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)

Twitchin, also called mini-titin, is structurally related to the giant elastic protein connectin/titin, and has been found in not only striated but also smooth muscles of bivalves. Many bivalve smooth muscles such as byssus retractor muscles and the opaque part of adductor muscles are known as catch muscles that can maintain high passive tension with little expenditure of energy after they have actively contracted. Twitchin is phosphorylated when this high-tension state (catch state) ceases. Our recent studies revealed that the catch tension is due to interactions between thick and thin filaments in the presence of MgATP at low free Ca2+ concentrations, which can be visualized in vitro under a light microscope (Yamada et al., 2001 Proc Natl Acad Sci USA 98: 6635-6640). We also found that twitchin is essential for the interactions of the catch state in mussel (Mytilus galloprovincialis) catch muscles. In the presence of twitchin, actin filaments bound to purified myosin filaments when twitchin was dephosphorylated by Ser/Thr protein phosphatase 2B, while they did not when it was phosphorylated by cAMP-dependent protein kinase. In the current study we demonstrate the same essential components of the catch state for another bivalve that exhibits catch, i.e., Japanese oyster (Crassostrea gigas).
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PMID:Twitchin purified from molluscan catch muscles regulates interactions between actin and myosin filaments at rest in a phosphorylation-dependent manner. 1645 60

Molluscan catch muscle can maintain tension for a long time with little energy consumption. This unique phenomenon is regulated by phosphorylation and dephosphorylation of twitchin, a member of the titin/connectin family. The catch state is induced by a decrease of intracellular Ca2+ after the active contraction and is terminated by the phosphorylation of twitchin by the cAMP-dependent protein kinase (PKA). Twitchin, from the well-known catch muscle, the anterior byssus retractor muscle (ABRM) of the mollusc Mytilus, incorporates three phosphates into two major sites D1 and D2, and some minor sites. Dephosphorylation is required for re-entering the catch state. Myosin, actin and twitchin are essential players in the mechanism responsible for catch during which force is maintained while myosin cross-bridge cycling is very slow. Dephosphorylation of twitchin allows it to bind to F-actin, whereas phosphorylation decreases the affinity of the two proteins. Twitchin has been also been shown to be a thick filament-binding protein. These findings raise the possibility that twitchin regulates the myosin cross-bridge cycle and force output by interacting with both actin and myosin resulting in a structure that connects thick and thin filaments in a phosphorylation-dependent manner.
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PMID:Twitchin as a regulator of catch contraction in molluscan smooth muscle. 1645 61

Titin is a giant protein responsible for passive-tension generation in muscle sarcomeres. Here, we used single-molecule AFM force spectroscopy to investigate the mechanical characteristics of a recombinant construct from the human cardiac-specific N2B-region, which harbors a 572-residue unique sequence flanked by two immunoglobulin (Ig) domains on either side. Force-extension curves of the N2B-construct revealed mean unfolding forces for the Ig-domains similar to those of a recombinant fragment from the distal Ig-region in titin (I91-98). The mean contour length of the N2B-unique sequence was 120 nm, but there was a bimodal distribution centered at approximately 95 nm (major peak) and 180 nm (minor peak). These values are lower than expected if the N2B-unique sequence were a permanently unfolded entropic spring, but are consistent with the approximately 100 nm maximum extension of that segment measured in isolated stretched cardiomyofibrils. A contour-length below 200 nm would be reasonable, however, if the N2B-unique sequence were stabilized by a disulphide bridge, as suggested by several disulphide connectivity prediction algorithms. Since the N2B-unique sequence can be phosphorylated by protein kinase A (PKA), which lowers titin-based stiffness, we studied whether addition of PKA (+ATP) affects the mechanical properties of the N2B-construct, but found no changes. The softening effect of PKA on N2B-titin may require specific conditions/factors present inside the cardiomyocytes.
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PMID:Mechanical properties of cardiac titin's N2B-region by single-molecule atomic force spectroscopy. 1668 30

