Estrogen delays the progression of salt-induced cardiac hypertrophy by influencing the renin-angiotensin system in heterozygous proANP gene-disrupted mice

Human SIRT2 is a cytoplasmic NAD-dependent deacetylase implicated in the mitotic regulation of microtubule dynamics by its association with the class II histone deacetylase 6 (HDAC6). We have previously reported that SIRT2 is multiply phosphorylated in a cell cycle dependent pattern. Here, we demonstrate that HDAC6 binds to both phosphorylated and unphosphorylated forms of SIRT2 and that tubulin binds only to the SIRT2-HDAC6 complex. Tubulin does not bind to either HDAC6 or SIRT2 individually. In addition, we show that replacement of specific serines with alanines in either isoform of SIRT2 regulates its enzymatic activity. We also found that overexpression of isoform2 was deleterious to cell survival. SIRT2 was found to be phosphorylated at serines 368 and 372, outside the conserved core domain of the Sir2 protein family. Double replacement of S368A and S372A reduced SIRT2 deacetylase activity by 44% compared to wildtype activity. Replacements of other serine, threonine, and tyrosine residues, which did not alter the phosphorylation pattern, had varying effects on SIRT2 deacetylase activity but no effect on tubulin/HDAC6 binding.     

JNK2-dependent regulation of SIRT1 protein stability

Mammalian SIRT1 is an NAD-dependent deacetylase with critical roles in the maintenance of homeostasis and cell survival. Elevated levels of SIRT1 protein are evident in cancer in which SIRT1 can function as a cancer-specific survival factor. Here we demonstrate that elevated SIRT1 protein in human cells is not attributable to increased SIRT1 mRNA levels but, instead, reflects SIRT1 protein stability. RNAi-mediated depletion of JNK2 reduced the half-life of SIRT1 protein from > 9h to < 2h and this correlated with lack of SIRT1 protein phosphorylation at serine 27. In contrast, depletion of JNK1 had no effect upon SIRT1 protein stability and SIRT1 phosphorylation at serine 47 showed no correlation with SIRT1 protein stability. Thus we show that JNK2 is linked, directly or indirectly, with SIRT1 protein stability and that this function is coupled with SIRT1 phosphorylation at serine 27. Our observations identify a route for therapeutic modulation of SIRT1 protein levels in SIRT1-linked diseases including cancer, neurodegeneration and diabetes.     

Sequence analysis of cell cycle control (cdc2) protein kinases among protein serine/threonine kinases

Among protein serine/threonine kinases, the CDC2 proteins are both well characterized as protein serine/threonine kinases and are functionally involved in the control of cell division. Protein serine/threonine kinase sequences were analysed using Fourier transform of the coded sequences. Characteristic code/frequency pairs were extracted from a set of well defined protein serine/threonine kinases. The characteristic frequencies 0.179, 0.250 and 0.408 distinguished protein serine/threonine kinases from proteins which did not have the biological activity. Pertinent patterns in the sequence, responsible for the code/frequency pairs detection were searched and found to be correlated with the putative catalytic domain of the proteins. Protein serine/threonine kinases involved in cell division control, CDC2 protein kinases, were compared to the other protein serine/threonine kinases. Specific code/frequency pairs were extracted from the sequences and could be related to the function or regulation of the kinases in cell division. Two CDC2 related proteins CDC2(Mm) from mice and CDC2(Gg) from chicken were shown to fit well with the CDC2 proteins, whereas KIN28, PHO85 and PSKJ3, which share sequence homology but not functional activity with the CDC2 proteins, were clearly excluded from the CDC2 proteins by the characteristic code/frequency pairs. Pertinent patterns in the CDC2 proteins were analysed and mapped on the CDC2 related protein sequences. Four patterns were correlated with the code/frequency detection and therefore, could be associated to the regulation of the CDC2-related proteins.     

