Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of protein kinase A (PKA) mediated phosphorylation on thin filament and cross-bridge function is not fully understood. To delineate the effects of troponin I (TnI) phosphorylation by PKA on contractile protein performance, reconstituted thin filaments were treated with PKA. With the use of the in vitro motility assay, PKA treated thin filament function was assessed relative to non-phosphorylated thin filaments in a calcium-regulated system. At maximal calcium activation, unloaded shortening velocity and force did not differ between the groups. However, at submaximal activation, an increase in calcium sensitivity of the thin filament was observed for velocity but a decrease in calcium sensitivity was observed for force. Activation of the thin filament by myosin strong-binding did not elicit a calcium-independent effect. The rightward shift in calcium sensitivity for force and the leftward shift in calcium sensitivity for velocity indicate that PKA phosphorylation of TnI directly modulates the kinetics of the myosin cross-bridge. In addition, the altered velocity dependence on thin filament length implicates reduced myosin cross-bridge binding with PKA treatment. These data highlight the importance of TnI serine 23 and 24 phosphorylation in the modulation of cardiac function.
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PMID:Protein kinase A mediated modulation of acto-myosin kinetics. 1562 28

We investigated the effect of protein kinase A (PKA) on passive force in skinned cardiac tissues that express different isoforms of titin, i.e., stiff (N2B) and more compliant (N2BA) titins, at different levels. We used rat ventricular (RV), bovine left ventricular (BLV), and bovine left atrial (BLA) muscles (passive force: RV > BLV > BLA, with the ratio of N2B to N2BA titin, approximately 90:10, approximately 40:60, and approximately 10:90%, respectively) and found that N2B and N2BA isoforms can both be phosphorylated by PKA. Under the relaxed condition, sarcomere length was increased and then held constant for 30 min and the peak passive force, stress-relaxation, and steady-state passive force were determined. Following PKA treatment, passive force was significantly decreased in all muscle types with the effect greatest in RV, lowest in BLA, and intermediate in BLV. Fitting the stress-relaxation data to the sum of three exponential decay functions revealed that PKA blunts the magnitude of stress-relaxation and accelerates its time constants. To investigate whether or not PKA-induced decreases in passive force result from possible alteration of titin-thin filament interaction (e.g., via troponin I phosphorylation), we conducted the same experiments using RV preparations that had been treated with gelsolin to extract thin filaments. PKA decreased passive force in gelsolin-treated RV preparations with a magnitude similar to that observed in control preparations. PKA was also found to decrease restoring force in skinned ventricular myocytes of the rat that had been shortened to below the slack length. Finally, we investigated the effect of the beta-adrenergic receptor agonist isoprenaline on diastolic force in intact rat ventricular trabeculae. We found that isoprenaline phosphorylated titin and that it reduced diastolic force to a degree similar to that found in skinned RV preparations. Taken together, these results suggest that during beta-adrenergic stimulation, PKA increases ventricular compliance in a titin isoform-dependent manner.
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PMID:Phosphorylation of titin modulates passive stiffness of cardiac muscle in a titin isoform-dependent manner. 1573 46

Here we demonstrate that type I protein kinase A is redoxactive, forming an interprotein disulfide bond between its two regulatory RI subunits in response to cellular hydrogen peroxide. This oxidative disulfide formation causes a subcellular translocation and activation of the kinase, resulting in phosphorylation of established substrate proteins. The translocation is mediated at least in part by the oxidized form of the kinase having an enhanced affinity for alpha-myosin heavy chain, which serves as a protein kinase A (PKA) anchor protein and localizes the PKA to its myofilament substrates troponin I and myosin binding protein C. The functional consequence of these events in cardiac myocytes is that hydrogen peroxide increases contractility independently of beta-adrenergic stimulation and elevations of cAMP. The oxidant-induced phosphorylation of substrate proteins and increased contractility is blocked by the kinase inhibitor H89, indicating that these events involve PKA activation. In essence, type I PKA contains protein thiols that operate as redox sensors, and their oxidation by hydrogen peroxide directly activates the kinase.
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PMID:Oxidant-induced activation of type I protein kinase A is mediated by RI subunit interprotein disulfide bond formation. 1675 66