Protein phosphorylation is an important modulator of many cellular processes, and identification of kinase substrates provides critical insights for signal transduction. However, this identification process is often difficult and many kinase substrates remain unexplored. Herein, a systematic proteomics approach solely depending on MS detection is reported for identifying substrates of PKA and PKG, which are suspected to have similar specificity determinants, in pregnant rat uteri. Instead of radioisotopes that are commonly used to couple with MS for substrate identification, this study developed an efficient in vitro kinase assay on depleted tissue homogenates to reveal substrate candidates directly by MS. To facilitate MS detection, exogenous phosphatases were added to remove intrinsic phosphorylation followed by a heating step to inactivate all enzymes. No observable interference caused by endogenous kinases or background phosphorylation was detected in the control experiment in which no kinase was externally added. A total of 61 and 12 substrate candidates were identified in vitro for PKA and PKG, respectively, and most of these identified sites contain consensus motifs of each kinase with only a few sites overlapped, indicating a good specificity. Moreover, differential phosphoproteomics analysis using stable isotope dimethyl labeling and MS was performed to detect the change of protein phosphorylation upon kinase stimulation in vivo. Four identified in vitro PKA substrates including three reported sites on HSP27 or filamin A were significantly phosphorylated in vivo, giving them high confidence as physiological substrates in pregnant rat uteri. Moreover, telokin, a known PKG substrate on S1880, and actin-binding proteins such as Arp 3, titin, and desmuslin were also identified to be in vitro PKG substrates in pregnant rat uteri. These proteins are all expected to be involved in the regulation of actin-mediated cytoskeletal remodeling.
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PMID:A systematic MS-based approach for identifying in vitro substrates of PKA and PKG in rat uteri. 1756 27

In myogenic C(2)C(12) cells, 5 mM creatine increased the incorporation of labeled [(35)S]methionine into sarcoplasmic (+20%, P < 0.05) and myofibrillar proteins (+50%, P < 0.01). Creatine also promoted the fusion of myoblasts assessed by an increased number of nuclei incorporated within myotubes (+40%, P < 0.001). Expression of myosin heavy chain type II (+1,300%, P < 0.001), troponin T (+65%, P < 0.01), and titin (+40%, P < 0.05) was enhanced by creatine. Mannitol, taurine, and beta-alanine did not mimic the effect of creatine, ruling out an osmolarity-dependent mechanism. The addition of rapamycin, the inhibitor of mammalian target of rapamycin/70-kDa ribosomal S6 protein kinase (mTOR/p70(s6k)) pathway, and SB 202190, the inhibitor of p38, completely blocked differentiation in control cells, and creatine did not reverse this inhibition, suggesting that the mTOR/p70(s6k) and p38 pathways could be potentially involved in the effect induced by creatine on differentiation. Creatine upregulated phosphorylation of protein kinase B (Akt/PKB; +60%, P < 0.001), glycogen synthase kinase-3 (+70%, P < 0.001), and p70(s6k) (+50%, P < 0.001). Creatine also affected the phosphorylation state of p38 (-50% at 24 h and +70% at 96 h, P < 0.05) as well as the nuclear content of its downstream targets myocyte enhancer factor-2 (-55% at 48 h and +170% at 96 h, P < 0.05) and MyoD (+60%, P < 0.01). In conclusion, this study points out the involvement of the p38 and the Akt/PKB-p70(s6k) pathways in the enhanced differentiation induced by creatine in C(2)C(12) cells.
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PMID:Creatine enhances differentiation of myogenic C2C12 cells by activating both p38 and Akt/PKB pathways. 1765 29

