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)

We tested the hypothesis that altered phosphorylation of myofibrillar proteins is involved in post-ischemic myocardial stunning. Myofibrillar proteins were isolated from Langendorff perfused control rabbit hearts, hearts submitted to 15 min normothermic ischemia and hearts submitted to 15 min ischemia followed by 10 min of reperfusion (stunned hearts). The in vivo level of phosphorylation of specific contractile proteins by protein kinases A and C was indirectly detected by the amount of 32P incorporated in vitro in the presence of these protein kinases and saturating concentration of [gamma-32P]-ATP (back-phosphorylation method). In control experiments the back-phosphorylation technique was able to detect PKA- or PKC-induced protein phosphorylation in hearts treated with isoproterenol and phorbol ester, respectively. In stunned hearts, contractile function was significantly suppressed compared to the period before ischemia. We found no difference in myofibrillar protein profile (on densitometry of the Coomassie-stained gels after SDS-PAGE) and in PKA mediated 32P incorporation when comparing control, ischemic and stunned myocardium. Three different PKCs were used for phosphorylation: commercial purified rat brain PKC, partially purified rat brain PKC or rabbit partially purified cardiac PKC. Cardiac PKC mainly phosphorylated troponin I, whereas brain PKC phosphorylated both troponin T and troponin I. No significant difference in 32P incorporation mediated by either brain or cardiac PKC was found between control, ischemic and ischemic/reperfused myofibrils. These data indicate that myocardial stunning does not cause changes in PKC- or PKA-mediated Pi incorporation into myofibrillar proteins detectable by the back-phosphorylation method.
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PMID:Phosphorylation by protein kinases A and C of myofibrillar proteins in rabbit stunned and non-stunned myocardium. 944 26

We have measured myocyte cell shortening, troponin-I (Tn-I) phosphorylation, Ca2+ dependence of actomyosin adenosinetriphosphatase (ATPase) activity, adenosine 3',5'-cyclic monophosphate (cAMP) levels, and myofibrillar isoform expression in the spontaneously hypertensive rat (SHR) during decompensated cardiac hypertrophy (76 wk old) and in age-matched Wistar-Kyoto rat (WKY) controls. The decreased inotropic response to beta-adrenergic stimulation previously observed in myocytes from 26-wk-old SHR was further reduced at 76 wk of age. In response to beta-adrenergic stimulation, Tn-I phosphorylation was greater in the 76-wk-old SHR than in the WKY, although cAMP-dependent protein kinase A (PKA)-dependent Tn-I phosphorylation in the SHR did not increase with progression from compensated (26 wk) to decompensated (76 wk) hypertrophy. We also observed a dissociation between the increased PKA-dependent Tn-I phosphorylation and decreased cAMP levels in the 76-wk-old SHR versus WKY during beta-adrenergic stimulation. Baseline Tn-I phosphorylation was significantly reduced in 76-wk-old SHR versus WKY and was associated with decreased basal cAMP levels and increased Ca2+ sensitivity of actomyosin ATPase activity. The change in myofilament Ca2+ sensitivity during beta-adrenergic stimulation in the 76-wk-old SHR (0.65 pCa units) was over twofold greater than in the 76-wk-old WKY (0.30 pCa units). We also determined whether embryonic troponin T isoforms were reexpressed in decompensated hypertrophy and observed significant reexpression of the embryonic cardiac troponin T isoforms in the 76-wk-old SHR. The significant decrease in Ca2+ sensitivity with beta-adrenergic stimulation in 76-wk-old SHR may contribute to the severely impaired inotropic response during decompensated hypertrophy in the SHR.
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PMID:Troponin I phosphorylation and myofilament calcium sensitivity during decompensated cardiac hypertrophy. 948 39

