Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Downstream regulation of the cAMP-dependent protein kinase (PKA) pathway is mediated by anchoring proteins (AKAPs) that sequester PKA to specific subcellular locations through binding to PKA regulatory subunits (RI or RII). The RII-binding domain of all AKAPs forms an amphipathic alpha-helix with similar secondary structure. However, the importance of sequence differences in the RII-binding domains of different AKAPs is unknown, and mechanisms that regulate AKAP-PKA affinity are not clearly defined. Using surface plasmon resonance (SPR) spectroscopy, we measured real-time kinetics of RII interaction with various AKAPs. Base-line equilibrium binding constants (K(d)) for RII binding to Ht31, mAKAP, and AKAP15/18 were 10 nm, 119 nm, and 6.6 microm, respectively. PKA stimulation of intact Chinese hamster ovary cells increased RIIalpha binding to AKAP100/mAKAP and AKAP15/18 by approximately 7- and 82-fold, respectively. These results suggest that differences in primary sequence of the RII-binding domain may be responsible for the selective affinity of RII for different AKAPs. Furthermore, RII autophosphorylation may provide additional localized regulation of kinase anchoring. In cardiac myocytes, disruption of RII-AKAP interaction decreased PKA phosphorylation of the PKA substrate, myosin-binding protein C. Thus, these mechanisms may be involved in adding additional specificity in intracellular signaling in diverse cell types and under conditions of cAMP/PKA activation.
 ...
PMID:Selectivity and regulation of A-kinase anchoring proteins in the heart. The role of autophosphorylation of the type II regulatory subunit of cAMP-dependent protein kinase. 1099 82

Myosin-binding protein-C (MyBP-C) is a component of all striated-muscle sarcomeres, with a well established structural role and a possible function for force regulation. Multiple mutations within the gene for cardiac MyBP-C, one of three known isoforms, have been linked to familial hypertrophic cardiomyopathy. Here we generated a knock-in mouse model that carries N-terminal-shortened cardiac MyBP-C. The mutant protein was designed to have a similar size as the skeletal MyBP-C isoforms, whereas known myosin and titin binding sites as well as the phosphorylatable MyBP-C motif were not altered. We have shown that mutant cardiac MyBP-C is readily incorporated into the sarcomeres of both heterozygous and homozygous animals and can still be phosphorylated by cAMP-dependent protein kinase. Although histological characterization of wild-type and mutant hearts did not reveal obvious differences in phenotype, left ventricular fibers from homozygous mutant mice exhibited an increased Ca(2+) sensitivity of force development, particularly at lower Ca(2+) concentrations, whereas maximal active force levels remained unchanged. The results allow us to propose a model of how cMyBP-C may affect myosin-head mobility and to rationalize why N-terminal mutations of the protein in some cases of familial hypertrophic cardiomyopathy could lead to a hypercontractile state.
 ...
PMID:Hypercontractile properties of cardiac muscle fibers in a knock-in mouse model of cardiac myosin-binding protein-C. 1109 95

"Catch," a state where some invertebrate muscles sustain high tension over long periods of time with little energy expenditure (low ATP hydrolysis rate) is similar to the "latch" state of vertebrate smooth muscles. Its induction and release involve Ca(2+)-dependent phosphatase and cAMP-dependent protein kinase, respectively. Molecular mechanisms for catch remain obscure. Here, we describe a quantitative microscopic in vitro assay reconstituting the catch state with proteins isolated from catch muscles. Thick filaments attached to glass coverslips and pretreated with approximately 10(-4) M free Ca(2+) and soluble muscle proteins bound fluorescently labeled native thin filaments tightly in catch at approximately 10(-8) M free Ca(2+) in the presence of MgATP. At approximately 10(-4) M free Ca(2+), the thin filaments moved at approximately 4 microm/s. Addition of cAMP and cAMP-dependent protein kinase at approximately 10(-8) M free Ca(2+) caused their release. Rabbit skeletal muscle F-actin filaments completely reproduced the results obtained with native thin filaments. Binding forces >500 pN/microm between thick and F-actin filaments were measured by glass microneedles, and were sufficient to explain catch tension in vivo. Synthetic filaments of purified myosin and twitchin bound F-actin in catch, showing that other components of native thick filaments such as paramyosin and catchin are not essential. The binding between synthetic thick filaments and F-actin filaments depended on phosphorylation of twitchin but not of myosin. Cosedimentation experiments showed that twitchin did not bind directly to F-actin in catch. These results show that catch is a direct actomyosin interaction regulated by twitchin phosphorylation.
 ...
PMID:An in vitro assay reveals essential protein components for the "catch" state of invertebrate smooth muscle. 1137 23

