A functional genetic screen identifies regions at the C-terminal tail and death-domain of death-associated protein kinase that are critical for its proapoptotic activity

Death-associated protein kinase (DAP-kinase) is a Ca+2/calmodulin-regulated serine/threonine kinase with a multidomain structure that participates in apoptosis induced by a variety of signals. To identify regions in this protein that are critical for its proapoptotic activity, we performed a genetic screen on the basis of functional selection of short DAP-kinase-derived fragments that could protect cells from apoptosis by acting in a dominant-negative manner. We expressed a library of randomly fragmented DAP-kinase cDNA in HeLa cells and treated these cells with IFN-γ to induce apoptosis. Functional cDNA fragments were recovered from cells that survived the selection, and those in the sense orientation were examined further in a secondary screen for their ability to protect cells from DAP-kinase-dependent tumor necrosis factor-α-induced apoptosis. We isolated four biologically active peptides that mapped to the ankyrin repeats, the “linker” region, the death domain, and the C-terminal tail of DAP-kinase. Molecular modeling of the complete death domain provided a structural basis for the function of the death-domain-derived fragment by suggesting that the protective fragment constitutes a distinct substructure. The last fragment, spanning the C-terminal serine-rich tail, defined a new regulatory region. Ectopic expression of the tail peptide (17 amino acids) inhibited the function of DAP-kinase, whereas removal of this region from the complete protein caused enhancement of the killing activity, indicating that the C-terminal tail normally plays a negative regulatory role. Altogether, this unbiased screen highlighted functionally important regions in the protein and revealed an additional level of regulation of DAP-kinase apoptotic function that does not affect the catalytic activity.

Activity-dependent CREB phosphorylation: Convergence of a fast, sensitive calmodulin kinase pathway and a slow, less sensitive mitogen-activated protein kinase pathway

The cAMP-responsive element binding protein (CREB), a key regulator of gene expression, is activated by phosphorylation on Ser-133. Several different protein kinases possess the capability of driving this phosphorylation, making it a point of potential convergence for multiple intracellular signaling cascades. Previous work in neurons has indicated that physiologic synaptic stimulation recruits a fast calmodulin kinase IV (CaMKIV)-dependent pathway that dominates early signaling to CREB. Here we show in hippocampal neurons that the fast, CaMK-dependent pathway can be followed by a slower pathway that depends on Ras/mitogen-activated protein kinase (MAPK), along with CaMK. This pathway was blocked by dominant-negative Ras and was specifically recruited by depolarizations that produced strong intracellular Ca2+ transients. When both pathways were recruited, phosphorylated CREB (pCREB) formation was overwhelmingly dominated by the CaMK pathway between 0 and 10 min, and by the MAPK pathway at 60 min, whereas the two pathways acted in concert at 30 min. The Ca2+ signals that produced only rapid CaMK signaling to pCREB or both rapid CaMK and slow MAPK signaling deviated significantly for only ≈1 min, yet their differential impact on pCREB extended over a much longer period, between 20 and 60 min and beyond, which is of likely significance for gene expression. The CaMK-dependent MAPK pathway may inform the nucleus about stimulus amplitude. In contrast, the CaMKIV pathway may be well suited to conveying information on the precise timing of localized synaptic stimuli, befitting its greater speed and sensitivity, whereas the previously described calcineurin pathway may carry information about stimulus duration.

Phosphorylation of MCM4 by cdc2 protein kinase inhibits the activity of the minichromosome maintenance complex.

