Biomedical subjects

Albert J Courey

Publications and source records attributed to Albert J Courey.

11 recordsLinked to original sources

PubMed2006

Uncoupling dorsal-mediated activation from dorsal-mediated repression in the Drosophila embryo.

The Rel family transcription factor Dorsal patterns the dorsoventral axis of the Drosophila embryo by activating genes such as twist and snail and repressing genes such as decapentaplegic and zerknüllt. Dorsal represses transcription by recruiting the co-repressor Groucho. However, repression occurs only when Dorsal-binding sites are close to binding sites for other factors that also bind Groucho. The need for additional factors to assist Dorsal in repression may result from the intrinsically weak interaction between Dorsal and Groucho. To test this idea, we generated a Dorsal variant containing a high-affinity Groucho recruitment motif at its C terminus. As predicted, this variant functions as a dedicated repressor, silencing decapentaplegic and zerknüllt while failing to activate twist and snail. We also converted Dorsal into a dedicated activator by replacing its weak Groucho-recruitment motif with heterologous activation domains. Although the dedicated activator alleles fail to repress decapentaplegic and zerknüllt in the syncytial blastoderm embryo, they are able to pattern the dorsoventral axis. This indicates that dorsoventral patterning is not dependent upon Dorsal-mediated repression, reflecting the existence of redundant mechanisms to block Decapentaplegic signaling.

Animals↗

PubMed2005

Mae inhibits Pointed-P2 transcriptional activity by blocking its MAPK docking site.

During Drosophila melanogaster eye development, signaling through receptor tyrosine kinases (RTKs) leads to activation of a mitogen activated protein tyrosine kinase, called Rolled. Key nuclear targets of Rolled are two antagonistic transcription factors: Yan, a repressor, and Pointed-P2 (Pnt-P2), an activator. A critical regulator of this process, Mae, can interact with both Yan and Pnt-P2 through their SAM domains. Although earlier work showed that Mae derepresses Yan-regulated transcription by depolymerizing the Yan polymer, the mechanism of Pnt-P2 regulation by Mae remained undefined. We find that efficient phosphorylation and consequent activation of Pnt-P2 requires a three-dimensional docking surface on its SAM domain for the MAP kinase, Rolled. Mae binding to Pnt-P2 occludes this docking surface, thereby acting to downregulate Pnt-P2 activity. Docking site blocking provides a new mechanism whereby the cell can precisely modulate kinase signaling at specific targets, providing another layer of regulation beyond the more global changes effected by alterations in the activity of the kinase itself.

Amino Acid Sequence↗

PubMed2005

Antagonistic regulation of Yan nuclear export by Mae and Crm1 may increase the stringency of the Ras response.

Phosphorylation of Yan, a major target of Ras signaling, leads to Crm1-dependent Yan nuclear export, a response that is regulated by Yan polymerization. Yan SAM (sterile alpha motif) domain mutations preventing polymerization result in Ras-independent, but Crm1-dependent Yan nuclear export, suggesting that polymerization prevents Yan export. Mae, which depolymerizes Yan, competes with Crm1 for binding to Yan. Phosphorylation of Yan favors Crm1 in this competition and counteracts inhibition of nuclear export by Mae. These findings suggest that, prior to Ras activation, the Mae/Yan interaction blocks premature nuclear export of Yan monomers. After activation, transcriptional up-regulation of Mae apparently leads to complete depolymerization and export of Yan.

Animals↗

PubMed2005

SUMO enhances vestigial function during wing morphogenesis.

The conjugation of the ubiquitin-like protein SUMO to lysine side chains plays widespread roles in the regulation of nuclear protein function. Since little information is available about the roles of SUMO in development, we have screened a collection of chromosomal deficiencies to identify developmental processes regulated by SUMO. We found that flies heterozygous for a deficiency uncovering vestigial (vg) and mutations in any of several genes encoding components of the SUMO conjugation machinery exhibit severe wing notching. This phenotype is due to an interaction between sumo and vg since it is suppressed by expression of Vg from a transgene, and is also observed in flies doubly heterozygous for vg hypomorphic alleles and sumo. In addition, the ability of Vg to direct the formation of ectopic wings when misexpressed in the eye field is enhanced by simultaneous misexpression of SUMO. In S2 cell transient transfection assays, overexpression of SUMO and the SUMO conjugating enzyme Ubc9, but not a catalytically inactive form of Ubc9, results in sumoylation of Vg and augments the activation of a Vg-responsive reporter. These findings are consistent with the idea that sumoylation stimulates Vg function during wing morphogenesis.

