Biomedical subjects
Publications and source records attributed to D Durocher.
The DNA damage signalling pathway is a core element of the cellular response to genotoxic insult, and its components play key roles in defending against neoplastic transformation. Recent work has indicated that the human ATM and ATR proteins, and their yeast homologues, are intimately involved in sensing DNA damage, suggesting parallels with the DNA double-strand break repair enzyme DNA-PK.
Forkhead-associated (FHA) domains are a class of ubiquitous signaling modules that appear to function through interactions with phosphorylated target molecules. We have used oriented peptide library screening to determine the optimal phosphopeptide binding motifs recognized by several FHA domains, including those within a number of DNA damage checkpoint kinases, and determined the X-ray structure of Rad53p-FHA1, in complex with a phospho-threonine peptide, at 1.6 A resolution. The structure reveals a striking similarity to the MH2 domains of Smad tumor suppressor proteins and reveals a mode of peptide binding that differs from SH2, 14-3-3, or PTB domain complexes. These results have important implications for DNA damage signaling and CHK2-dependent tumor suppression, and they indicate that FHA domains play important and unsuspected roles in S/T kinase signaling mechanisms in prokaryotes and eukaryotes.
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FHA domains are conserved sequences of 65-100 amino acid residues found principally within eukaryotic nuclear proteins, but which also exist in certain prokaryotes. The FHA domain is thought to mediate protein-protein interactions, but its mode of action has yet to be elucidated. Here, we show that the two highly divergent FHA domains of Saccharomyces cerevisiae Rad53p, a protein kinase involved in cell cycle checkpoint control, possess phosphopeptide-binding specificity. We also demonstrate that other FHA domains bind peptides in a phospho-dependent manner. These findings indicate that the FHA domain is a phospho-specific protein-protein interaction motif and have important implications for mechanisms of intracellular signaling in both eukaryotes and prokaryotes.
In vertebrates, heart development is a multistep process that starts with formation and patterning of the primitive heart tube and is followed by complex morphological events to give rise to the mature four-chambered heart. These various stages are characterized by distinct patterns of gene expression. Although chamber specificity and developmental regulation can be demonstrated in transgenic mice using short promoter fragments, the mechanism underlying spatial and temporal specificity within the heart remains largely unclear. Combinatorial interaction between a limited number of cardiac-specific and ubiquitous transcription factors may account for the diverse genetic inputs required to generate the complex transcriptional patterns that characterize the developing myocardium. We have used the cardiac atrial natriuretic peptide (ANP) promoter to test this hypothesis. The ANP gene is transcribed in a spatial- and temporal-specific manner in the heart, and a 500 bp promoter fragment is sufficient to recapitulate both chamber and developmental specificity. This promoter is composed of three modules, a "basal" cardiac promoter that is essential for transcription in embryonic and postnatal atrial and ventricular myocytes and two other independent modules that behave as chamber-specific enhancers. The basal cardiac promoter is the target of two cardiac-specific transcription factors, the zinc finger GATA-4 protein and the Nkx2-5 homeodomain, which bind to contiguous elements within this region. At low concentrations--a situation that likely occurs during the very first stages of cardiac cell fate determination--the two proteins synergistically activate transcription from the ANP promoter. This functional synergy requires physical interaction between the GATA-4 protein and an extended C-terminal homeodomain on Nkx2-5. This interaction, which unmasks an activation domain present just N-terminal of the homeodomain, is specific for GATA-4 and-5, but is not observed with the other cardiac GATA factor, GATA-6. Optimal synergy requires binding of both proteins to their cognate sites, although modest synergy also could be observed on heterologous promoters containing only multimerized Nkx binding sites, suggesting that Nkx2-5 is able to recruit GATA-4 into a transcriptionally active complex. The GATA/Nkx interaction, which appears to have been evolutionary conserved in nematode, fly, and mammals, provides a paradigm for analyzing transcription factor interaction during organogenesis. The data are also discussed in the context of our present knowledge of the roles of GATA and NK2 proteins in cardiac development.
