binding of ITSN1 to

PI3KC2beta results in a conformation change leading to release of Ras which then immediately binds GTP to become active. In addition, loss of nfRas binding to PI3KC2beta results in activation of the lipid kinase of this

enzyme. We are currently working to further understand the physiological importance of this pathway.

2. Isolation of novel inhibitors of the Ras oncogene. Ras is one of the most frequently mutated genes in human cancers, with nearly 30% of human tumors bearing activating mutations in one of the three Ras genes. However, some tumors such as pancreatic tumors have a much higher incidence of Ras mutations, e.g., >90% KRas mutations in pancreatic tumors. However, pharmacological inhibition of Ras has proven disappointing thus far. We have recently isolated unique, highly specific inhibitors of HRas. These genetically encodable reagents have provided novel insight into unique approaches to therapeutically target Ras inhibition. We are currently working to further understand the mechanism by which these agents target Ras as well as isolate small molecules which mimic their biochemical activity.

3. Determining the role of ITSN1 in human disease. ITSN1 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 [9]. These findings suggest that ITSN1 overexpression contributes in part to the sequelae associated with Dwon Syndrome. Given its role in endocytosis and trafficking, we have begun exploring the role of ITSN1 in Down Syndrome through alteraction of these processes in patients. In particular, we are interested in the potential role of ITSN1 in the trafficking and processing a amyloid precursor protein (APP) which contributes to both Alzheimer Disease and an early onset Alzheimer-like neuropathology in Down SYndrome patients.

ITSN1 also contributes to oncogenic transformation of cells, playing a role in the tumorigenic properties of a number of cancers including neuroblastoma and glioblastoma [10-12]. Thus, we are currently working to define specific ITSN1-regulates pathways that contribute to oncogenesis.

Spencer-Smith R, Koide A, Zhou Y, Eguchi RR, Sha F, Gajwani P, Santana D, Gupta A, Jacobs M, Herrero-Garcia E, Cobbert J, Lavoie H, Smith M, Rajakulendran T, Dowdell E, Okur MN, Dementieva I, Sicheri F, Therrien M, Hancock JF, Ikura M, Koide S, O'Bryan JP. Inhibition of RAS function through targeting an allosteric regulatory site. Nat Chem Biol. 2017 Jan;13(1):62-68.

Hunter MP, Russo A, O’Bryan JP. Emerging Roles for Intersectin (ITSN) in Regulating Signaling and Disease Pathways. Int J Mol Sci. 14, 7829-7852, 2013.

O’Bryan, JP. Intersecting pathways in cell biology. Sci Signal. 3: re10, 2010.

Okur MN, Russo A, O’Bryan JP. Receptor tyrosine kinase ubiquitylation involves the dynamic regulation of Cbl-Spry2 by intersectin 1 and the Shp2 tyrosine phosphatase. Mol Cell Biol. 34, 271-279, 2014.

Okur MN, et al. Intersectin 1 enhances Cbl ubiquitylation of epidermal growth factor receptor through regulation of Sprouty2-Cbl interaction. Mol Cell Biol. 32: 817-825, 2012.

Martin NP, et al. Intersection regulates epidermal growth factor receptor endocytosis, ubiquitylation, and signaling. Mol Pharmacol. 70, 1643-1653, 2006.

Mohney RP, et al. Intersection activates Ras but stimulates transcription through an independent pathway involving JNK. J Biol Chem. 278: 47038-47045, 2003.

Wong KA, et al. A new dimension to Ras function: a novel role for nucleotide-free Ras in Class II phosphatidylinsitol 3-kinase beta (PI3K-C2b) regulation. PLOS ONE. 7: e45360, 2012.

Das M, et al. Regulation of neuron survival through an intersectin-phosphoinsotide 3'-kinase C2beta-AKT pathway. Mol Cell Biol. 27: 7906-7917, 2007.

Hunter MP, et al. Intersectin 1 contributes to phenotypes in vivo: implications for Down's syndrome. Neuroreport. 22, 767-772, 2011.

Russo A, Okur MN, Bosland M, O'Bryan JP. Phosphatidylinositol 3-kinase, class 2 beta (PI3KC2beta) isoform contributes to neuroblastoma tumorigenesis. Cancer Lett. 359: 262-268, 2015.

Russo A, O'Bryan JP. Intersectin 1 is required for neuroblastoma tumorigenesis. Oncogene. 31: 4828-4834, 2012.

Adams A, Thorn JM, Yamabhai M, Kay BK, O'Bryan JP. Intersectin, an adaptor protein involved in clathrin-mediated endocytosis, activates mitogenic signaling pathways. J Biol Chem. 275: 27414-27420, 2000.