In eukaryotes, tight regulatory mechanisms ensure the ordered progression through the cell cycle phases. The mechanisms that prevent chromosomal DNA replication from taking place more than once each cell cycle are thought to involve the function of proteins of the minichromosome maintenance (MCM) family. Here, we demonstrate that Xenopus MCM4, a member of the MCM protein family related to Spcdc21/ ScCDC54, is part of a large protein complex comprising several other MCM proteins. MCM4 undergoes cell cycle-dependent phosphorylation both in cleaving embryos and in cell-free extracts. MCM4 phosphorylation starts concomitantly with the clearing of the MCM complex from the chromatin during S phase. Phosphorylation is carried out by cdc2/cyclinB protein kinase, which phosphorylates MCM4 in vitro at identical sites as the ones phosphorylated in vivo. Phosphorylation is specific for cdc2 protein kinase since MCM4 is not a substrate for other members of the cdk family. Furthermore, phosphorylation of MCM4 dramatically reduces its affinity for the chromatin. We propose that the cell cycle-dependent phosphorylation of MCM4 is a mechanism which inactivates the MCM complex from late S phase through mitosis, thus preventing illegitimate DNA replication during that period of the cell cycle.

Regulation of mitochondrial pyruvate dehydrogenase activity by tau protein kinase I/glycogen synthase kinase 3beta in brain.

According to the amyloid hypothesis for the pathogenesis of Alzheimer disease, beta-amyloid peptide (betaA) directly affects neurons, leading to neurodegeneration and tau phosphorylation. In rat hippocampal culture, betaA exposure activates tau protein kinase I/glycogen synthase kinase 3beta (TPKI/GSK-3beta), which phosphorylates tau protein into Alzheimer disease-like forms, resulting in neuronal death. To elucidate the mechanism of betaA-induced neuronal death, we searched for substrates of TPKI/GSK-3beta in a two-hybrid system and identified pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA in mitochondria. PDH was phosphorylated and inactivated by TPKI/GSK-3beta in vitro and also in betaA-treated hippocampal cultures, resulting in mitochondrial dysfunction, which would contribute to neuronal death. In cholinergic neurons, betaA impaired acetylcholine synthesis without affecting choline acetyltransferase activity, which suggests that PDH is inactivated by betaA-induced TPKI/GSK-3beta. Thus, TPKI/GSK-3beta regulates PDH and participates in energy metabolism and acetylcholine synthesis. These results suggest that TPKI/GSK-3beta plays a key role in the pathogenesis of Alzheimer disease.

Catalytic activity of the mouse guanine nucleotide exchanger mSOS is activated by Fyn tyrosine protein kinase and the T-cell antigen receptor in T cells.

mSOS, a guanine nucleotide exchange factor, is a positive regulator of Ras. Fyn tyrosine protein kinase is a potential mediator in T-cell antigen receptor signal transduction in subsets of T cells. We investigated the functional and physical interaction between mSOS and Fyn in T-cell hybridoma cells. Stimulation of the T-cell antigen receptor induced the activation of guanine nucleotide exchange activity in mSOS immunoprecipitates. Overexpression of Fyn mutants with an activated kinase mutation and with a Src homology 2 deletion mutation resulted in a stimulation and suppression of the mSOS activity, respectively. The complex formations of Fyn-Shc, Shc-Grb2, and Grb2-mSOS were detected in the activated Fyn-transformed cells, whereas the SH2 deletion mutant of Fyn failed to form a complex with mSOS. Moreover, tyrosine phosphorylation of Shc was induced by the overexpression of the activated Fyn. These findings support the idea that Fyn activates the activity of mSOS bound to Grb2 through tyrosine phosphorylation of Shc. Unlike the current prevailing model, Fyn-induced activation of Ras might involve the stimulation of the catalytic guanine nucleotide exchange activity of mSOS.

Role of phospholipids containing docosahexaenoyl chains in modulating the activity of protein kinase C.