Cardiac growth and contractility is profoundly altered in response to various hormones and neurotransmitters as well as by changes in electrolyte balance, blood volume, and blood pressure. In turn, the endocrine heart contributes to body homeostasis by secreting a number of biologically active peptide hormones that act on several target tissues. Atrial natriuretic peptide (ANP) and B-type natiuretic peptide (BNP) are the major secretory products of the endocrine myocardium. These peptide hormones act on many target organs via guanylate cyclase-linked membrane receptors to produce natriuresis, diuresis, vasodilatation, and hypotension. ANP and BNP receptors are found on most organs involved in cardiovascular homeostasis (e.g., kidney, adrenal, vasculature, brain). They are also present in gonads and in pituitary, where they alter steroidogenesis and pituitary hormone secretion. Not surprisingly, changes in blood pressure, volume, or hormone status influence ANP and BNP expression, which is also altered in almost all diseases that affect cardiac function. Thus, studies of ANP and BNP gene expression are relevant for many clinical settings. Moreover, transcription of the ANP and BNP genes characterizes cardiac cells at very early stages of development and is tightly linked to cardiac growth--be it proliferation, as in the fetal heart, or trophic growth, as in postnatal ventricular hypertrophy. Thus, analysis of the molecular circuitry that controls ANP and BNP expression might shed important insight into the complex regulatory pathways that underlie normal and pathologic heart development. Indeed, over the past few years, we have analysed transcriptional control of the ANP and BNP genes in embryonic and postnatal cardiomyocytes and in cardiomyocytes treated with hormones and neurotransmitters that cause cardiomyocyte hypertrophy. These studies have lead to the identification of distinct cis-acting DNA elements that modulate basal and hormone-stimulated transcription. Most importantly, the work also resulted in the isolation and characterization of cardiac-specific transcription factors that play critical roles for cardiac cell differentiation and survival.
The tissue-restricted GATA-4 transcription factor and Nkx2-5 homeodomain protein are two early markers of precardiac cells. Both are essential for heart formation, but neither can initiate cardiogenesis. Overexpression of GATA-4 or Nkx2-5 enhances cardiac development in committed precursors, suggesting each interacts with a cardiac cofactor. We tested whether GATA-4 and Nkx2-5 are cofactors for each other by using transcription and binding assays with the cardiac atrial natriuretic factor (ANF) promoter_the only known target for Nkx2-5. Co-expression of GATA-4 and Nkx2-5 resulted in synergistic activation of the ANF promoter in heterologous cells. The synergy involves physical Nkx2-5-GATA-4 interaction, seen in vitro and in vivo, which maps to the C-terminal zinc finger of GATA-4 and a C-terminus extension; similarly, a C-terminally extended homeodomain of Nkx2-5 is required for GATA-4 binding. Structure/function studies suggest that binding of GATA-4 to the C-terminus autorepressive domain of Nkx2-5 may induce a conformational change that unmasks Nkx2-5 activation domains. GATA-6 cannot substitute for GATA-4 for interaction with Nkx2-5. This interaction may impart functional specificity to GATA factors and provide cooperative crosstalk between two pathways critical for early cardiogenesis. Given the co-expression of GATA proteins and NK2 class members in other tissues, the GATA/Nkx partnership may represent a paradigm for transcription factor interaction during organogenesis.
The recently described NK2 family of homeodomain proteins are key developmental regulators. In Drosophila melanogaster, two members of this family, bagpipe and tinman, are required for visceral and cardiac mesoderm formation, respectively. In vertebrates, tinman appears to represent a family of closely related NK2 genes, including Nkx-2.5, that are expressed at an early stage in precardiac cells. Consistent with a role for Nkx-2.5 in heart development, inactivation of the Nkx-2.5 gene in mice causes severe cardiac malformations and embryonic lethality. However, little is known about the molecular action of Nkx-2.5 and its targets in cardiac muscle. In this paper, we report the identification and characterization of a functional and highly conserved Nkx-2.5 response element, termed the NKE, in the proximal region of the cardiac atrial natriuretic factor (ANF) promoter. The NKE is composed of two near-consensus NK2 binding sites that are each able to bind purified Nkx-2.5. The NKE is sufficient to confer cardiac cell-specific activity to a minimal TATA-containing promoter and is required for Nkx-2.5 activation of the ANF promoter in heterologous cells. Interestingly, in primary cardiocyte cultures, the NKE contributes to ANF promoter activity in a chamber- and developmental stage-specific manner, suggesting that Nkx-2.5 and/or other related cardiac proteins may play a role in chamber specification. This work provides the identification of a direct target for NK2 homeoproteins in the heart and lays the foundation for further molecular analyses of the role of Nkx-2.5 and other NK2 proteins in cardiac development.
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