Effects of 18-glycyrrhetinic acid on serine 368 phosphorylation of connexin43 in rat neonatal cardiomyocytes

18Beta-glycyrrhetinic acid (18beta-GA) regulates serine/threonine dephosphorylation of connexin43 (Cx43). Phospho-specific antibodies were used here to determine the effect of 18beta-GA on serine 368-phosphorylated Cx43 (pSer368Cx43) in cultured rat neonatal cardiomyocytes by immunofluorescence microscopy and immunoblot analyses. 18beta-GA caused a time-dependent increase in pSer368Cx43 levels and induced gap junction disassembly, shown by a change in pSer368Cx43 immunostaining from large aggregates to dispersed punctates at cell-cell contact areas. 18beta-GA also induced a time-dependent increase in the levels of serine 729-phosphorylated PKCepsilon, the active form of PKCepsilon. The 18beta-GA-induced increase in pSer368Cx43 levels and changes in pSer368Cx43 staining pattern were abolished by the PKC inhibitor, chelerythrine. Furthermore, 18beta-GA increased the co-immunoprecipitation of Cx43 with PKCepsilon. However, the 18beta-GA-induced increase in pSer368Cx43 levels and increased association of Cx43 with PKCepsilon were inhibited by co-treatment with the protein phosphatase type 1 and type 2A inhibitor, calyculin A. We conclude that 18beta-GA induces Ser368 phosphorylation of Cx43 via PKCepsilon.     

Nuclear localization of CDC25B1 and serine 146 integrity are required for induction of mitosis

CDC25B phosphatases are essential regulators that control cyclin-dependent kinases activities at the entry into mitosis. In this study, we demonstrate that serine 146 is required for two crucial features of CDC25B1. It is essential for CDC25B1 to function as a mitotic inducer and to prevent CDC25B1 export from the nucleus. We also show that serine 146 is phosphorylated in vitro by CDK1-cyclin B. However, phosphorylation of CDC25B does not stimulate its phosphatase activity, and mutation of serine 146 had no effect on its catalytic activity. Serine 146 phosphorylation is proposed to be a key event in the regulation of the CDC25B function in the initiation of mammalian mitosis.     

Discovery of XL413, a potent and selective CDC7 inhibitor

CDC7 is a serine/threonine kinase that has been shown to be required for the initiation and maintenance of DNA replication. Up-regulation of CDC7 is detected in multiple tumor cell lines, with inhibition of CDC7 resulting in cell cycle arrest. In this paper, we disclose the discovery of a potent and selective CDC7 inhibitor, XL413 (14), which was advanced into Phase 1 clinical trials. Starting from advanced lead 3, described in a preceding communication, we optimized the CDC7 potency and selectivity to demonstrate in vitro CDC7 dependent cell cycle arrest and in vivo tumor growth inhibition in a Colo-205 xenograft model.     

SIRT2 is a negative regulator of anoxia-reoxygenation tolerance via regulation of 14-3-3 zeta and BAD in H9c2 cells

Knockdown or inhibition of SIRT2 enhances biological stress-tolerance. We extend this phenotype showing that SIRT2 knockdown reduces anoxia-reoxygenation injury in H9c2 cells. Gene array analysis following SIRT2 siRNA knockdown identifies 14-3-3 zeta as the most robustly induced gene. SIRT2 knockdown evokes induction of this chaperone, facilitating cytosolic sequestration of BAD with a corresponding reduction in mitochondrial BAD localization. Concurrent siRNA against SIRT2 and 14-3-3 zeta abolishes the SIRT2-depleted cytoprotective phenotype. SIRT2 functions to moderate cellular stress-tolerance, in part, by modulating the levels of 14-3-3 zeta with the concordant control of BAD subcellular localization.     

