Ilaria Rebay, Ph.D.

Receptor Tyrosine Kinase Signal Transduction; Transcriptional Regulatory Circuitries in Development and Disease

Research Summary

The long term goal of my research is to understand how complex developmental decisions are controlled in time and space by multiple signaling pathways. Current research in my laboratory centers on the premise that transcription factors provide critical nodes of information integration and that elaborate layers of regulation have evolved to modulate and coordinate their activities. Using a multifaceted experimental approach that bridges genetics, molecular genetics, cell biology, biochemistry and genomics, our strategy has been first to identify the individual genes comprising the regulatory networks, and then to unravel the complex functional relationships between the components at a mechanistic level of detail. In particular we have focused on identifying the post-translational control mechanisms that provide powerful strategies for translating diverse signaling inputs into appropriately modified patterns of gene expression.

Our current work focuses on elucidating the function and regulation of two independent but interconnected nuclear circuitries operating downstream of the receptor tyrosine kinase (RTK) pathway: the ETS network, comprised of MAPK, the transcription factors Yan and Pointed which are Drosophila homologs of the human oncogenes Tel and Ets1, and a fourth protein called Mae; and the Retinal Determination (RD) network, an interactive set of conserved transcriptional regulators that cooperatively direct the formation of many tissues and organs, including the eye.

The RD network: Within the RD network, an elaborate hierarchy of transcriptional induction and protein-protein interactions provides multiple nodes of regulation and feedback control (Figure 1). Adding further complexity, and making it an ideal system for studying nuclear information integration, genetic studies from numerous laboratories have indicated that proper regulation of the network depends on inputs from an intricate meshwork of major signaling pathways. At present, the only molecularly defined node of cross-talk derives from our work demonstrating that one member of the network, Eyes absent (Eya), is directly regulated by MAPK-mediated phosphorylation at two sites in response to RTK-initiated signals. Eya has been best studied in its role as a transcriptional coactivator that binds to the homeodomain containing Six protein, and our work suggests that this activity is potentiated by MAPK-mediated phosphorylation.

Adding a new layer of complexity to the RD, and possibly other networks, we have recently discovered that Eya, traditionally known as a transcriptional coactivator, also operates as the prototype of a new family of protein tyrosine phosphatases (PTPs). In contrast to classical PTPs which employ a thiol-based reaction mechanism, Eya-type PTPs use an aspartate-based catalytic mechanism first characterized in a heterogeneous group of prokaryotic enzymes referred to as the Haloacid Dehalogenase (HAD) superfamily. This work has raised awareness both of a novel collection of enzymes that had been previously overlooked in higher eukaryotes and of an unexpected new layer of dynamic nuclear regulation, namely the addition of tyrosine phosphorylation to the growing list of post-translational modifications that may influence transcription factor activity. Major efforts in the lab are currently geared toward identifying the substrates of Eya's phosphatase activity and more generally, toward investigating how Eya's two functions as transactivator and phosphatase are coordinated and coregulated. In this context, we are exploring how and why disruption in either function results in an autosomal dominant disorder in humans, termed Branchio-Oto-Renal (BOR) syndrome, that is characterized by craniofacial abnormalities, deafness, impaired kidney function, and eye defects.

The ETS network: The ETS network comprises four components connected via multiple levels of transcriptional regulation, protein-protein interactions, and post-translational modifications (Figure 2). A S/T kinase, Mitogen Activated Protein Kinase (MAPK), in response to activation of the RTK signaling pathway, directly phosphorylates a pair of functionally antagonistic ETS family transcription factors, Yan and Pointed. This attenuates the repressor function of Yan and stimulates the activation ability of Pointed. Mae, a direct transcriptional target, provides dual positive and negative regulation by binding directly and inhibiting both Yan and Pointed. We have shown that phosphorylation of Yan by MAPK results in abrogation of transcriptional repression, export from the nucleus, and very likely rapid degradation in the cytoplasm, with at least the first two events facilitated by Mae. Our current work is directed toward exploring in further mechanistic detail, how this complex interplay between Yan, Pointed an Mae results in proper transcriptional responses during development. In addition we are investigating the extent to which the regulatory mechanisms we have uncovered in our Drosophila system are conserved with respect to the human homologs Tel and Ets1, and in the long-term hope to apply this knowledge to treating the malignancies that result from impaired expression and regulation of Tel and Ets1.

Selected Papers

Rebay I, Chen F, Hsiao F, Kolodziej PA, Kuang BH, Laverty T, Suh C, Voas M, Williams A and Rubin GM. (2000). "A genetic screen for novel components of the Ras/Mitogen-activated protein kinase signaling pathway that interact with the yan gene of Drosophila identifies split ends, a new RNA recognition motif-containing protein," Genetics 154: 695-712.

Chen F and Rebay I. (2000). "split ends, a new component of the Drosophila EGF receptor pathway, regulates development of midline glial cells," Curr Biol 10: 943-946.

Hsiao FC, Williams A, Davies EL and Rebay I. (2001). "Eyes absent mediates cross-talk between retinal determination genes and the receptor tyrosine kinase signaling pathway," Dev Cell 1: 51-61.

Rebay I. (2002). "Keeping the Receptor Tyrosine Kinase Signaling Pathway in Check: Lessons from Drosophila," Developmental Biology 251: 1-17.

Silver SJ, Davies EL, Doyon L and Rebay I. (2003). "A functional dissection of Eyes absent reveals new modes of regulation within the retinal determination gene network," Molecular and Cellular Biology 23: 5989-5999.

Tootle TL, Lee PS and Rebay I. (2003). "CRM1-mediated nuclear export and regulated activity of the Receptor Tyrosine Kinase antagonist YAN require specific interactions with MAE," Development 130: 845-857.

Tootle TL, Silver SJ, Davies EL, Newman V, Latek RR, Mills IA, Selengut JD, Parlikar BEW and Rebay I. (2003). "The transcription factor Eyes absent is a protein tyrosine phosphatase," Nature 426: 299-302.

Voas MG and Rebay I. (2003). "The Novel Plant Homeodomain Protein Rhinoceros Antagonizes Ras Signaling in the Drosophila Eye," Genetics 165: 1993-2006.

Voas MG and Rebay I. (2003). "Signal integration during development: insights from the Drosophila eye," Developmental Dynamics, 229: 162-175.

Qiao F, Song H, Kim CA, Sawaya MR, Hunter JB, Gingery M, Courey AJ, Rebay I and Bowie JU. (2004). "Derepression by Depolymerization: Structural Insights into the Regulation of Yan by Mae," Cell 118: 163-173.

Vivekanand P, Tootle TL and Rebay I. (2004). "MAE, a dual regulator of the EGFR signaling pathway, is a target of the Ets transcription factors PNT and YAN," Mech Dev 121: 1469-1479.

Tootle TL and Rebay I. (2005). "Post-translational modifications influence transcription factor activity: A view from the ETS superfamily," Bioessays 27: 285-298.

Silver S and Rebay I. (2005). "Signaling circuitries in development: Insights from the Retinal Determination Gene Network," Development 132: 3-13.

Rebay I, Silver S and Tootle TL. (2005). "New vision from Eyes absent: transcription factors as enzymes," Trends in Genetics in press.