New Role for hPar-1 Kinases EMK and C-TAK1 in Regulating Localization and Activity of Class IIa Histone Deacetylases

ABSTRACT Class IIa histone deacetylases (HDACs) are found both in the cytoplasm and in the nucleus where they repress genes involved in several major developmental programs. In response to specific signals, the repressive activity of class IIa HDACs is neutralized through their phosphorylation on multiple N-terminal serine residues and 14-3-3-mediated nuclear exclusion. Here, we demonstrate that class IIa HDACs are subjected to signal-independent nuclear export that relies on their constitutive phosphorylation. We identify EMK and C-TAK1, two members of the microtubule affinity-regulating kinase (MARK)/Par-1 family, as regulators of this process. We further show that EMK and C-TAK1 phosphorylate class IIa HDACs on one of their multiple 14-3-3 binding sites and alter their subcellular localization and repressive function. Using HDAC7 as a paradigm, we extend these findings by demonstrating that signal-independent phosphorylation of the most N-terminal serine residue by the MARK/Par-1 kinases, i.e., Ser155, is a prerequisite for the phosphorylation of the nearby 14-3-3 site, Ser181. We propose that this multisite hierarchical phosphorylation by a variety of kinases allows for sophisticated regulation of class IIa HDACs function.

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Phosphorylation of histone deacetylase 7 by protein kinase D mediates T cell receptor–induced Nur77 expression and apoptosis

Journal of Experimental Medicine ◽

10.1084/jem.20042034 ◽

2005 ◽

Vol 201 (5) ◽

pp. 793-804 ◽

Cited By ~ 121

Author(s):

Franck Dequiedt ◽

Johan Van Lint ◽

Emily Lecomte ◽

Viktor Van Duppen ◽

Thomas Seufferlein ◽

...

Keyword(s):

T Cell ◽

Protein Kinase ◽

T Cell Receptor ◽

Nuclear Export ◽

Histone Deacetylases ◽

Negative Selection ◽

Cell Receptor ◽

Protein Kinase D ◽

Orphan Nuclear Receptor ◽

Nuclear Exclusion

The molecular basis of thymocyte negative selection, a crucial mechanism in establishing central tolerance, is not yet resolved. Histone deacetylases (HDACs) have emerged as key transcriptional regulators in several major developmental programs. Recently, we showed that the class IIa member, HDAC7, regulates negative selection by repressing expression of Nur77, an orphan nuclear receptor involved in antigen-induced apoptosis of thymocytes. Engagement of the T cell receptor (TCR) alleviates this repression through phosphorylation-dependent nuclear exclusion of HDAC7. However, the identity of the TCR-activated kinase that phosphorylates and inactivates HDAC7 was still unknown. Here, we demonstrate that TCR-induced nuclear export of HDAC7 and Nur77 expression is mediated by activation of protein kinase D (PKD). Indeed, active PKD stimulates HDAC7 nuclear export and Nur77 expression. In contrast, inhibition of PKD prevents TCR-mediated nuclear exclusion of HDAC7 and associated Nur77 activation. Furthermore, we show that HDAC7 is an interaction partner and a substrate for PKD. We identify four serine residues in the NH2 terminus of HDAC7 as targets for PKD. More importantly, a mutant of HDAC7 specifically deficient in phosphorylation by PKD, inhibits TCR-mediated apoptosis of T cell hybridomas. These findings indicate that PKD is likely to play a key role in the signaling pathways controlling negative selection.

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The Carboxy Terminus of Prospero Regulates Its Subcellular Localization

Molecular and Cellular Biology ◽

10.1128/mcb.23.3.1014-1024.2003 ◽

2003 ◽

Vol 23 (3) ◽

pp. 1014-1024 ◽

Cited By ~ 13

Author(s):

Xiaolin Bi ◽

Andrey V. Kajava ◽

Tamara Jones ◽

Zoya N. Demidenko ◽

Mark A. Mortin

Keyword(s):

Subcellular Localization ◽

Cell Fate ◽

Nuclear Export ◽

System Development ◽

Nuclear Export Signal ◽

Nervous System Development ◽

Carboxy Terminus ◽

Nuclear Exclusion ◽

Multiple Processes ◽

Embryonic Nervous System

ABSTRACT Subcellular localization of the transcription factor Prospero is dynamic. For example, the protein is cytoplasmic in neuroblasts, nuclear in sheath cells, and degraded in newly formed neurons. The carboxy terminus of Prospero, including the homeodomain and Prospero domain, plays roles in regulating these changes. The homeodomain has two distinct subdomains, which exclude proteins from the nucleus, while the intact homeo/Prospero domain masks this effect. One subdomain is an Exportin-dependent nuclear export signal requiring three conserved hydrophobic residues, which models onto helix 1. Another, including helices 2 and 3, requires proteasome activity to degrade nuclear protein. Finally, the Prospero domain is missing in prosI13 embryos, thus unmasking nuclear exclusion, resulting in constitutively cytoplasmic protein. Multiple processes direct Prospero regulation of cell fate in embryonic nervous system development.