Amino acids C-terminal to the 14-3-3 binding motif in CDC25B affect the efficiency of 14-3-3 binding

The phospho-site adapter protein 14-3-3 binds to target proteins at amino acid sequences matching the consensus motif Arg-X-X-Ser/Thr-X-Pro, where the serine or threonine residue is phosphorylated and X is any amino acid. The dual-specificity phosphatase CDC25B, which is involved in cell cycle regulation, contains five 14-3-3 binding motifs, but 14-3-3 preferentially binds to the motif at Ser309 in CDC25B1 (or Ser323 in CDC25B3). In the present study, we demonstrate that amino acid residues C-terminal to the 14-3-3 binding motif strongly affect the efficiency of 14-3-3 binding. Alanine substitutions at residues downstream of the Ser309 motif dramatically reduced 14-3-3 binding, although phosphorylation of Ser309 was unaffected. We also observed that binding of endogenous 14-3-3 to mutant CDC25B occurred less efficiently than to the wild type. Mutants to which 14-3-3 cannot bind efficiently tend to be located in the nucleus, although not as specifically as the alanine substitution mutant of Ser309. These results indicate that amino acid sequences C-terminal to the consensus binding site have an important role in the efficient binding of 14-3-3 to at least CDC25B, which may partly explain why some consensus sequences are inactive as 14-3-3 binding sites.     

CDC25B Phosphorylated by pEg3 Localizes to the Centrosome and the Spindle Poles at Mitosis

The phosphatase CDC25B is one of the key regulators that control entry into mitosis throughthe dephosphorylation and subsequent activation of the cyclin-dependent kinases. Here westudy the phosphorylation of CDC25B at mitosis by the kinase pEg3, a member of theKIN1/PAR-1/MARK family. Using mass spectrometry analysis we demonstrate thatCDC25B is phosphorylated in vitro by pEg3 on serine 169, a residue that lies within the Bdomain. Moreover, using phosphoepitope-specific antibodies we show that serine 169 isphosphorylated in vivo, that this phosphorylated form of CDC25B accumulates duringmitosis, and is localized to the centrosomes. This labelling is abrogated when pEg3expression is repressed by RNA interference. Taken together, these results support a model inwhich pEg3 contributes to the control of progression through mitosis by phosphorylation ofthe CDC25 phosphatases.     

CDC25B Phosphorylation by Aurora A Occurs at the G2/M Transition and is Inhibited by DNA Damage

CDC25B is one of the three human dual-specificity phosphatases involved in the activation ofcyclin-dependent kinases at key stages of the cell division cycle. CDC25B that is responsiblefor the activation of CDK1-cyclin B1 is regulated by phosphorylation. The STK15/Aurora-Akinase locally phosphorylates CDC25B on serine 353 at the centrosome during the G2/Mtransition. Here we have investigated this phosphorylation event during the cell cycle, and inresponse to activation of the G2 DNA damage checkpoint. We show that accumulation of theS353-phosphorylated form of CDC25B at the centrosome correlates with the relocalisation ofcyclin B1 to the nucleus and the activation of CDK1 at entry into mitosis. Upon activation ofthe G2/M checkpoint by DNA damage, we demonstrate that Aurora-A is not activated andconsequently CDC25B is not phosphorylated. We show that ectopic expression of Aurora-Aresults in a bypass of the checkpoint that partially overcome by a S353A mutant of CDC25B.Finally, we show that bypass of the G2/M checkpoint by the CHK1 kinase inhibitor UCN-01results in the activation of Aurora-A and phosphorylation of CDC25B on S353. These resultsstrongly suggest that Aurora-A-mediated phosphorylation of CDC25B at the centrosome is animportant step contributing to the earliest events inducing mitosis, upstream of CDK1-cyclinB1 activation.     

Critical role of RelB serine 368 for dimerization and p100 stabilization

In mature B cells RelB-containing complexes are constitutively present in the nucleus, and they are less susceptible to inhibitory kappaB proteins. In most other cell types inhibitory kappaB proteins prevent nuclear translocation and activation of NFkappaB. We reasoned that this characteristic might be because of post-translational modifications of RelB. In Drosophila, signal-dependent phosphorylation of the Rel homologue Dorsal at serine 317 has been shown to be critical for nuclear import. The evolutionary conservation of this serine prompted us to analyze the function of the corresponding site in RelB. As a model system we used the murine S107 plasmacytoma cell line, which lacks endogenous RelB expression. Analysis of S107 cells expressing wild type RelB and serine 368 mutants reveals that serine 368 is not required for nuclear import but that it is critical for RelB dimerization with other members of the NFkappaB family. Similar effects were obtained when the conserved serine in RelA was mutated. We further demonstrate that expression of functional RelB, but not of serine 368 mutants, severely reduces p52 generation and strongly increases expression of the p52 precursor, p100. Wild type RelB, but not mutant RelB, prolonged p100 half-life. We therefore suggest an inhibitory effect of RelB on p100 processing, which is possibly regulated in a signal-dependent manner.     