Phosphorylation of cardiac myofibrils by cAMP-dependent protein kinase (PKA) can increase the intrinsic rate of myofibrillar relaxation, which may contribute to the shortening of the cardiac twitch during beta-adrenoceptor stimulation. However, it is not known whether the acceleration of myofibrillar relaxation is due to phosphorylation of troponin I (TnI) or of myosin binding protein-C (MyBP-C). To distinguish between these possibilities, we used transgenic mice that overexpress the nonphosphorylatable, slow skeletal isoform of TnI in the myocardium and do not express the normal, phosphorylatable cardiac TNI: The intrinsic rate of relaxation of myofibrils from wild-type and transgenic mice was measured using flash photolysis of diazo-2 to rapidly decrease the [Ca(2+)] within skinned muscles from the mouse ventricles. Incubation with PKA nearly doubled the intrinsic rate of myofibrillar relaxation in muscles from wild-type mice (relaxation half-time fell from approximately 150 to approximately 90 ms at 22 degrees C) but had no effect on the relaxation rate of muscles from the transgenic mice. In parallel studies with intact muscles, we assessed crossbridge kinetics indirectly by determining f(min) (the frequency for minimum dynamic stiffness) during tetanic contractions. Stimulation of beta-adrenoceptors with isoproterenol increased f(min) from 1.9 to 3.1 Hz in muscles from wild-type mice but had no effect on f(min) in muscles from transgenic mice. We conclude that the acceleration of myofibrillar relaxation rate by PKA is due to phosphorylation of TnI, rather than MyBP-C, and that this may be due, at least in part, to faster crossbridge cycle kinetics.
 ...
PMID:Phosphorylation of troponin I by protein kinase A accelerates relaxation and crossbridge cycle kinetics in mouse ventricular muscle. 1137 76

1. The Ca(2+) sensitivity of smooth muscle contractility is modulated via regulation of phosphatase activity. Protein phosphatase inhibitor-1 (I-1) is the classic type-1 phosphatase inhibitor, but its presence and role in cAMP-dependent protein kinase (PKA) modulation of smooth muscle is unclear. To address the relevance of I-1 in vivo, we investigated smooth muscle function in a mouse model lacking the I-1 protein (I-1((-/-)) mice). 2. Significant amounts of I-1 protein were detected in the wild-type (WT) mouse aorta and could be phosphorylated by PKA, as indicated by (32)P-labelled aortic extracts from WT mice. 3. Despite the significant presence of I-1 in WT aorta, phenylephrine and KCl concentration- isometric force relations in the presence or absence of the PKA pathway activator isoproterenol (isoprenaline) were unchanged compared to I-1((-/-)) aorta. cGMP-dependent protein kinase (PKG) relaxation pathways were also not different. Consistent with these findings, dephosphorylation rates of the 20 kDa myosin light chains (MLC(20)), measured in aortic extracts, were nearly identical between WT and I-1((-/-)) mice. 4. In the portal vein, I-1 protein ablation was associated with a significant (P < 0.05) rightward shift in the EC(50) of isoproterenol relaxation (EC(50) = 10.4 +/- 1.4 nM) compared to the WT value (EC(50) = 3.5 +/- 0.2 nM). Contraction in response to acetylcholine as well as Ca(2+) sensitivity were similar between WT and I-1((-/-)) aorta. 5. Despite the prevalence of I-1 and its activation by PKA in the aorta, I-1 does not appear to play a significant role in contractile or relaxant responses to any pharmacomechanical or electromechanical agonists used. I-1 may play a role as a fine-tuning mechanism involved in regulating portal vein responsiveness to beta-adrenergic agonists.
 ...
PMID:Is myosin phosphatase regulated in vivo by inhibitor-1? Evidence from inhibitor-1 knockout mice. 1145 56