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Mechanism for Nucleocytoplasmic Shuttling of Histone Deacetylase 7

Journal of Biological Chemistry ◽

10.1074/jbc.m107631200 ◽

2001 ◽

Vol 276 (50) ◽

pp. 47496-47507 ◽

Cited By ~ 171

Author(s):

Hung-Ying Kao ◽

André Verdel ◽

Chih-Cheng Tsai ◽

Cynthia Simon ◽

Henry Juguilon ◽

...

Keyword(s):

Subcellular Localization ◽

Hela Cells ◽

Nuclear Export ◽

Histone Deacetylases ◽

Direct Interaction ◽

Binding Activity ◽

Nucleocytoplasmic Shuttling ◽

Nih3t3 Cells ◽

Dna Binding Activity ◽

A Cell

Here we show that HDAC7, a member of the class II histone deacetylases, specifically targets several members of myocyte enhancer factors, MEF2A, -2C, and -2D, and inhibits their transcriptional activity. Furthermore, we demonstrate that DNA-bound MEF2C is capable of recruiting HDAC7, demonstrating that the HDAC7-dependent repression of transcription is not due to the inhibition of the MEF2 DNA binding activity. The data also suggest that the promoter bound MEF2 is potentially capable of remodeling adjacent nucleosomes via the recruitment of HDAC7. We have also observed a nucleocytoplasmic shuttling of HDAC7 and dissected the mechanism involved. In NIH3T3 cells, HDAC7 was primarily localized in the cytoplasm, essentially due to an active CRM1-dependent export of the protein from the nucleus. Interestingly, in HeLa cells, HDAC7 was predominantly nuclear. In these cells we could restore the cytoplasmic localization of HDAC7 by expressing CaMK I. This CaMK I-induced nuclear export of HDAC7 was abolished when three critical serines, Ser-178, Ser-344, and Ser-479, of HDAC7 were mutated. We show that these serines are involved in the direct interaction of HDAC7 with 14-3-3. Mutations of these serine residues weakened the association with 14-3-3 and dramatically enhanced the repression activity of HDAC7 in NIH3T3 cells, but not in HeLa cells. Data presented in this work clearly show that the signal dependent subcellular localization of HDAC7 is essential in controlling its activities. The data also show that the cellular concentration of factors such as 14-3-3, CaMK I, and other yet unknown molecules may determine the subcellular localization of an individual HDAC member in a cell type and HDAC-specific manner.

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Novel late-stage radiosynthesis of 5-[18F]-trifluoromethyl-1,2,4-oxadiazole (TFMO) containing molecules for PET imaging

Scientific Reports ◽

10.1038/s41598-021-90069-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Nashaat Turkman ◽

Daxing Liu ◽

Isabella Pirola

Keyword(s):

Late Stage ◽

Histone Deacetylases ◽

Radiochemical Purity ◽

Radiochemical Yield ◽

Single Step ◽

The Novel ◽

Molar Activity ◽

The Central Nervous System ◽

Class Iia Hdacs ◽

18F Fluoride

AbstractSmall molecules that contain the (TFMO) moiety were reported to specifically inhibit the class-IIa histone deacetylases (HDACs), an important target in cancer and the disorders of the central nervous system (CNS). However, radiolabeling methods to incorporate the [18F]fluoride into the TFMO moiety are lacking. Herein, we report a novel late-stage incorporation of [18F]fluoride into the TFMO moiety in a single radiochemical step. In this approach the bromodifluoromethyl-1,2,4-oxadiazole was converted into [18F]TFMO via no-carrier-added bromine-[18F]fluoride exchange in a single step, thus producing the PET tracers with acceptable radiochemical yield (3–5%), high radiochemical purity (> 98%) and moderate molar activity of 0.33–0.49 GBq/umol (8.9–13.4 mCi/umol). We validated the utility of the novel radiochemical design by the radiosynthesis of [18F]TMP195, which is a known TFMO containing potent inhibitor of class-IIa HDACs.

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Bioinformatic and experimental survey of 14-3-3-binding sites

Biochemical Journal ◽

10.1042/bj20091834 ◽

2010 ◽

Vol 427 (1) ◽

pp. 69-78 ◽

Cited By ~ 203

Author(s):

Catherine Johnson ◽

Sandra Crowther ◽

Margaret J. Stafford ◽

David G. Campbell ◽

Rachel Toth ◽

...