It is known that the phospholipids of the brain cells of fish are altered during cold adaptation. In particular, the 1-monounsaturated 2-polyunsaturated phosphatidylethanolamines (PEs) increase 2- to 3-fold upon adaptation to cold. One of the most striking changes is in the 18:1/22:6 species of PE. We determined how this lipid affected the bilayer-to-hexagonal-phase transition temperature of 16:1/16:1 PE. We found that it was more effective in lowering this transition temperature than were other, less unsaturated, PE species. In addition, it was not simply the presence of the 18:1/22:6 acyl chains which caused this effect, since the 18:1/22:6 species of phosphatidylcholine had the opposite effect on this transition temperature. Zwitterionic substances that lower the bilayer-to-hexagonal-phase transition temperature often cause an increase in the activity of protein kinase C (PKC). Indeed, the 18:1/22:6 PE caused an increase in the rate of histone phosphorylation by PKC which was greater than that caused by other, less unsaturated, PEs. The 18:1/22:6 phosphatidylcholine had no effect on this enzyme. The stimulation of the activity of PKC by the 18:1/22:6 PE is a consequence of this lipid's increasing the partitioning of PKC to the membrane.

Involvement of the double-stranded-RNA-dependent kinase PKR in interferon expression and interferon-mediated antiviral activity.

The signaling mechanisms responsible for the induced expression of interferon (IFN) genes by viral infection or double-stranded RNA (dsRNA) are not well understood. Here we investigate the role of the interferon-induced dsRNA-dependent protein kinase PKR in the regulation of IFN induction. Biological activities attributed to PKR include regulating protein synthesis, mediating IFN actions, and functioning as a possible tumor suppressor. Since binding of dsRNA is required for its activation, PKR has been considered as a candidate signal transducer for regulating IFN expression. To examine this role of PKR, loss-of-function phenotypes in stable transformants of promonocytic U-937 cells were achieved by two different strategies, overexpression of an antisense PKR transcript or a dominant negative PKR mutant gene. Both types of PKR-deficient cells were more permissive for viral replication than the control U-937 cells. As the result of PKR loss, they also showed impaired induction of IFN-alpha and IFN-beta genes in response to several inducers--specifically, encephalomyocarditis virus, lipopolysaccharide, and phorbol 12-myristate 13-acetate. Interestingly, while IFN-alpha induction by dsRNA was impaired in PKR-deficient cells, IFN-beta induction remained intact. Loss of PKR function also resulted in decreased antiviral activity as elicited by IFN-alpha and, to a greater extent, by IFN-gamma. These results implicate PKR in the regulation of several antiviral activities.

Basal expression of the cystic fibrosis transmembrane conductance regulator gene is dependent on protein kinase A activity.

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel that becomes activated after phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrate that PKA also plays a crucial role in maintaining basal expression of the CFTR gene in the human colon carcinoma cell line T84. Inhibition of PKA activity by expression of a dominant-negative regulatory subunit or treatment with the PKA-selective inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) caused a complete suppression of CFTR gene expression without affecting other constitutively active genes. Basal expression of a 2.2-kb region of the CFTR promoter linked to a luciferase reporter gene (CFTR-luc) exhibited the same dependence on PKA. The ability of cAMP to induce CFTR over basal levels is cell-type specific. In T84 cells, both the endogenous CFTR gene and CFTR-luc exhibited only a modest inducibility (approximately 2-fold), whereas in the human choriocarcinoma cell line JEG-3, CFTR-luc could be induced at least 4-fold. A variant cAMP-response element is present at position -48 to -41 in the CFTR promoter, and mutation of this sequence blocks basal expression. We conclude that cAMP, acting through PKA, is an essential regulator of basal CFTR gene expression and may mediate an induction of CFTR in responsive cell types.

Constitutive glycogen synthase kinase-3α/β activity protects against chronic β-adrenergic remodelling of the heart

