Nemo phosphorylates Eyes absent and enhances output from the Eya-Sine oculis transcriptional complex during Drosophila retinal determination

AbstractThe retinal determination gene network comprises a collection of transcription factors that respond to multiple signaling inputs to direct Drosophila eye development. Previous genetic studies have shown that nemo (nmo), a gene encoding a proline-directed serine/threonine kinase, can promote retinal specification through interactions with the retinal determination gene network, although the molecular point of cross-talk was not defined. Here, we report that the Nemo kinase positively and directly regulates Eyes absent (Eya). Genetic assays show that Nmo catalytic activity enhances Eya-mediated ectopic eye formation and potentiates induction of the Eya-Sine oculis (So) transcriptional targets dachshund and lozenge. Biochemical analyses demonstrate that Nmo forms a complex with and phosphorylates Eya at two consensus mitogen-activated protein kinase (MAPK) phosphorylation sites. These same sites appear crucial for Nmo-mediated activation of Eya function in vivo. Thus, we propose that Nmo phosphorylation of Eya potentiates its transactivation function to enhance transcription of Eya-So target genes during eye specification and development

The SAM Domain of Human TEL2 Can Abrogate Transcriptional Output from TEL1 (ETV-6) and ETS1/ETS2

Regulation of gene expression downstream of the Receptor Tyrosine Kinase signaling pathway in Drosophila relies on a transcriptional effector network featuring two conserved Ets family proteins, Yan and Pointed, known as TEL1 (ETV6) and ETS1/ETS2, respectively, in mammals. As in Drosophila, both TEL1 and ETS1/ETS2 operate as Ras pathway transcriptional effectors and misregulated activity of either factor has been implicated in many human leukemias and solid tumors. Providing essential regulation to the Drosophila network, direct interactions with the SAM domain protein Mae attenuate both Yan-mediated repression and PointedP2-mediated transcriptional activation. Given the critical contributions of Mae to the Drosophila circuitry, we investigated whether the human Ets factors TEL1 and ETS1/ETS2 could be subject to analogous regulation. Here we demonstrate that the SAM domain of human TEL2 can inhibit the transcriptional activities of ETS1/2 and TEL1. Drosophila Mae can also attenuate human ETS1/ETS2 function, suggesting there could be cross-species conservation of underlying mechanism. In contrast, Mae is not an effective inhibitor of TEL1, suggesting the mode of TEL2SAM-mediated inhibition of TEL1 may be distinct from how Drosophila Mae antagonizes Yan. Together our results reveal both further similarities and new differences between the mammalian and Drosophila networks and more broadly suggest that SAM domain-mediated interactions could provide an effective mechanism for modulating output from the TEL1 and ETS1/2 oncogenes

Keeping the Receptor Tyrosine Kinase Signaling Pathway in Check: Lessons from Drosophila

AbstractThe receptor tyrosine kinase (RTK) signaling network plays a central role in regulating cellular differentiation, proliferation, and survival in all metazoan animals. Excessive or continuous activation of the RTK pathway has been linked to carcinogenesis in mammals, underscoring the importance of preventing uncontrolled signaling. This review will focus on the inhibitory mechanisms that keep RTK-mediated signals in check, with emphasis on conserved principles discerned from studies using Drosophila as a model system. Two general strategies of inhibition will be discussed. The first, threshold regulation, postulates that an effective way of antagonizing RTK signaling is to erect and maintain high threshold barriers that prevent inappropriate responses to moderate signaling levels. Activation of the pathway above this level overcomes the inhibitory blocks and shifts the balance to allow a positive flow of inductive information. A second layer of negative regulation involving induction of negative feedback loops that limit the extent, strength, or duration of the signal prevents runaway signaling in response to the high levels of activation required to surmount the threshold barriers. Such autoinhibitory mechanisms attenuate signaling at critical points throughout the network, from the receptor to the downstream effectors

The SAM Domain of Human TEL2 Can Abrogate Transcriptional Output from TEL1 (ETV-6) and ETS1/ETS2