Contraction and relaxation of cardiac muscle are regulated by the inhibitory and regulatory regions of troponin I (cTnI). Our previous FRET studies showed that the inhibitory region of cTnI in isolated troponin experiences a structural transition from a beta-turn/coil motif to an extended conformation upon Ca(2+) activation. During the relaxation process, the kinetics of the reversal of this conformation is coupled to the closing of the Ca(2+)-induced open conformation of the N-domain of troponin C (cTnC) and an interaction between cTnC and cTnI in their interface. We have since extended the structural kinetic study of the inhibitory region to fully regulated thin filament. Single-tryptophan and single-cysteine mutant cTnI(L129W/S151C) was labeled with 1,5-IAEDANS at Cys151, and the tryptophan-AEDANS pair served as a donor-acceptor pair. Labeled cTnI mutant was used to prepare regulated thin filaments. Ca(2+)-induced conformational changes in the segment of Trp129-Cys151 of cTnI were monitored by FRET sensitized acceptor (AEDANS) emission in Ca(2+) titration and stopped-flow measurements. Control experiments suggested energy transfer from endogenous tryptophan residues of actin and myosin S1 to AEDANS attached to Cys151 of cTnI was very small and Ca(2+) independent. The present results show that the rate of Ca(2+)-induced structural transition and Ca(2+) sensitivity of the inhibitory region of cTnI were modified by (1) thin filament formation, (2) the presence of strongly bound S1, and (3) PKA phosphorylation of the N-terminus of cTnI. Ca(2+) sensitivity was not significantly changed by the presence of cTm and actin. However, the cTn-cTm interaction decreased the cooperativity and kinetics of the structural transition within cTnI, while actin filaments elicited opposite effects. The strongly bound S1 significantly increased the Ca(2+) sensitivity and slowed down the kinetics of structural transition. In contrast, PKA phosphorylation of cTnI decreased the Ca(2+) sensitivity and accelerated the structural transition rate of the inhibitory region of cTnI on thin filaments. These results support the idea of a feedback mechanism by strong cross-bridge interaction with actin and provide insights on the molecular basis for the fine tuning of cardiac function by beta-adrenergic stimulation.
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PMID:Structural transition of the inhibitory region of troponin I within the regulated cardiac thin filament. 1696 89

Beta-adrenergic agonists induce protein kinase A (PKA) phosphorylation of the cardiac myofilament proteins myosin binding protein C (cMyBP-C) and troponin I (cTnI), resulting in enhanced systolic function, but the relative contributions of cMyBP-C and cTnI to augmented contractility are not known. To investigate possible roles of cMyBP-C in this response, we examined the effects of PKA treatment on the rate of force redevelopment and the stretch activation response in skinned ventricular myocardium from both wild-type (WT) and cMyBP-C null (cMyBP-C(-/-)) myocardium. In WT myocardium, PKA treatment accelerated the rate of force redevelopment and the stretch activation response, resulting in a shorter time to the peak of delayed force development when the muscle was stretched to a new isometric length. Ablation of cMyBP-C accelerated the rate of force redevelopment and stretch activation response to a degree similar to that observed in PKA treatment of WT myocardium; however, PKA treatment had no effect on the rate of force development and the stretch activation response in null myocardium. These results indicate that ablation of cMyBP-C and PKA treatment of WT myocardium have similar effects on cross-bridge cycling kinetics and suggest that PKA phosphorylation of cMyBP-C accelerates the rate of force generation and thereby contributes to the accelerated twitch kinetics observed in living myocardium during beta-adrenergic stimulation.
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PMID:Protein kinase A-mediated acceleration of the stretch activation response in murine skinned myocardium is eliminated by ablation of cMyBP-C. 1703 48

Impaired function of the phospholamban (PLB)-regulated sarcoplasmic reticulum Ca(2+) pump (SERCA2a) contributes to cardiac dysfunction in heart failure (HF). PLB downregulation may increase SERCA2a activity and improve cardiac function. Small interfering (si)RNAs mediate efficient gene silencing by RNA interference (RNAi). However, their use for in vivo gene therapy is limited by siRNA instability in plasma and tissues, and by low siRNA transfer rates into target cells. To address these problems, we developed an adenoviral vector (AdV) transcribing short hairpin (sh)RNAs against rat PLB and evaluated its potential to silence the PLB gene and to modulate SERCA2a-mediated Ca(2+) sequestration in primary neonatal rat cardiomyocytes (PNCMs). Over a period of 13 days, vector transduction resulted in stable > 99.9% ablation of PLB-mRNA at a multiplicity of infection of 100. PLB protein gradually decreased until day 7 (7+/-2% left), whereas SERCA, Na(+)/Ca(2+) exchanger (NCX1), calsequestrin and troponin I protein remained unchanged. PLB silencing was associated with a marked increase in ATP-dependent oxalate-supported Ca(2+) uptake at 0.34 microM of free Ca(2+), and rapid loss of responsiveness to protein kinase A-dependent stimulation of Ca(2+) uptake was maintained until day 7. In summary, these results indicate that AdV-derived PLB-shRNA mediates highly efficient, specific and stable PLB gene silencing and modulation of active Ca(2+) sequestration in PNCMs. The availability of the new vector now enables employment of RNAi for the treatment of HF in vivo.
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PMID:Highly efficient and specific modulation of cardiac calcium homeostasis by adenovector-derived short hairpin RNA targeting phospholamban. 1702 1