Aims - Glycogen synthase kinase 3 (GSK-3) signalling is implicated in the growth of the heart during development and in response to stress. However, its precise role remains unclear. We set out to characterize developmental growth and response to chronic isoproterenol (ISO) stress in knockin (KI) mice lacking the critical N-terminal serines, 21 of GSK-3 and 9 of GSK-3 respectively, required for inactivation by upstream kinases. Methods and results - Between 5 and 15 weeks, KI mice grew more rapidly, but normalized heart weight and contractile performance were similar to wild-type (WT) mice. Isolated hearts of both genotypes responded comparably to acute ISO infusion with increases in heart rate and contractility. In WT mice, chronic subcutaneous ISO infusion over 14 days resulted in cardiac hypertrophy, interstitial fibrosis, and impaired contractility, accompanied by foetal gene reactivation. These effects were all significantly attenuated in KI mice. Indeed, ISO-treated KI hearts demonstrated reversible physiological remodelling traits with increased stroke volume and a preserved contractile response to acute adrenergic stimulation. Furthermore, simultaneous pharmacological inhibition of GSK-3 in KI mice treated with chronic subcutaneous ISO recapitulated the adverse remodelling phenotype seen in WT hearts. Conclusion - Expression of inactivation-resistant GSK-3/does not affect eutrophic myocardial growth but protects against pathological hypertrophy induced by chronic adrenergic stimulation, maintaining cardiac function and attenuating interstitial fibrosis. Accordingly, strategies to prevent phosphorylation of Ser-21/9, and consequent inactivation of GSK-3/, may enable a sustained cardiac response to chronic-agonist stimulation while preventing pathological remodelling. © 2010 The Author.

Cyclin-dependent kinase 9 activity regulates neutrophil spontaneous apoptosis

Neutrophils are the most abundant leukocyte and play a central role in the immune defense against rapidly dividing bacteria. However, they are also the shortest lived cell in the blood with a lifespan in the circulation of 5.4 days. The mechanisms underlying their short lifespan and spontaneous entry into apoptosis are poorly understood. Recently, the broad range cyclin-dependent kinase (CDK) inhibitor R-roscovitine was shown to increase neutrophil apoptosis, implicating CDKs in the regulation of neutrophil lifespan. To determine which CDKs were involved in regulating neutrophil lifespan we first examined CDK expression in human neutrophils and found that only three CDKs: CDK5, CDK7 and CDK9 were expressed in these cells. The use of CDK inhibitors with differing selectivity towards the various CDKs suggested that CDK9 activity regulates neutrophil lifespan. Furthermore CDK9 activity and the expression of its activating partner cyclin T1 both declined as neutrophils aged and entered apoptosis spontaneously. CDK9 is a component of the P-TEFb complex involved in transcriptional regulation and its inhibition will preferentially affect proteins with short half-lives. Treatment of neutrophils with flavopiridol, a potent CDK9 inhibitor, increased apoptosis and caused a rapid decline in the level of the anti-apoptotic protein Mcl-1, whilst Bcl2A was unaffected. We propose that CDK9 activity is a key regulator of neutrophil lifespan, preventing apoptosis by maintaining levels of short lived anti-apoptotic proteins such as Mcl-1. Furthermore, as inappropriate inhibition of neutrophil apoptosis contributes to chronic inflammatory diseases such as Rheumatoid Arthritis, CDK9 represents a novel therapeutic target in such diseases.

Functional analysis of the 5′ flanking region of the human G6PC3 gene: regulation of promoter activity by glucose, pyruvate, AMP kinase and the pentose phosphate pathway

G6PC3 is a widely expressed isoform of glucose-6-phosphatase, found in many foetal and adult tissues. Mutations in this gene cause developmental abnormalities and severe neutropenia due to abolition of glucose recycling between the cytoplasm and endoplasmic reticulum. Low G6PC3 expression as a result of promoter polymorphisms or dysregulation could produce similar outcomes. Here we investigated the regulation of human G6PC3 promoter activity. HeLa and H4IIE cells were transiently transfected with G6PC3 promoter coupled to the firefly luciferase gene, and promoter activity was measured by dual luciferase assay. Activity was highest in a 453 bp segment of the G6PC3 promoter, from − 455 to − 3 relative to the transcriptional start site. This promoter was unresponsive to glucostatic hormones. Its activity increased significantly between 1 and 5.5 mM glucose, and was not elevated further by glucose concentrations up to 25 mM. Pyruvate increased its activity, but β-hydroxybutyrate and sodium acetate did not. Promoter activity was reduced by inhibitors of hexokinase, glyceraldehyde phosphate dehydrogenase and the oxidative branch of the pentose phosphate pathway, but not by a transketolase inhibitor. Deletion of two adjacent Enhancer-boxes (− 274 to − 279 and − 299 to − 304) reduced promoter activity and abolished the glucose effect, suggesting they could function as a glucose response element. Deletion of an additional downstream 140 bp (− 140 to − 306) restored activity, but not the glucose response, suggesting the presence of repressor elements in this region. 5-Aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR) reduced promoter activity, showing dependence on AMP-kinase. Regulation of the G6PC3 promoter is thus radically different to that of the hepatic isoform, G6PC. It is sensitive to carbohydrate, but not to fatty acid metabolites, and at much lower physiological concentrations. Based on these findings, we speculate that reduced G6PC3 expression could occur during hypoglycemic episodes in vivo, which are common in utero and in the postnatal period. If such episodes lower G6PC3 expression they could place the foetus or infant at risk of impaired immune function and development, and this possibility requires further examination both in vitro and in vivo.

