Class IIa histone deacetylases (HDACs 4/5/7/9) are transcriptional regulators with critical roles in cardiac disease, cancer, and viral infection. HDAC inhibitors are promising anti-cancer agents, and while they are known to disrupt mitotic progression, the underlying mechanisms of mitotic regulation by HDACs are not fully understood. Here we provide the first identification of histone deacetylases as substrates of Aurora B kinase (AurB). Our study identifies class IIa HDACs as a novel family of AurB targets and provides the first evidence that HDACs are temporally and spatially regulated by phosphorylation during the cell cycle. We define the precise sites of AurB-mediated phosphorylation as a conserved serine within the nuclear localization signals of HDAC4, HDAC5, and HDAC9 at Ser265, Ser278, and Ser242, respectively. We establish that AurB interacts with these HDACs in vivo, and that this association increases upon disruption of 14-3-3 binding. We observe co-localization of endogenous, phosphorylated HDACs with AurB at the mitotic midzone in late anaphase and the midbody during cytokinesis, complemented by a reduction in HDAC interactions with components of the nuclear co-repressor (NCoR) complex. We propose that AurB-dependent phosphorylation of HDACs induces sequestration within a phosphorylation gradient at the midzone, maintaining separation from re-forming nuclei and contributing to transcriptional control.
"These are Aurora kinase B (Aurora B), Dual specificity protein kinase CLK3 and cGMP-dependent protein kinase 1 (PKG1). Recently, Aurora B was shown to phosphorylate certain histone deacetylases (HDACs) with critical roles in cardiac disease and thereby likely regulate their localization . PKG1 is involved in vascular smooth muscle relaxation, where it influences actin binding by the phosphorylation of proteins important for cytoskeletal reorganisation  which fits well with regulation by Ang II. "
[Show abstract][Hide abstract] ABSTRACT: Recent progress in the understanding of seven-transmembrane receptor (7TMR) signalling has promoted the development of a new generation of pathway selective ligands. The angiotensin II type I receptor (AT1aR) is one of the most studied 7TMRs with respect to selective activation of the β-arrestin dependent signalling. Two complimentary global phosphoproteomics studies have analyzed the complex signalling induced by the AT1aR. Here we integrate the data sets from these studies and perform a joint analysis using a novel method for prediction of differential kinase activity from phosphoproteomics data. The method builds upon NetworKIN, which applies sophisticated linear motif analysis in combination with contextual network modelling to predict kinase-substrate associations with high accuracy and sensitivity. These predictions form the basis for subsequently nonparametric statistical analysis to identify likely activated kinases. This suggested that AT1aR-dependent signalling activates 48 of the 285 kinases detected in HEK293 cells. Of these, Aurora B, CLK3 and PKG1 have not previously been described in the pathway whereas others, such as PKA, PKB and PKC, are well known. In summary, we have developed a new method for kinase-centric analysis of phosphoproteomes to pinpoint differential kinase activity in large-scale data sets.
PLoS ONE 04/2014; 9(4):e94672. DOI:10.1371/journal.pone.0094672 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Proteins play a fundamental role in establishing the diversity of cellular processes in health or disease systems. This diversity is accomplished by a vast array of protein functions. In fact, a protein rarely has a single function. The majority of proteins are involved in numerous cellular processes, and these multiple functions are made possible by interactions with other molecules. The complexity of interactions is substantially increased by the spatial and temporal diversity of proteins. For example, proteins can be part of distinct complexes within different subcellular compartments or at different stages of the cell cycle. Posttranslational modifications can regulate and further expand the ability of proteins to establish localization- or temporal-dependent interactions. This complexity and functional divergence of interactions is further increased by the simultaneous presence of stable, transient, direct, and indirect protein interactions. Thus, an understanding of protein functions cannot be fully accomplished without knowledge of its interactions. Characterizing these interactions is therefore critical to understanding the biology of health and disease systems.
[Show abstract][Hide abstract] ABSTRACT: The past few years have seen significant advances in the use of modern proteomics approaches for biological discoveries. Among the fields impacted by proteomics is that of epigenetics, as mass spectrometry-based approaches have allowed the identification and characterization of transcriptional regulators, epigenetic marks, and the constantly evolving epigenetic landscape of a cell in health and disease states. These studies have substantially expanded our understanding of critical genes that mediate cell processes, such as differentiation, cell cycle regulation, and apoptosis. Not surprisingly, a great emphasis has been placed on defining factors that are de-regulated in cancers, in an attempt to define new and specific targets for therapeutic design. Differential gene expression observed during carcinogenesis can be induced by aberrant activities of transcription factors and chromatin remodeling enzymes. Through a series of recent mass spectrometry studies of histone deacetylases and nuclear receptors, Deleted in Breast Cancer 1 (DBC1) has emerged as a master regulator of transcriptional processes. DBC1 acts as a modulator of cellular epigenetic mechanisms and is frequently associated with human metastasis. Through its negative regulation of SIRT1 and HDAC3 deacetylation activities, DBC1 has a broad impact on gene expression, downstream cellular pathways, and associated human diseases. Here, we review the identified roles of DBC1, highlighting the critical contribution of mass spectrometry to these findings. Additionally, we provide a perspective of integrative proteomics approaches that can continue to shed light on the interplay between DBC1 and its protein targets, helping to further define its role in epigenetic modifications and to identify novel targets for cancer therapy.
Journal of Proteomics & Bioinformatics 04/2013; Suppl 2.
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