Keyword(s):

Protein Kinase ◽

Protein Kinases ◽

Binding Sites ◽

Histone Deacetylases ◽

Cognitive Disorders ◽

Logic Gates ◽

Protein Families ◽

Kinase C ◽

Dependent Protein ◽

Physiological Demands

More than 200 phosphorylated 14-3-3-binding sites in the literature were analysed to define 14-3-3 specificities, identify relevant protein kinases, and give insights into how cellular 14-3-3/phosphoprotein networks work. Mode I RXX(pS/pT)XP motifs dominate, although the +2 proline residue occurs in less than half, and LX(R/K)SX(pS/pT)XP is prominent in plant 14-3-3-binding sites. Proline at +1 is rarely reported, and such motifs did not stand up to experimental reanalysis of human Ndel1. Instead, we discovered that 14-3-3 interacts with two residues that are phosphorylated by basophilic kinases and located in the DISC1 (disrupted-in-schizophrenia 1)-interacting region of Ndel1 that is implicated in cognitive disorders. These data conform with the general findings that there are different subtypes of 14-3-3-binding sites that overlap with the specificities of different basophilic AGC (protein kinase A/protein kinase G/protein kinase C family) and CaMK (Ca2+/calmodulin-dependent protein kinase) protein kinases, and a 14-3-3 dimer often engages with two tandem phosphorylated sites, which is a configuration with special signalling, mechanical and evolutionary properties. Thus 14-3-3 dimers can be digital logic gates that integrate more than one input to generate an action, and coincidence detectors when the two binding sites are phosphorylated by different protein kinases. Paired sites are generally located within disordered regions and/or straddle either side of functional domains, indicating how 14-3-3 dimers modulate the conformations and/or interactions of their targets. Finally, 14-3-3 proteins bind to members of several multi-protein families. Two 14-3-3-binding sites are conserved across the class IIa histone deacetylases, whereas other protein families display differential regulation by 14-3-3s. We speculate that 14-3-3 dimers may have contributed to the evolution of such families, tailoring regulatory inputs to different physiological demands.

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Distinct Mechanisms Involving Diverse Histone Deacetylases Repress Expression of the Two Gonadotropin β-Subunit Genes in Immature Gonadotropes, and Their Actions Are Overcome by Gonadotropin-Releasing Hormone

Molecular and Cellular Biology ◽

10.1128/mcb.00248-07 ◽

2007 ◽

Vol 27 (11) ◽

pp. 4105-4120 ◽

Cited By ~ 54

Author(s):

Stefan Lim ◽

Min Luo ◽

Mingshi Koh ◽

Meng Yang ◽

Mohammed Nizam bin Abdul Kadir ◽

...

Keyword(s):

Histone Deacetylases ◽

Reproductive Function ◽

Gonadotropin Releasing Hormone ◽

Β Subunit ◽

Releasing Hormone ◽

Calcium Calmodulin Dependent ◽

Gnrh Release ◽

Dependent Protein ◽

Reproductive Cycles ◽

Class Iia Hdacs

ABSTRACT The gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are produced in the embryonic pituitary in response to delivery of the hypothalamic gonadotropin releasing hormone (GnRH). GnRH has a pivotal role in reestablishing gonadotropin levels at puberty in primates, and for many species with extended reproductive cycles, these are reinitiated in response to central nervous system-induced GnRH release. Thus, a clear role is evident for GnRH in overcoming repression of these genes. Although the mechanisms through which GnRH actively stimulates LH and FSH β-subunit (FSHβ) gene transcription have been described in some detail, there is currently no information on how GnRH overcomes repression in order to terminate reproductively inactive stages. We show here that GnRH overcomes histone deacetylase (HDAC)-mediated repression of the gonadotropin β-subunit genes in immature gonadotropes. The repressive factors associated with each of these genes comprise distinct sets of HDACs and corepressors which allow for differentially regulated derepression of these two genes, produced in the same cell by the same regulatory hormone. We find that GnRH activation of calcium/calmodulin-dependent protein kinase I (CaMKI) plays a crucial role in the derepression of the FSHβ gene involving phosphorylation of several class IIa HDACs associated with both the FSHβ and Nur77 genes, and we propose a model for the mechanisms involved. In contrast, derepression of the LH β-subunit gene is not CaMK dependent. This demonstration of HDAC-mediated repression of these genes could explain the temporal shut-down of reproductive function at certain periods of the life cycle, which can easily be reversed by the actions of the hypothalamic regulatory hormone.

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