KRP (telokin), an independently expressed C-terminal myosin-binding domain of smooth muscle myosin light chain kinase (MLCK), has been reported to have two related functions. First, KRP stabilizes myosin filaments (Shirinsky et al., 1993, J. Biol. Chem. 268, 16578-16583) in the presence of ATP. Secondly, KRP can modulate the level of myosin light chain phosphorylation. In this latter role, multiple mechanisms have been suggested. One hypothesis is that light chain phosphorylation is diminished by the direct competition of KRP and MLCK for myosin, resulting in a loss of contraction. Alternatively, KRP, through an unidentified mechanism, accelerates myosin light chain dephosphorylation in a manner possibly enhanced by KRP phosphorylation. Here, we demonstrate that KRP is a major phosphoprotein in smooth muscle, and use a comparative approach to investigate how its phosphorylation correlates with sustained contraction and forskolin-induced relaxation. Forskolin relaxation of precontracted artery strips caused little increase in KRP phosphorylation, while treatment with phorbol ester increased the level of KRP phosphorylation without a subsequent change in contractility. Although phorbol ester does not induce contraction of phasic tissues, the level of KRP phosphorylation is increased. Phosphopeptide maps of KRP from both tissues revealed multiple sites of phosphorylation within the N-terminal region of KRP. Phosphopeptide maps of KRP from gizzard were more complex than those for KRP from artery consistent with heterogeneity at the amino terminus and/or additional sites. We discovered through analysis of KRP phosphorylation in vitro that Ser12, Ser15 and Ser15 are phosphorylated by cAMP-dependent protein kinase, mitogen-activated protein (MAP) kinase and glycogen synthase kinase 3 (GSK3), respectively. Phosphorylation by GSK3 was dependent upon prephosphorylation by MAP kinase. This appears to be the first report of conditional or hierarchical phosphorylation of KRP. Peptides consistent with such multiple phosphorylations were found on the in vivo phosphopeptide maps of avian KRP. Collectively, the available data indicate that there is a complex relationship between the in vivo phosphorylation states of KRP and its effects on relaxation in smooth muscle.
 ...
PMID:Phosphorylation of kinase-related protein (telokin) in tonic and phasic smooth muscles. 1196 68

A mechanism proposed for regulation of myosin phosphatase (MP) activity is phosphorylation of the myosin phosphatase target subunit (MYPT1). Integrin-linked kinase (ILK) is associated with the contractile machinery and can phosphorylate myosin at the myosin light-chain kinase sites. The possibility that ILK may also phosphorylate and regulate MP was investigated. ILK was associated with the MP holoenzyme, shown by Western blots and in-gel kinase assays. MYPT1 was phosphorylated by ILK and phosphorylation sites in the N- and C-terminal fragments of MYPT1 were detected. From sequence analyses, three sites were identified: a primary site at Thr(709), and two other sites at Thr(695) and Thr(495). One of the sites for cAMP-dependent protein kinase (PKA) was Ser(694). Assays with the catalytic subunit of type 1 phosphatase indicated that only the C-terminal fragment of MYPT1 phosphorylated by zipper-interacting protein kinase, and ILK inhibited activity. The phosphorylated N-terminal fragment activated phosphatase activity and phosphorylation by PKA was without effect. Using full-length MYPT1 constructs phosphorylated by various kinases it was shown that Rho kinase gave marked inhibition; ILK produced an intermediate level of inhibition, which was considerably reduced for the Thr(695)-->Ala mutant; and PKA had no effect. In summary, phosphorylation of the various sites indicated that Thr(695) was the major inhibitory site, Thr(709) had only a slight inhibitory effect and Ser(694) had no effect. The findings that ILK phosphorylated both MYPT1 and myosin and the association of ILK with MP suggest that ILK may influence cytoskeletal structure or function.
 ...
PMID:Phosphorylation of the myosin phosphatase target subunit by integrin-linked kinase. 1203 Aug 46