Neuromuscular Activity Of Bothrops Fonsecai Snake Venom In Vertebrate Preparations.

The neuromuscular activity of venom from Bothrops fonsecai, a lancehead endemic to southeastern Brazil, was investigated. Chick biventer cervicis (CBC) and mouse phrenic nerve-diaphragm (PND) preparations were used for myographic recordings and mouse diaphragm muscle was used for membrane resting potential (RP) and miniature end-plate potential (MEPP) recordings. Creatine kinase release and muscle damage were also assessed. In CBC, venom (40, 80 and 160μg/ml) produced concentration- and time-dependent neuromuscular blockade (50% blockade in 85±9 min and 73±8 min with 80 and 160μg/ml, respectively) and attenuated the contractures to 110μM ACh (78-100% inhibition) and 40mM KCl (45-90% inhibition). The venom-induced decrease in twitch-tension in curarized, directly-stimulated preparations was similar to that in indirectly stimulated preparations. Venom (100 and 200μg/ml) also caused blockade in PND preparations (50% blockade in 94±13 min and 49±8 min with 100 and 200μg/ml, respectively) but did not alter the RP or MEPP amplitude. In CBC, venom caused creatine kinase release and myonecrosis. The venom-induced decrease in twitch-tension and in the contractures to ACh and K(+) were abolished by preincubating venom with commercial antivenom. These findings indicate that Bothrops fonsecai venom interferes with neuromuscular transmission essentially through postsynaptic muscle damage that affects responses to ACh and KCl. These actions are effectively prevented by commercial antivenom.

Kinase Inhibitor Profile For Human Nek1, Nek6, And Nek7 And Analysis Of The Structural Basis For Inhibitor Specificity.

Human Neks are a conserved protein kinase family related to cell cycle progression and cell division and are considered potential drug targets for the treatment of cancer and other pathologies. We screened the activation loop mutant kinases hNek1 and hNek2, wild-type hNek7, and five hNek6 variants in different activation/phosphorylation statesand compared them against 85 compounds using thermal shift denaturation. We identified three compounds with significant Tm shifts: JNK Inhibitor II for hNek1(Δ262-1258)-(T162A), Isogranulatimide for hNek6(S206A), andGSK-3 Inhibitor XIII for hNek7wt. Each one of these compounds was also validated by reducing the kinases activity by at least 25%. The binding sites for these compounds were identified by in silico docking at the ATP-binding site of the respective hNeks. Potential inhibitors were first screened by thermal shift assays, had their efficiency tested by a kinase assay, and were finally analyzed by molecular docking. Our findings corroborate the idea of ATP-competitive inhibition for hNek1 and hNek6 and suggest a novel non-competitive inhibition for hNek7 in regard to GSK-3 Inhibitor XIII. Our results demonstrate that our approach is useful for finding promising general and specific hNekscandidate inhibitors, which may also function as scaffolds to design more potent and selective inhibitors.

Antitumor Activity Of Irradiated Riboflavin On Human Renal Carcinoma Cell Line 786-o.