Regulation of gene expression downstream of the Receptor Tyrosine Kinase signaling pathway in Drosophila relies on a transcriptional effector network featuring two conserved Ets family proteins, Yan and Pointed, known as TEL1 (ETV6) and ETS1/ETS2, respectively, in mammals. As in Drosophila, both TEL1 and ETS1/ETS2 operate as Ras pathway transcriptional effectors and misregulated activity of either factor has been implicated in many human leukemias and solid tumors. Providing essential regulation to the Drosophila network, direct interactions with the SAM domain protein Mae attenuate both Yan-mediated repression and PointedP2-mediated transcriptional activation. Given the critical contributions of Mae to the Drosophila circuitry, we investigated whether the human Ets factors TEL1 and ETS1/ETS2 could be subject to analogous regulation. Here we demonstrate that the SAM domain of human TEL2 can inhibit the transcriptional activities of ETS1/2 and TEL1. Drosophila Mae can also attenuate human ETS1/ETS2 function, suggesting there could be cross-species conservation of underlying mechanism. In contrast, Mae is not an effective inhibitor of TEL1, suggesting the mode of TEL2SAM-mediated inhibition of TEL1 may be distinct from how Drosophila Mae antagonizes Yan. Together our results reveal both further similarities and new differences between the mammalian and Drosophila networks and more broadly suggest that SAM domain-mediated interactions could provide an effective mechanism for modulating output from the TEL1 and ETS1/2 oncogenes.</p

Pleiotropy in Drosophila organogenesis: Mechanistic insights from Combgap and the retinal determination gene network

Master regulatory transcription factors cooperate in networks to shepherd cells through organogenesis. In the Drosophila eye, a collection of master control proteins known as the retinal determination gene network (RDGN) switches the direction and targets of its output to choreograph developmental transitions, but the molecular partners that enable such regulatory flexibility are not known. We recently showed that two RDGN members, Eyes absent (Eya) and Sine oculis (So), promote exit from the terminal cell cycle known as the second mitotic wave (SMW) to permit differentiation. A search for co-factors identified the ubiquitously expressed Combgap (Cg) as a novel transcriptional partner that impedes cell cycle exit and interferes with Eya-So activity specifically in this context. Here, we argue that Cg acts as a flexible transcriptional platform that contributes to numerous gene expression outcomes by a variety of mechanisms. For example, Cg provides repressive activities that dampen Eya-So output, but not by recruiting Polycomb chromatin-remodeling complexes as it does in other contexts. We propose that master regulators depend on both specifically expressed co-factors that assemble the combinatorial code and broadly expressed partners like Cg that recruit the diverse molecular activities needed to appropriately regulate their target enhancers

TEL2SAM inhibits transcriptional activation by ETS1/2 in HeLa cells.

<p>Ras^V12 enhanced and Mae or TEL2SAM suppressed activation of (A) the MMP9-luciferase reporter and (B) the dEts-luciferase reporter. (C) Mutations in the EH or ML surfaces of TEL2SAM do not alter its ability to suppress ETS2.</p

TEL2SAM inhibits transcriptional repression by TEL1 of the E74tkluciferase reporter.

<p>(A) Repression by TEL1 is suppressed by TEL2SAM but <i>Drosophila</i> Mae has only a weak effect. The full-length TEL2 alone lane derives from an independent experiment in which repression by TEL1 was almost identical to that shown here. Using the TEL1 alone values to normalize between experiments, the % transcriptional activity for TEL2 was adjusted by a factor of 0.82. (B) TEL2SAM effectively inhibits repression by TEL1 at decreasing TEL2SAM concentrations. The ratio of TEL2SAM DNA to TEL1 DNA that was used for titration ranged from 2∶1 to 0.25∶1.</p

Mae suppresses transcriptional activation by ETS1/2 in <i>Drosophila</i> S2 cells.

<p>(A) Activation of the argos-luciferase reporter by ETS1/2 is enhanced by expression of Ras^V12 and inhibited by Mae. (B) Myc-Mae coimmunoprecipitated with Flag-ETS1/2 from lysates of cotransfected <i>Drosophila</i> S2 cells cotransfected (lanes 2 and 3) but not from lysates of cells transfected with Myc-Mae alone (lane 1). Myc-Mae runs below the IgG light chain (strong band marked with asterisk). Flag-ETS1/2 run as doublets.</p

Inhibition of TEL1 repression by TEL2SAM is alleviated by mutations that prevent SAM domain-polymerization.

<p>(A) Communoprecipitation of myc-TEL2SAM with HA-TEL1 from cotransfected HeLa cells (lane 2) but not from cells transfected with HA-TEL1 alone (lane 1). Top and bottom panels were from the same gel, as were the middle two panels. (B) TEL2SAM can inhibit transcriptional repression by Yan, although not as effectively as Mae. (C) Repression of the E74tkluciferase reporter by TEL1 is suppressed by TEL2SAM but not by TEL2SAM^EHmut or TEL2SAM^MLmut.</p