Much is known about the anatomy of Limulus retinal efferent neurons and the structural and functional consequences of their activation. Retinal efferent axons arise from cell bodies located in the cheliceral ganglia of the brain, and they project out all of the optic nerves. Their unique neurosecretory-like terminals contact all cell types in lateral eye ommatidia, the retinular cells of the median eye, and the internal rhabdom of ventral photoreceptors. Lateral and median rudimentary photoreceptors are also innervated. The activity of the efferents is circadian. They are active during the subjective night and inactive during the subjective day. Activation of the efferents drives dramatic and diverse changes in the structure and function of Limulus eyes and causes the sensitivity and responsiveness of the eyes to light to increase at night. Relatively little is known about the molecular mechanisms that produce these structural and functional changes, but one efferent-activated biochemical cascade has been identified. The biogenic amine octopamine is released from efferent terminals, and an octopamine-stimulated rise in cAMP in photoreceptors, with a subsequent activation of cAMP-dependent protein kinase, mediates many of the known effects of efferent input. A photoreceptor-specific protein, myosin III, is phosphorylated in response to efferent input; this protein may play a role in the efferent stimulated changes in photoreceptor structure and function. Anatomical, biophysical, biochemical, and molecular approaches are now being effectively combined in studies of Limulus eyes; thus, this preparation should be particularly useful for further detailed investigations of mechanisms underlying the modulation of primary sensory cells by efferent input.
 ...
PMID:Circadian efferent input to Limulus eyes: anatomy, circuitry, and impact. 1221 1

cAMP-dependent protein kinase (PKA)-dependent phosphorylation of the two serine residues in the amino terminal region unique to cardiac troponin I (cTnI) is known to cause two effects: (i) decrease of the maximum Ca2+-controlled thin filament-activated myosin S1-ATPase (actoS1-ATPase) activity and mean sliding velocity of reconstituted thin filaments; (ii) rightward shift of the Ca2+ activation curves of actoS1-ATPase activity, filament sliding velocity, and force generation. We have studied the influence of phosphorylation of human wild-type cTnI and of two mutant cTnI (G203S and K206Q) causing familial hypertrophic cardiomyopathy (fHCM) on the secondary structure by circular dichroism spectroscopy and on the Ca2+ regulation of actin-myosin interaction using actoS1-ATPase activity and in vitro motility assays. Both mutations slightly influence the backbone structure of cTnI but only the secondary structure of cTnI-G203S is also affected by bis-phosphorylation of cTnI. In functional studies, cTnI-G203S behaves similarly to wild-type cTnI, i.e. the mutation itself has no measurable effect and bis-phosphorylation alters the actoS1-ATPase activity and the in vitro thin filament motility in the same way as does bis-phosphorylation of wild-type cTnI. In contrast, the mutation K206Q leads to a considerable increase in the maximum actoS1-ATPase activity as well as filament motility compared to wild-type cTnI. Bis-phosphorylation of this mutant cTnI still suppresses the maximum actoS1-ATPase activity and filament sliding velocity but does no longer affect the Ca2+ sensitivity of these processes. Thus, these two fHCM-linked cTnI mutations, although reflecting similar pathological situations, exert different effects on the actomyosin system per se and in response to bis-phosphorylation of cTnI.
 ...
PMID:Phosphorylation of human cardiac troponin I G203S and K206Q linked to familial hypertrophic cardiomyopathy affects actomyosin interaction in different ways. 1459 93

Myosin binding protein-C (MyBP-C) is a thick filament-associated protein localized to the crossbridge-containing C zones of striated muscle sarcomeres. The cardiac isoform is composed of eight immunoglobulin I-like domains and three fibronectin 3-like domains and is known to be a physiological substrate of cAMP-dependent protein kinase. MyBP-C contributes to thick filament structure via interactions at its C-terminus with the light meromyosin section of the myosin rod and with titin. The protein also has a role in the regulation of contraction, due to the binding of its N-terminus to the subfragment-2 portion of myosin, which reduces actomyosin ATPase activity; phosphorylation abolishes this interaction, resulting in release of the "brake" on crossbridge cycling. Several structural models of the interaction of MyBP-C with myosin have been proposed, although its precise arrangement on the thick filament remains to be elucidated. Mutations in the gene encoding cardiac MyBP-C are a common cause of hypertrophic cardiomyopathy, and this has led to increased interest in the protein's function. Investigation of disease-causing mutations in domains with unknown function has led to further insights into the mechanism of cMyBP-C action. This Review aims to collate the published data on those aspects of MyBP-C that are well characterized and to consider new and emerging data that further define its structural and regulatory roles and its arrangement in the sarcomere. We also speculate on the mechanisms by which hypertrophic cardiomyopathy-causing truncation and missense mutations affect the normal functioning of the sarcomere.
 ...
PMID:Cardiac myosin binding protein C: its role in physiology and disease. 1516 15


<< Previous 1 2 3 4 5 6 7 Next >>