The involvement of the double-stranded RNA-activated protein kinase PKR in the regulation of the myogenic process was investigated. For this purpose, the murine myogenic cell line C2C12 was used. The cells were first cultivated in either growth medium or differentiation medium (DM), and the activation of PKR during differentiation was determined by monitoring its enzymatic activity and by immunoblot analysis. A significant increase in both parameters was detected already at 24 h in DM, whereas in cells grown in growth medium, the increase was evident only after 96 h, when spontaneous differentiation was observed in highly crowded cultures. Consequently, we established the direct effect of PKR activation on the myogenic process. C2C12 cells were transfected with an expression vector harboring a cDNA molecule encoding human PKR fused to the inducible metallothionein promoter. One of the clones (clone 8) expressing high levels of PKR was selected and further analyzed. In the presence of ZnCl2, which activates the promoter, the rate of cell growth of the transfected cells was clearly reduced compared to that of wild-type C2C12 cells transfected with only the neomycin-resistant gene (C2-NEO). In addition, altered morphology with partial fusion was observed. Biochemically, an increase in creatine kinase activity accompanied by an increased rate of expression of the myogenic protein troponin T and the myogenic transcription factors myoD and myogenin was detected in clone 8 cells exposed to ZnCl2. Most importantly, an induction in the level of cyclin-dependent kinase inhibitor p21WAF1 and an increase in the level of the underphosphorylated active form of the tumor suppressor protein pRb concomitant with the down-regulation of cyclin D1 and c-myc were also evident in the transfected clones. These changes were similar to those observed in normal C2C12 cells cultivated in DM. We conclude that PKR is an important regulatory protein participating in the myogenic process.
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PMID:Involvement of PKR in the regulation of myogenesis. 1009 34

A splice donor site mutation in intron 15 of the cardiac troponin T (TnT) gene has been shown to cause familial hypertrophic cardiomyopathy (HCM). In this study, two truncated human cardiac TnTs expected to be produced by this mutation were expressed in Escherichia coli and partially (50-55%) exchanged into rabbit permeabilized cardiac muscle fibers. The fibers into which a short truncated TnT, which lacked the COOH-terminal 21 amino acids because of the replacement of 28 amino acids with 7 novel residues, had been exchanged generated a Ca(2+)-activated maximum force that was slightly, but statistically significantly, lower than that generated by fibers into which wild-type TnT had been exchanged when troponin I (TnI) was phosphorylated by cAMP-dependent protein kinase. A long truncated TnT simply lacking the COOH-terminal 14 amino acids had no significant effect on the maximum force-generating capability in the fibers with either phosphorylated or dephosphorylated TnI. Both these two truncated TnTs conferred a lower cooperativity and a higher Ca(2+) sensitivity on the Ca(2+)-activated force generation than did wild-type TnT, independent of the phosphorylation of TnI by cAMP-dependent protein kinase. The results demonstrate that the splice donor site mutation in the cardiac TnT gene impairs the regulatory function of the TnT molecule, leading to an increase in the Ca(2+) sensitivity, and a decrease in the cooperativity, of cardiac muscle contraction, which might be involved in the pathogenesis of HCM.
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PMID:Functional changes in troponin T by a splice donor site mutation that causes hypertrophic cardiomyopathy. 1044 98

cGMP-dependent protein kinase (cGK) is a major intracellular receptor of cGMP and is implicated in several signal transduction pathways. To identify proteins that participate in the cGMP/cGK signaling pathway, we employed the yeast two-hybrid system with cGK Ialpha as bait. cDNAs encoding slow skeletal troponin T (skTnT) were isolated from both mouse embryo and human skeletal muscle cDNA libraries. The skTnT protein interacted with cGK Ibeta but not with cGK II nor cAMP-dependent protein kinase. The yeast two-hybrid and in vitro binding assays revealed that the N-terminal region of cGK Ialpha, containing the leucine zipper motif, is sufficient for the association with skTnT. In vivo analysis, mutations in cGK Ialpha, which disrupted the leucine zipper motif, were shown to completely abolish the binding to skTnT. Furthermore, cGK I also interacted with cardiac TnT (cTnT) but not with cardiac troponin I (cTnI). Together with the observations that cTnI is a good substrate for cGK I and is effectively phosphorylated in the presence of cTnT in vitro, these findings suggest that TnT functions as an anchoring protein for cGK I and that cGK I may participate in the regulation of muscle contraction through phosphorylation of TnI.
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PMID:A novel interaction of cGMP-dependent protein kinase I with troponin T. 1060 15