Animals↗

PubMed2004

EGFR signaling attenuates Groucho-dependent repression to antagonize Notch transcriptional output.

Crosstalk between signaling pathways is crucial for the generation of complex and varied transcriptional networks. Antagonism between the EGF-receptor (EGFR) and Notch pathways in particular is well documented, although the underlying mechanism is poorly understood. The global corepressor Groucho (Gro) and its transducin-like Enhancer-of-split (TLE) mammalian homologs mediate repression by a myriad of repressors, including effectors of the Notch, Wnt (Wg) and TGF-beta (Dpp) signaling cascades. Given that there are genetic interactions between gro and components of the EGFR pathway (ref. 9 and P.H. et al., unpublished results), we tested whether Gro is at a crossroad between this and other pathways. Here we show that phosphorylation of Gro in response to MAPK activation weakens its repressor capacity, attenuating Gro-dependent transcriptional silencing by the Enhancer-of-split proteins, effectors of the Notch cascade. Thus, Gro is a new junction between signaling pathways, enabling EGFR signaling to antagonize transcriptional output by Notch and potentially other Gro-dependent pathways.

Animals↗

PubMed2004

Drosophila Ulp1, a nuclear pore-associated SUMO protease, prevents accumulation of cytoplasmic SUMO conjugates.

SUMO is a small ubiquitin-like protein that becomes covalently conjugated to a variety of target proteins, the large majority of which are found in the nucleus. Ulp1 is a member of a family of proteases that control SUMO function positively, by catalyzing the proteolytic processing of SUMO to its mature form, and negatively, by catalyzing SUMO deconjugation. In Drosophila S2 cells, depletion of Ulp1 by RNA interference results in a dramatic change in the overall spectrum of SUMO conjugates, indicating that SUMO deconjugation is substrate-specific and plays a critical role in determining the steady state targets of SUMO conjugation. Ulp1 normally serves to prevent the accumulation of SUMO-conjugated forms of a number of proteins, including the aminoacyl-tRNA synthetase EPRS. In the presence of Ulp1, most SUMO conjugates reside in the nucleus. However, in its absence, SUMO-conjugated EPRS accumulates in the cytoplasm, contributing to an overall shift of SUMO from the nucleus to the cytoplasm. The ability of Ulp1 to restrict SUMO conjugates to the nucleus is independent of its role as a SUMO-processing enzyme because Ulp1-dependent nuclear localization of SUMO is even observed when SUMO is expressed in a preprocessed form. Studies of a Ulp1-GFP fusion protein suggest that Ulp1 localizes to the nucleoplasmic face of the nuclear pore complex. We hypothesize that, as a component of the nuclear pore complex, Ulp1 may prevent proteins from leaving the nucleus with SUMO still attached.

Active Transport, Cell Nucleus↗

PubMed2004

Derepression by depolymerization; structural insights into the regulation of Yan by Mae.

Yan, an ETS family transcriptional repressor, is regulated by receptor tyrosine kinase signaling via the Ras/MAPK pathway. Phosphorylation and downregulation of Yan is facilitated by a protein called Mae. Yan and Mae interact through their SAM domains. We find that repression by Yan requires the formation of a higher order structure mediated by Yan-SAM polymerization. Moreover, a crystal structure of the Yan-SAM/Mae-SAM complex shows that Mae-SAM specifically recognizes a surface on Yan-SAM that is also required for Yan-SAM polymerization. Mae-SAM binds to Yan-SAM with approximately 1000-fold higher affinity than Yan-SAM binds to itself and can effectively depolymerize Yan-SAM. Mutations on Mae that specifically disrupt its SAM domain-dependent interactions with Yan disable the derepression function of Mae in vivo. Depolymerization of Yan by Mae represents a novel mechanism of transcriptional control that sensitizes Yan for regulation by receptor tyrosine kinases.

Amino Acid Sequence↗

PubMed2004

Groucho oligomerization is required for repression in vivo.