Mitotic phosphatases: no longer silent partners

Recent work has highlighted the important role played by protein phosphatase complexes in the regulation of mitosis from yeast to mammals. There have been important advances in defining the roles of the protein serine/threonine phosphatases PP1 and PP2A and the dual specificity protein tyrosine phosphatases CDC25 and Cdc14. Three independent studies defined a regulatory role for PP2A in the control of sister chromatid cohesion, involving a direct interaction with shugoshin. A chromatin targeting subunit has been identified for PP1 and the complex shown to play an essential role in chromosome segregation. Key regulatory residues within CDC25 have been mapped and its activity tied both to the initial activation of cyclin-dependent kinases at the centrosome and to DNA damage checkpoints. Novel roles have been defined for Cdc14, including regulation of rDNA and telomere segregation and participation in spindle assembly. These exciting advances show that protein phosphatases are not merely silent partners to kinases in regulating the control of cell division.     

PKB/Akt phosphorylates the CDC25B phosphatase and regulates its intracellular localisation

Regulation of the intracellular localisation of its actors is one of the key mechanisms underlying cell cycle control. CDC25 phosphatases are activators of Cyclin-Dependent Kinases (CDK) that undergo nucleo-cytoplasmic shuttling during the cell cycle and in response to checkpoint activation. Here we report that the protein kinase PKB/Akt phosphorylates CDC25B on serine 353, resulting in a nuclear export-dependent cytoplasmic accumulation of the phosphatase. Oxidative stress activates PKB/Akt and reproduces the effect on CDC25B phosphorylation and localisation. However, inhibition of PKB/Akt activity only partially reverted the effect of oxidative stress on CDC25B localisation and mutation of serine 353 abolishes phosphorylation but only delays nuclear exclusion. These results indicate that additional mechanisms are also involved in preventing nuclear import of CDC25B. Our findings identify CDC25B as a target of PKB/Akt and provide new insight into the regulation of its localisation in response to stress-activated signalling pathways.     

Regulation of the stability and activity of CDC25A and CDC25B by protein phosphatase PP2A and 14-3-3 binding

Cyclin-dependent kinase (CDK)-activating phosphatases, CDC25A and CDC25B, are labile proteins, and their levels vary in a cell cycle-dependent manner. Immediate-early response IER5 protein negatively regulates the cellular CDC25B levels, and stress-induced IER5 expression potentiates G2/M arrest. IER5 binds to protein phosphatase PP2A and regulates the PP2A substrate specificity. We show that IER5 binds to CDC25B and assists PP2A to convert CDC25B to hypophosphorylated forms. Hypophosphorylation at Ser323 results in the dissociation of CDC25B from 14‐3-3 phospho-binding proteins. In IER5 expressing cells, CDC25B dissociated from 14‐3-3 is unstable but slightly activated, because 14‐3-3 inhibits CDC25B polyubiquitination and CDC25B binding to CDK1. The 14‐3-3 binding to CDC25A also impedes CDC25A degradation and CDC25A-CDK2 interaction. We propose that 14‐3-3 is an important regulator of CDC25A and CDC25B and that PP2A/IER5 controls the stability and activity of CDC25B through regulating the interaction of CDC25B and 14‐3-3.     