The sarcomeric titin springs influence myocardial distensibility and passive stiffness. Titin isoform composition and protein kinase (PK)A-dependent titin phosphorylation are variables contributing to diastolic heart function. However, diastolic tone, relaxation speed, and left ventricular extensibility are also altered by PKG activation. We used back-phosphorylation assays to determine whether PKG can phosphorylate titin and affect titin-based stiffness in skinned myofibers and isolated myofibrils. PKG in the presence of 8-pCPT-cGMP (cGMP) phosphorylated the 2 main cardiac titin isoforms, N2BA and N2B, in human and canine left ventricles. In human myofibers/myofibrils dephosphorylated before mechanical analysis, passive stiffness dropped 10% to 20% on application of cGMP-PKG. Autoradiography and anti-phosphoserine blotting of recombinant human I-band titin domains established that PKG phosphorylates the N2-B and N2-A domains of titin. Using site-directed mutagenesis, serine residue S469 near the COOH terminus of the cardiac N2-B-unique sequence (N2-Bus) was identified as a PKG and PKA phosphorylation site. To address the mechanism of the PKG effect on titin stiffness, single-molecule atomic force microscopy force-extension experiments were performed on engineered N2-Bus-containing constructs. The presence of cGMP-PKG increased the bending rigidity of the N2-Bus to a degree that explained the overall PKG-mediated decrease in cardiomyofibrillar stiffness. Thus, the mechanically relevant site of PKG-induced titin phosphorylation is most likely in the N2-Bus; phosphorylation of other titin sites could affect protein-protein interactions. The results suggest that reducing titin stiffness by PKG-dependent phosphorylation of the N2-Bus can benefit diastolic function. Failing human hearts revealed a deficit for basal titin phosphorylation compared to donor hearts, which may contribute to diastolic dysfunction in heart failure.
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PMID:Protein kinase G modulates human myocardial passive stiffness by phosphorylation of the titin springs. 1911 83

In healthy human myocardium a tight balance exists between receptor-mediated kinases and phosphatases coordinating phosphorylation of regulatory proteins involved in cardiomyocyte contractility. During heart failure, when neurohumoral stimulation increases to compensate for reduced cardiac pump function, this balance is perturbed. The imbalance between kinases and phosphatases upon chronic neurohumoral stimulation is detrimental and initiates cardiac remodelling, and phosphorylation changes of regulatory proteins, which impair cardiomyocyte function. The main signalling pathway involved in enhanced cardiomyocyte contractility during increased cardiac load is the beta-adrenergic signalling route, which becomes desensitized upon chronic stimulation. At the myofilament level, activation of protein kinase A (PKA), the down-stream kinase of the beta-adrenergic receptors (beta-AR), phosphorylates troponin I, myosin binding protein C and titin, which all exert differential effects on myofilament function. As a consequence of beta-AR down-regulation and desensitization, phosphorylation of the PKA-target proteins within the cardiomyocyte may be decreased and alter myofilament function. Here we discuss involvement of altered PKA-mediated myofilament protein phosphorylation in different animal and human studies, and discuss the roles of troponin I, myosin binding protein C and titin in regulating myofilament dysfunction in cardiac disease. Data from the different animal and human studies emphasize the importance of careful biopsy procurement, and the need to investigate localization of kinases and phosphatases within the cardiomyocyte, in particular their co-localization with cardiac myofilaments upon receptor stimulation.
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PMID:Myofilament dysfunction in cardiac disease from mice to men. 1914 19