In a tail suspension rat model, we investigated changes in myofilament protein during cardiac adaptation in simulated microgravity. Contractile force and velocity of cardiac muscle were decreased in the tail suspension rats as compared with the control. Ca(2+)-dependent actomyosin ATPase activity was also decreased; however, sensitivity of cardiac muscle to Ca(2+) activation was unchanged. There was no change in expression of myosin heavy chain, tropomyosin, troponin T, or troponin I isoforms in hearts of tail suspension rats. A novel finding is a fragment of cardiac troponin I (cTnI) that had increased amounts in the heart of tail suspension rats. Binding of this cTnI fragment by a monoclonal antibody that specifically recognizes the COOH terminus indicates an intact COOH terminus. NH(2)-terminal sequence analysis of the cTnI fragment revealed truncations primarily of amino acids 1-26 and 1-27 and smaller amounts of 1-30, including Ser(23) and Ser(24), which are substrates of protein kinase A phosphorylation. This cTnI fragment is present in normal cardiac muscle and incorporated into myofibrils, indicating a role in regulating contractility. This proteolytic modification of cTnI up-regulated during simulated microgravity suggests a potential role of the NH(2)-terminal segment of cTnI in functional adaptations of cardiac muscle.
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PMID:A proteolytic NH2-terminal truncation of cardiac troponin I that is up-regulated in simulated microgravity. 1127 23

Contractility of the myocardium is altered in end-stage heart failure. We investigated whether this was related to functional changes in troponin. We isolated troponin from 1 g samples of end-stage failing, non-failing and foetal human heart and studied its regulation of actin-tropomyosin movement over immobilised HMM by in vitro motility assay. At pCa5.4 the sliding velocity of thin filaments reconstituted with non-failing heart troponin was 52+/-4% more than actin-tropomyosin, with failing heart troponin velocity increased by 35+/-2% and with foetal heart troponin velocity increased by 11+/-4%. Thin filaments containing troponin from failing hearts were more Ca(2+)-sensitive than non-failing heart troponin. EC(50) for the fraction of filaments motile and filament velocity decreased 1.76+/-0.20 and 1.89+/-0.62-fold respectively relative to non-failing heart troponin. With foetal heart troponin the EC(50) decreased 2.16+/-0.23 and 3.50+/-1.73-fold for fraction and velocity respectively. Western blots revealed no difference in troponin T or troponin I isoform expression in troponin from failing and non-failing adult hearts but foetal isoforms of troponin I and T were observed in troponin from foetal heart. The level of PKA phosphorylation of troponin from failing and non-failing heart was not significantly different, however, complete non-specific dephosphorylation of troponin abolished most of the difference between failing and non-failing heart troponin. These findings show functional alterations in troponin in failing hearts which could account for the reduced contractile function but there is no change in troponin isoform expression or PKA phosphorylation. Differential phosphorylation by other kinases may account for altered troponin function.
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PMID:In vitro motility analysis of thin filaments from failing and non-failing human heart: troponin from failing human hearts induces slower filament sliding and higher Ca(2+) sensitivity. 1209 6