Riboflavin (vitamin B2) is a precursor for coenzymes involved in energy production, biosynthesis, detoxification, and electron scavenging. Previously, we demonstrated that irradiated riboflavin (IR) has potential antitumoral effects against human leukemia cells (HL60), human prostate cancer cells (PC3), and mouse melanoma cells (B16F10) through a common mechanism that leads to apoptosis. Hence, we here investigated the effect of IR on 786-O cells, a known model cell line for clear cell renal cell carcinoma (CCRCC), which is characterized by high-risk metastasis and chemotherapy resistance. IR also induced cell death in 786-O cells by apoptosis, which was not prevented by antioxidant agents. IR treatment was characterized by downregulation of Fas ligand (TNF superfamily, member 6)/Fas (TNF receptor superfamily member 6) (FasL/Fas) and tumor necrosis factor receptor superfamily, member 1a (TNFR1)/TNFRSF1A-associated via death domain (TRADD)/TNF receptor-associated factor 2 (TRAF) signaling pathways (the extrinsic apoptosis pathway), while the intrinsic apoptotic pathway was upregulated, as observed by an elevated Bcl-2 associated x protein/B-cell CLL/lymphoma 2 (Bax/Bcl-2) ratio, reduced cellular inhibitor of apoptosis 1 (c-IAP1) expression, and increased expression of apoptosis-inducing factor (AIF). The observed cell death was caspase-dependent as proven by caspase 3 activation and poly(ADP-ribose) polymerase-1 (PARP) cleavage. IR-induced cell death was also associated with downregulation of v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homologue (avian)/protein serine/threonine kinase B/extracellular signal-regulated protein kinase 1/2 (Src/AKT/ERK1/2) pathway and activation of p38 MAP kinase (p38) and Jun-amino-terminal kinase (JNK). Interestingly, IR treatment leads to inhibition of matrix metalloproteinase-2 (MMP-2) activity and reduced expression of renal cancer aggressiveness markers caveolin-1, low molecular weight phosphotyrosine protein phosphatase (LMWPTP), and kinase insert domain receptor (a type III receptor tyrosine kinase) (VEGFR-2). Together, these results show the potential of IR for treating cancer.

Selective Decrease of Components of the Creatine Kinase System and ATP Synthase Complex in Chronic Chagas Disease Cardiomyopathy

Background: Chronic Chagas disease cardiomyopathy (CCC) is an inflammatory dilated cardiomyopathy with a worse prognosis than other cardiomyopathies. CCC occurs in 30 % of individuals infected with Trypanosoma cruzi, endemic in Latin America. Heart failure is associated with impaired energy metabolism, which may be correlated to contractile dysfunction. We thus analyzed the myocardial gene and protein expression, as well as activity, of key mitochondrial enzymes related to ATP production, in myocardial samples of end-stage CCC, idiopathic dilated (IDC) and ischemic (IC) cardiomyopathies. Methodology/Principal Findings: Myocardium homogenates from CCC (N = 5), IC (N = 5) and IDC (N = 5) patients, as well as from heart donors (N = 5) were analyzed for protein and mRNA expression of mitochondrial creatine kinase (CKMit) and muscular creatine kinase (CKM) and ATP synthase subunits aplha and beta by immunoblotting and by real-time RT-PCR. Total myocardial CK activity was also assessed. Protein levels of CKM and CK activity were reduced in all three cardiomyopathy groups. However, total CK activity, as well as ATP synthase alpha chain protein levels, were significantly lower in CCC samples than IC and IDC samples. CCC myocardium displayed selective reduction of protein levels and activity of enzymes crucial for maintaining cytoplasmic ATP levels. Conclusions/Significance: The selective impairment of the CK system may be associated to the loss of inotropic reserve observed in CCC. Reduction of ATP synthase alpha levels is consistent with a decrease in myocardial ATP generation through oxidative phosphorylation. Together, these results suggest that the energetic deficit is more intense in the myocardium of CCC patients than in the other tested dilated cardiomyopathies.