Drosophila Groucho (Gro) is a member of a family of metazoan corepressors with widespread roles in development. Previous studies indicated that a conserved domain in Gro, termed the Q domain, was required for repression in cultured cells and mediated homotetramerization. Evidence presented here suggests that the Q domain contains two coiled-coil motifs required for oligomerization and repression in vivo. Mutagenesis of the putative hydrophobic faces of these motifs, but not of the hydrophilic faces, prevents the formation of both tetramers and higher order oligomers. Mutagenesis of the hydrophobic faces of both coiled-coil motifs in the context of a Gal4-Gro fusion protein prevents repression of a Gal4-responsive reporter in S2 cells, while mutagenesis of a single motif weakens repression. The finding that the repression directed by the single mutants depends on endogenous wild-type Gro further supports the idea that oligomerization plays a role in repression. Overexpression in the fly of forms of Gro able to oligomerize, but not of a form of Gro unable to oligomerize, results in developmental defects and ectopic repression of Gro target genes in the wing disk. Although the function of several corepressors is suspected to involve oligomerization, these studies represent one of the first direct links between corepressor oligomerization and repression in vivo.

Amino Acid Motifs↗

PubMed2002

The MADF-BESS domain factor Dip3 potentiates synergistic activation by Dorsal and Twist.

The transcription factors Dorsal and Twist regulate dorsoventral axis formation during Drosophila embryogenesis. Dorsal and Twist bind to closely linked DNA elements in a number of promoters and synergistically activate transcription. We have identified a novel protein named Dorsal-interacting protein 3 (Dip3) that may play a role in this synergy. Dip3 functions as a coactivator to stimulate synergistic activation by Dorsal and Twist, but does not stimulate simple activation of promoters containing only Dorsal or only Twist binding sites. In addition, Dip3 is able to bind DNA in a sequence specific manner and activate transcription directly. Dip3 possesses an N-terminal MADF domain and a C-terminal BESS domain, an architecture that is conserved in at least 14 Drosophila proteins, including Adf-1 and Stonewall. The MADF domain directs sequence specific DNA binding to a site consisting of multiple trinucleotide repeats, while the BESS domain directs a variety of protein-protein interactions, including interactions with itself, with Dorsal, and with a TBP-associated factor. We assess the possibility that the MADF and BESS domains are related to the SANT domain, a well-characterized motif found in many transcriptional regulators and coregulators.

Amino Acid Sequence↗

PubMed2002

The Dorsal Rel homology domain plays an active role in transcriptional regulation.

The Dorsal morphogen directs formation of the Drosophila dorsoventral axis by both activating and repressing transcription. It contains an N-terminal Rel homology domain (RHD), which is responsible for DNA binding and regulated nuclear import, and a C-terminal domain (CTD) that contains activation and repression motifs. To determine if the RHD has a direct role in transcriptional control, we analyzed a series of RHD mutations in S2 cells and embryos. Two classes of mutations (termed class I and class II mutations) that alter activation without affecting DNA binding or nuclear import were identified. The two classes appear to define distinct protein interaction surfaces on opposite faces of the RHD. Class I mutations enhance an apparently inhibitory interaction between the RHD and the CTD and eliminate both activation and repression by Dorsal. In contrast, class II mutations result in increased activation in S2 cells but severely decreased activation in embryos and have little effect on repression. Analysis of the cuticles of class II mutant embryos suggests that, in the absence of Dorsal-mediated activation, Dorsal-mediated repression is not sufficient to pattern the embryo. These results provide some of the first evidence that the RHD plays an active role in transcriptional regulation in intact multicellular organisms.

Amino Acid Sequence↗

PubMed2002

Conjugation of Smt3 to dorsal may potentiate the Drosophila immune response.

A variety of transcription factors are targets for conjugation to the ubiquitin-like protein Smt3 (also called SUMO). While many such factors exhibit enhanced activity under conditions that favor conjugation, the mechanisms behind this enhancement are largely unknown. We previously showed that the Drosophila melanogaster rel family factor, Dorsal, is a substrate for Smt3 conjugation. The conjugation machinery was found to enhance Dorsal activity at least in part by counteracting the Cactus-mediated inhibition of Dorsal nuclear localization. In this report, we show that Smt3 conjugation occurs at a single site in Dorsal (lysine 382), requires just the Smt3-activating and -conjugating enzymes, and is reversed by the deconjugating enzyme Ulp1. Mutagenesis of the acceptor lysine eliminates the response of Dorsal to the conjugation machinery and results in enhanced levels of synergistic transcriptional activation. Thus, in addition to controlling Dorsal localization, Smt3 also appears to regulate Dorsal-mediated activation, perhaps by modulating an interaction with a negatively acting nuclear factor. Finally, since Dorsal contributes to innate immunity, we examined the role of Smt3 conjugation in the immune response. We find that the conjugation machinery is required for lipopolysaccharide-induced expression of antimicrobial peptides in cultured cells and larvae, suggesting that Smt3 regulates Dorsal function in vivo.

Amino Acid Sequence↗