N-cadherin, a member of the Ca(2+)-dependent cell-cell adhesion molecule family, plays an essential role in skeletal muscle cell differentiation. We show that inhibition of N-cadherin-dependent adhesion impairs the upregulation of the two cyclin-dependent kinase inhibitors p21 and p27, the expression of the muscle-specific genes myogenin and troponin T, and C2C12 myoblast fusion. To determine the nature of N-cadherin-mediated signals involved in myogenesis, we investigated whether N-cadherin-dependent adhesion regulates the activity of Rac1, Cdc42Hs, and RhoA. N-cadherin-dependent adhesion decreases Rac1 and Cdc42Hs activity, and as a consequence, c-jun NH2-terminal kinase (JNK) MAPK activity but not that of the p38 MAPK pathway. On the other hand, N-cadherin-mediated adhesion increases RhoA activity and activates three skeletal muscle-specific promoters. Furthermore, RhoA activity is required for beta-catenin accumulation at cell-cell contact sites. We propose that cell-cell contacts formed via N-cadherin trigger signaling events that promote the commitment to myogenesis through the positive regulation of RhoA and negative regulation of Rac1, Cdc42Hs, and JNK activities.
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PMID:N-cadherin-dependent cell-cell contact regulates Rho GTPases and beta-catenin localization in mouse C2C12 myoblasts. 1221 39

Phosphorylation of myofilament proteins by kinases such as cAMP-dependent protein kinase and protein kinase C has been shown to lead to altered thin-filament protein-protein interactions and modulation of cardiac function in vitro. In the present study, we report that a small GTPase-dependent kinase, p21-activated kinase (PAK), increases the calcium sensitivity of Triton-skinned cardiac muscle fiber bundles. Constitutively active PAK3 caused an average 1.25-fold (25.0+/-6.0%, n=6) increase in force at pCa 5.75, 1.44-fold (44.0+/-7.78%, n=6) at pCa 6.25, and 2.41-fold (141.2+/-23.7%, n=4) at pCa 6.5, representing a change in pCa50 value of approximately 0.25. Constitutively active PAK3 produced no change in force under conditions of relaxation (pCa 8.0) or maximal contraction (pCa 4.5). Furthermore, an inactive, kinase-dead form of PAK3 failed to produce any change in force development at any pCa value. The myofilament proteins phosphorylated by PAK3, at pCa 6.5, are desmin, troponin T, troponin I, and an unidentified 70-kDa protein. Importantly, cardiac troponin I was found to be phosphorylated at serine 149 of human cardiac troponin I, representing a novel phosphorylation site. These findings suggest a novel mechanism of modulating the calcium sensitivity of cardiac muscle contraction.
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PMID:p21-activated kinase increases the calcium sensitivity of rat triton-skinned cardiac muscle fiber bundles via a mechanism potentially involving novel phosphorylation of troponin I. 1224 69

In failing human myocardium changes occur, in particular, in isoform composition and phosphorylation level of the troponin T (TnT) and troponin I (TnI) subunits of the actin filament and the myosin light chains (MLC-1 and -2), but it is unclear to what extent they influence cardiac performance. This overview concentrates on the relation between contractile function, contractile protein composition and phosphorylation levels in small biopsies from control (donor) hearts, from biopsies obtained during open heart surgery (NYHA Class I-IV) and from end-stage failing (explanted, NYHA class IV) hearts. Furthermore, attention is paid to the effect of the catalytic subunit of protein kinase A on isometric force development in single Triton-skinned human cardiomyocytes isolated from donor and end-stage failing left ventricular myocardium at different resting sarcomere lengths. A reduction in sarcomere length from 2.2 to 1.8 microm caused reductions in maximum isometric force by approximately 35% both in donor and in failing cardiomyocytes. The midpoints of the calcium sensitivity curves (pCa50) of donor and end-stage failing hearts differed markedly at all sarcomere lengths (mean delta pCa50 = 0.22). Our findings indicate that 1) TnI phosphorylation contributes to the differences in calcium sensitivity between donor and end-stage failing hearts, 2) human ventricular myocardium is heterogeneous with respect of the phosphorylation of TnT, MLC-2 and the isoform distribution of MLC-1 and MLC-2, and 3) the Frank-Starling mechanism is preserved in end-stage failing myocardium.
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PMID:Calcium sensitivity of force in human ventricular cardiomyocytes from donor and failing hearts. 1247 45


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