We made quantitative measurements of phosphorylation in troponin isolated from 6 non-failing donor hearts and 6 explanted hearts with end-stage heart failure in SDS-PAGE gels using Pro-Q Diamond phosphoprotein stain. The troponin T phosphorylation level was the same in troponin from failing and non-failing heart (3.1 mol Pi/mol). However, troponin I phosphorylation was significantly lower in failing (0.37+/-0.18 mol Pi/mol) compared with non-failing heart troponin (2.25+/-0.36 mol Pi/mol). Levels of troponin I PKA-dependent phosphorylation, measured with a phosphoserine 23/24-specific antibody, were also significantly lower in failing heart troponin (0.19+/-0.06 mol Pi/mol) compared to non-failing troponin (1.14+/-0.09 mol Pi/mol). We calculate that there is phosphorylation in addition to serine 23/24 of 1.11+/-0.34 mol Pi/mol in non-failing reduced to 0.18+/-0.17 mol Pi/mol in failing heart troponin, attributed to phosphorylation on the PKC sites. To test for the functional role of troponin I phosphorylation, the native troponin I from either non-failing or failing heart troponin was exchanged for a recombinant (unphosphorylated) human cardiac troponin I. Thin filament Ca(2+)-regulatory function was studied with the quantitative in vitro motility assay: thin filaments containing the replaced troponin I resulted in a failing phenotype of a 17-26% reduced sliding speed and an increased Ca(2+)-sensitivity relative to non-failing troponin (EC(50) TnI-exchanged/non-failing=0.57, p<0.001). When exchanged with troponin I phosphorylated with PKA motility parameters reverted to a pattern indistinguishable from non-failing troponin (p=0.35-0.75). We suggest that changes in troponin function can account for the contractile abnormality in failing heart muscle and that the functional changes in troponin are due to reduced phosphorylation of troponin I at the PKA sites.
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PMID:Troponin phosphorylation and regulatory function in human heart muscle: dephosphorylation of Ser23/24 on troponin I could account for the contractile defect in end-stage heart failure. 1708 61

Activation of either coexisting beta1- or beta2 -adrenoceptors with noradrenaline or adrenaline, respectively, causes maximum increases of contractility of human atrial myocardium. Previous biochemical work with the beta2 -selective agonist zinterol is consistent with activation of the cascade beta2 -adrenoceptors-->Gsalpha-protein-->adenylyl cyclase-->cAMP-->protein kinase (PKA)-->phosphorylation of phospholamban, troponin I, and C-protein-->hastened relaxation of human atria from nonfailing hearts. However, in feline and rodent myocardium, catecholamines and zinterol usually do not hasten relaxation through activation of beta2 -adrenoceptors, presumably because of coupling of the receptors to Gi protein. It is unknown whether the endogenously occurring beta2 -adrenoceptor agonist adrenaline acts through the above cascade in human atrium and whether its mode of action could be changed in heart failure. We assessed the effects of (-)-adrenaline, mediated through beta2 -adrenoceptors (in the presence of CGP 20712A 300 nM to block beta1 -adrenoceptors), on contractility and relaxation of right atrial trabecula obtained from nonfailing and failing human hearts. Cyclic AMP levels were measured as well as phosphorylation of phospholamban, troponin I, and protein C with Western blots and the back-phosphorylation procedure. For comparison, beta1 -adrenoceptor-mediated effects of (-)-noradrenaline were investigated in the presence of ICI 118,551 (50 nM to block beta2 -adrenoceptors). The positive inotropic effects of both (-)-noradrenaline and (-)-adrenaline were accompanied by reductions in time to peak force and time to reach 50% relaxation. (-)-Adrenaline caused similar positive inotropic and lusitropic effects in atrial trabeculae from failing hearts. However, the inotropic potency, but not the lusitropic potency, of (-)-noradrenaline was reduced fourfold in atrial trabeculae from heart failure patients. Both (-)-adrenaline and (-)-noradrenaline enhanced cyclic AMP levels and produced phosphorylation of phospholamban, troponin I, and C-protein to a similar extent in atrial trabeculae from nonfailing hearts. The hastening of relaxation caused by (-)-adrenaline together with the PKA-catalyzed phosphorylation of the three proteins involved in relaxation, indicate coupling of beta2 -adrenoceptors to Gs protein. The phosphorylation of phospholamban at serine16 and threonine17 evoked by (-)-adrenaline through beta2 -adrenoceptors and by (-)-noradrenaline through beta1 -adrenoceptors was not different in atria from nonfailing and failing hearts. Activation of beta2 -adrenoceptors caused an increase in phosphorylase a activity in atrium from failing hearts further emphasizing the presence of the beta2 -adrenoceptor-Gsalpha-protein pathway in human heart. The positive inotropic and lusitropic potencies of (-)-adrenaline were conserved across Arg16Gly- and Gln27Glu-beta2 -adrenoceptor polymorphisms in the right atrium from patients undergoing coronary artery bypass surgery, chronically treated with beta1 -selective blockers. The persistent relaxant and biochemical effects of (-)-adrenaline through beta2 -adrenoceptors and of (-)-noradrenaline through beta1 -adrenoceptors in heart failure are inconsistent with an important role of coupling of beta2 -adrenoceptors with Gialpha-protein in human atrial myocardium.
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PMID:(-)-Adrenaline elicits positive inotropic, lusitropic, and biochemical effects through beta2 -adrenoceptors in human atrial myocardium from nonfailing and failing hearts, consistent with Gs coupling but not with Gi coupling. 1729 24