Nodal points of mechanotransduction are found along the cardiac sarcomere, notably in the Z-disc/I-band and M-band regions. A major integrating component of these mechanosensitive complexes is the giant protein titin, which is anchored at the Z-disc, spans the I-band as an elastic spring and enters the A-band bound to myosin, then reaching all the way to the M-band. Passive-force generation and transmission of stress via the titin filaments may be central to the mechanosensory function of the myofibrillar signalosome complexes. This review discusses recent findings shedding light on mechanisms by which titin elasticity is regulated dynamically. Adjustment of titin stiffness occurs during heart development and disease through a shift in the expression ratio of the two main titin isoforms in cardiac sarcomeres, N2BA (compliant) and N2B (stiffer). Titin-isoform switching in favor of the stiffer N2B-titin can be triggered by thyroid hormone (T3)activating the phosphatidylinositol-3-kinase (PI3K)/AKT pathway. Conversely, low T3 promotes the compliant N2BA-titin. In addition, titin stiffness can be tuned acutely by protein kinase (PK)A-or PKG-mediated phosphorylation of a cardiac-specific I-band titin segment, the N2-B domain. Beta-adrenergic agonists, nitric oxide, or natriuretic peptides thus trigger a softening of the titin springs, thereby modulating diastolic function. Failing human hearts can have elevated passive stiffness in part because of a titin phosphorylation deficit, which may contribute to mechanical dysfunction. Altered titin phosphorylation could also affect protein-protein interactions in the mechanosensory complexes associated with the sarcomere. In this context, the review highlights novel links between titin and stress-signalling pathways in the cardiomyocyte.
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PMID:Titin-based mechanical signalling in normal and failing myocardium. 1963 76

In sarcomeres of striated muscles the middle parts of adjacent thick filaments are connected to each other by the M-band proteins. To understand the role of the M-band in sarcomere mechanics a model of forces which pull a thick filament to opposite Z-disks of a sarcomere is considered. Forces of actin-myosin cross-bridges, I-band titin segments and the M-band are accounted for. A continual expression for the M-band force is obtained assuming that the M-band proteins which connect neighbor thick filaments have nonlinear elastic properties. On the ascending and descending limbs of the force-length diagram cross-bridge forces tend to destabilize sarcomere while titin tries to restore its symmetric configuration. When destabilizing cross-bridge force exceeds a critical limit, symmetric configuration of a sarcomere becomes unstable and the M-band buckles. Stiffness of the M-band increases stability only if the M-band is anchored to the extra-sarcomere cytoskeleton. Realistic magnitudes of the M-band buckling require that the M-band proteins have essentially nonlinear elasticity. The buckling may explain the M-band bending and axial misalignment of the thick filaments observed in contracting muscle. We hypothesize that the buckling stretches the titin protein kinase domain localized in the M-band being the signal for mechanical control of gene expression and protein turnover in striated muscle.
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PMID:Proposed role of the M-band in sarcomere mechanics and mechano-sensing: a model study. 1966 83

S100A1 is a member of the S100 family of calcium-binding proteins. As with most S100 proteins, S100A1 undergoes a large conformational change upon binding calcium as necessary to interact with numerous protein targets. Targets of S100A1 include proteins involved in calcium signaling (ryanidine receptors 1 & 2, Serca2a, phopholamban), neurotransmitter release (synapsins I & II), cytoskeletal and filament associated proteins (CapZ, microtubules, intermediate filaments, tau, mocrofilaments, desmin, tubulin, F-actin, titin, and the glial fibrillary acidic protein GFAP), transcription factors and their regulators (e.g. myoD, p53), enzymes (e.g. aldolase, phosphoglucomutase, malate dehydrogenase, glycogen phosphorylase, photoreceptor guanyl cyclases, adenylate cyclases, glyceraldehydes-3-phosphate dehydrogenase, twitchin kinase, Ndr kinase, and F1 ATP synthase), and other Ca2+-activated proteins (annexins V & VI, S100B, S100A4, S100P, and other S100 proteins). There is also a growing interest in developing inhibitors of S100A1 since they may be beneficial for treating a variety of human diseases including neurological diseases, diabetes mellitus, heart failure, and several types of cancer. The absence of significant phenotypes in S100A1 knockout mice provides some early indication that an S100A1 antagonist could have minimal side effects in normal tissues. However, development of S100A1-mediated therapies is complicated by S100A1's unusual ability to function as both an intracellular signaling molecule and as a secreted protein. Additionally, many S100A1 protein targets have only recently been identified, and so fully characterizing both these S100A1-target complexes and their resulting functions is a necessary prerequisite.
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PMID:S100A1: Structure, Function, and Therapeutic Potential. 1989 Apr 75


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