Enhanced PKCε mediated phosphorylation of connexin43 at serine 368 by a carboxyl-terminal mimetic peptide is dependent on injury

The gap junction (GJ) protein connexin (Cx43) is important for organized action potential propagation between mammalian cardiomyocytes. Disruption of the highly ordered distribution of Cx43 GJs is characteristic of cardiac tissue after ischemic injury. We recently demonstrated that epicardial administration of a peptide mimetic of the Cx43 carboxyl-terminus reduced pathologic remodeling of Cx43 GJs and protected against induced arrhythmias following ventricular injury. Treatment of injuries with the carboxyl-terminal peptide was associated with an increase in phosphorylation at serine 368 of the Cx43 carboxyl-terminus. Here, we report that Cx43 peptide treatment of uninjured hearts does not prompt a similar increase in phosphorylation. Moreover, we show that peptide treatment of undisturbed cultured HeLa cells expressing a Cx43 construct also exhibit no changes in Cx43 phosphorylation at serine 368. However, in parallel with the results in vivo, a trend of increasing phosphorylation at serine 368 was observed in Cx43-expressing HeLa cells following scratch wounding of cultured monolayers. These results suggest that peptide-enhanced phosphorylation of the Cx43 carboxyl-terminus is dependent on injury-mediated cellular responses.     

A novel CDC25A/DYRK2 regulatory switch modulates cell cycle and survival

The cell division cycle 25A (CDC25A) phosphatase is a key regulator of cell cycle progression that acts on the phosphorylation status of Cyclin–Cyclin-dependent kinase complexes, with an emergent role in the DNA damage response and cell survival control. The regulation of CDC25A activity and its protein level is essential to control the cell cycle and maintain genomic integrity. Here we describe a novel ubiquitin/proteasome-mediated pathway negatively regulating CDC25A stability, dependent on its phosphorylation by the serine/threonine kinase DYRK2. DYRK2 phosphorylates CDC25A on at least 7 residues, resulting in its degradation independent of the known CDC25A E3 ubiquitin ligases. CDC25A in turn is able to control the phosphorylation of DYRK2 at several residues outside from its activation loop, thus affecting DYRK2 localization and activity. An inverse correlation between DYRK2 and CDC25A protein amounts was observed during cell cycle progression and in response to DNA damage, with CDC25A accumulation responding to the manipulation of DYRK2 levels or activity in either physiological scenario. Functional data show that the pro-survival activity of CDC25A and the pro-apoptotic activity of DYRK2 could be partly explained by the mutual regulation between both proteins. Moreover, DYRK2 modulation of CDC25A expression and/or activity contributes to the DYRK2 role in cell cycle regulation. Altogether, we provide evidence suggesting that DYRK2 and CDC25A mutually control their activity and stability by a feedback regulatory loop, with a relevant effect on the genotoxic stress pathway, apoptosis, and cell cycle regulation.Subject terms: Proteins, Cell biology, Proteomics     

Enhanced PKCε mediated phosphorylation of connexin43 at serine 368 by a carboxyl-terminal mimetic peptide is dependent on injury

The gap junction (GJ) protein connexin (Cx43) is important for organized action potential propagation between mammalian cardiomyocytes. Disruption of the highly ordered distribution of Cx43 GJs is characteristic of cardiac tissue after ischemic injury. We recently demonstrated that epicardial administration of a peptide mimetic of the Cx43 carboxyl-terminus reduced pathologic remodeling of Cx43 GJs and protected against induced arrhythmias following ventricular injury. Treatment of injuries with the carboxyl-terminal peptide was associated with an increase in phosphorylation at serine 368 of the Cx43 carboxyl-terminus. Here, we report that Cx43 peptide treatment of uninjured hearts does not prompt a similar increase in phosphorylation. Moreover, we show that peptide treatment of undisturbed cultured HeLa cells expressing a Cx43 construct also exhibit no changes in Cx43 phosphorylation at serine 368. However, in parallel with the results in vivo, a trend of increasing phosphorylation at serine 368 was observed in Cx43-expressing HeLa cells following scratch wounding of cultured monolayers. These results suggest that peptide-enhanced phosphorylation of the Cx43 carboxyl-terminus is dependent on injury-mediated cellular responses.