It is not well-known yet how granulocyte colony-stimulating factor (G-CSF) affects nonischemic cardiomyopathy, though its beneficial effects on acute myocardial infarction are well-established. We hypothesize that G-CSF beneficially might affect nonischemic cardiomyopathy through the direct cardioprotective effects. Here, we show that a single injection of doxorubicin (DOX, 15 mg/kg) induced left ventricular dilatation and dysfunction in mice within 2 weeks, and that these effects were significantly attenuated by human recombinant G-CSF (100 microg/kg/day for 5 days). G-CSF also protected hearts against DOX-induced cardiomyocyte atrophy/degeneration, fibrosis, inflammatory cell infiltration and down regulation of GATA-4 and sarcomeric proteins, myosin heavy chain, troponin I and desmin, both in vivo and in vitro. Cardiac cyclooxygenase-2 was upregulated and G-CSF receptor was downregulated in DOX-induced cardiomyopathy, but both of those effects were largely reversed by G-CSF. No DOX-induced apoptotic effects were seen, nor were there any changes in tumor necrosis factor-alpha or transforming growth factor-beta1 levels. Among downstream mediators of G-CSF receptor signaling, DOX-induced cardiomyopathy involved inactivation of extracellular signal-regulated protein kinase (ERK); the ERK inactivation was reversed by G-CSF. Inhibition of ERK activation, but not cyclooxygenase-2 inhibition, completely abolished beneficial effect of G-CSF on cardiac function. G-CSF did not promote differentiation of bone marrow cells into cardiomyocytes according to the experiment using green fluorescent protein-chimeric mice, and inhibition of CXCR4+ cell homing using AMD3100 did not diminish the effect of G-CSF. Finally, G-CSF was also effective when administered after cardiomyopathy was established. In conclusion, these findings imply the therapeutic usefulness of G-CSF mainly through restoring ERK activation against DOX-induced nonischemic cardiomyopathy.
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PMID:Granulocyte colony-stimulating factor improves left ventricular function of doxorubicin-induced cardiomyopathy. 1733 14

The phosphorylation status of myofibrillar proteins influences the Ca(2+) responsiveness of the myofilaments,but the contribution of and the interaction between the individual components is poorly characterized. Therefore, in Langendorff perfused rat hearts (n=30), the phosphorylation levels of cardiac myosin binding protein-C (cMyBP-C), troponin I and T (cTnI, cTnT) and myosin light chain 1 and 2 (MLC-1, MLC-2) were determined by 1- and 2-dimensional gel electrophoresis. Isometric force development, its Ca(2+)-sensitivity, the rate of tension redevelopment (k(tr)) and passive force (F(pas)) were studied at optimal sarcomere length (2.2 microm) in mechanically isolated,permeabilized cardiomyocytes at 15 degrees C. Protein phosphorylation was varied by: 1) blocking spontaneous cardiac activity by lidocaine (0.35 mM; Quiescence); 2) electrical stimulation of the hearts at 5 Hz (Contraction) and 3. treatment of contracting hearts with Isoprenaline (1 microM). MLC-2 phosphorylation was increased in the Contraction group almost 2-fold, relative to the Quiescence group, whereas cMyBP-C and cTnI phosphorylation remained the same. Isoprenaline resulted in 3.7-fold increases in both cMyBP-C and cTnI phosphorylation, but did not result in a further increase in MLC-2 phosphorylation. No significant differences were found in maximum force and k(tr) between groups, both before and after protein kinase A (PKA) treatment. Ca(2+)-sensitivity in the Contraction and Isoprenaline groups was significantly reduced in comparison to the Quiescence group. These differences were largely abolished by PKA and F(pas) was reduced. These results highlight the impact of PKA-dependent phosphorylation on Ca(2+)-sensitivity and provide evidence for an interaction between the effects of TnI and MLC-2 phosphorylation.
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PMID:Effects of contractile protein phosphorylation on force development in permeabilized rat cardiac myocytes. 1754 28


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