Phosphorylation of a bovine cardiac actin complex

Bailin, Gary

doi:10.1152/ajpcell.1979.236.1.c41

Cited by 29 publications

(12 citation statements)

References 23 publications

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“…Studies from other groups also report that acid desulphation does not alter uPAR or PAI-1 binding to Vn [29]. It is also worth noting that any phosphorylated amino acids present (such as threonine 50 and 57) may not be affected by the acid-treatment employed as O-linked phosphates are highly resistant to acid hydrolysis [42], [43] (as also demonstrated in Figure S1). …”

Section: Discussionmentioning

confidence: 64%

“…In addition, we also subjected the peptides to mild acid-hydrolysis to remove the sulphate residues from VA-26S. Such a treatment (<20 minutes in 1 M HCl at 80°C) was not expected to affect the phosphorylation of the VA-26P peptide as prolonged hydrolysis >1.5 h in 6 M HCl at 110°C in vacuum has been shown to be required to extract intact phosphothreonines and phosphoserines from proteins [42], [43]. Accordingly, mAb 8E6 binding was abrogated but the phosphate residues on VA-26P were not at all affected by acid-treatment and the anti-phosphothreonine antibody binding remained unchanged (Figures 6B and S1).…”

Section: Resultsmentioning

confidence: 99%

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Neisseria meningitidis Opc Invasin Binds to the Sulphated Tyrosines of Activated Vitronectin to Attach to and Invade Human Brain Endothelial Cells

2010

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The host vasculature is believed to constitute the principal route of dissemination of Neisseria meningitidis (Nm) throughout the body, resulting in septicaemia and meningitis in susceptible humans. In vitro, the Nm outer membrane protein Opc can enhance cellular entry and exit, utilising serum factors to anchor to endothelial integrins; but the mechanisms of binding to serum factors are poorly characterised. This study demonstrates that Nm Opc expressed in acapsulate as well as capsulate bacteria can increase human brain endothelial cell line (HBMEC) adhesion and entry by first binding to serum vitronectin and, to a lesser extent, fibronectin. This study also demonstrates that Opc binds preferentially to the activated form of human vitronectin, but not to native vitronectin unless the latter is treated to relax its closed conformation. The direct binding of vitronectin occurs at its Connecting Region (CR) requiring sulphated tyrosines Y56 and Y59. Accordingly, Opc/vitronectin interaction could be inhibited with a conformation-dependent monoclonal antibody 8E6 that targets the sulphotyrosines, and with synthetic sulphated (but not phosphorylated or unmodified) peptides spanning the vitronectin residues 43–68. Most importantly, the 26-mer sulphated peptide bearing the cell-binding domain 45RGD47 was sufficient for efficient meningococcal invasion of HBMECs. To our knowledge, this is the first study describing the binding of a bacterial adhesin to sulphated tyrosines of the host receptor. Our data also show that a single region of Opc is likely to interact with the sulphated regions of both vitronectin and of heparin. As such, in the absence of heparin, Opc-expressing Nm interact directly at the CR but when precoated with heparin, they bind via heparin to the heparin-binding domain of the activated vitronectin, although with a lower affinity than at the CR. Such redundancy suggests the importance of Opc/vitronectin interaction in meningococcal pathogenesis and may enable the bacterium to harness the benefits of the physiological processes in which the host effector molecule participates.

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Section: Discussionmentioning

confidence: 64%

Section: Resultsmentioning

confidence: 99%

Neisseria meningitidis Opc Invasin Binds to the Sulphated Tyrosines of Activated Vitronectin to Attach to and Invade Human Brain Endothelial Cells

2010

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“…PKA phosphorylation of CTnI causes a decrease in the Ca 2ϩ dependence of force development or of the myofibrillar ATPase of cardiac muscle and has been observed by many investigators (8,9,20,21,36). We have recently found that CTnI phosphorylation increases the rate of Ca 2ϩ dissociation from CTnC, thus contributing to the observed faster relaxation (22,23).…”

Section: Discussionmentioning

confidence: 64%

Phosphorylation of Both Serine Residues in Cardiac Troponin I Is Required to Decrease the Ca2+ Affinity of Cardiac Troponin C

Zhang

Zhao

Potter

1995

Journal of Biological Chemistry

152

147

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) have correlated this increase in Ca 2؉ dissociation with a reduced Ca 2؉ sensitivity of force development and a faster rate of cardiac muscle relaxation in a PKA phosphorylated skinned cardiac muscle preparation. To further determine the role of the two PKA phosphorylation sites in mouse CTnI (serine 22 and 23), serine 22 or 23, or both were mutated to alanine. The wild type and the mutated CTnIs were expressed in Escherichia coli and purified. Using these mutants, it was found that serine 23 was phosphorylated more rapidly than serine 22 and that both serines are required to be phosphorylated in order to observe the characteristic reduction in the Ca 2؉ sensitivity of force development seen in a skinned cardiac muscle preparation. The latter result confirms that PKA phosphorylation of CTnI, and not other proteins, is responsible for this change in Ca 2؉ sensitivity. The results also suggest that one of the serines (23) may be constitutively phosphorylated and that serine 22 may be functionally more important.Several lines of evidence have led to a general understanding of how ␤-agonist stimulation leads to positive inotropic and chronotropic effects by phosphorylation of cellular substrates through cAMP-dependent protein kinase (PKA) 1 (1, 2). It is generally agreed that phosphorylation of sarcolemmal Ca 2ϩ channels (3) and phospholamban (4, 5), a sarcoplasmic reticular protein regulating the Ca 2ϩ pump, are responsible for the changes seen in the intracellular Ca 2ϩ transient. The resulting increases in intracellular Ca 2ϩ and the rate of Ca 2ϩ resequestration contribute to an increase in cardiac muscle contractility and to faster rates of force development and relaxation. PKA can also phosphorylate contractile machinery proteins, such as C-protein and cardiac troponin I (CTnI) (6 -9). Many laboratories, including ours, have focused on the mechanism by which phosphorylation of CTnI may be involved in the inotropic effect brought about by ␤-agonist stimulation.Troponin I (TnI), a subunit of the troponin complex, inhibits the actomyosin ATPase activity when muscle is in the resting state. Binding of Ca 2ϩ to the low affinity site(s) of troponin C (TnC) releases the TnI inhibition on actomyosin ATPase through protein-protein interactions among the troponin complex, tropomyosin and actin, and leads to muscle contraction (10). In comparison with skeletal TnI, CTnI has an additional 32-33 amino acids at its N terminus, and this segment also contains two adjacent serine residues at positions 22 and 23 or 23 and 24, depending on the species (11-13). By converting the phosphoserine into the stable S-ethylcysteine derivative on bovine CTnI, Swiderek et al. (14,15) demonstrated that these two serines were able to be fully phosphorylated by PKA. Biochemical studies have shown that CTnI can be phosphorylated to the level of 2 mol of phosphate/mol of protein (16), consistent with the sequence prediction. Considerable effort has been made with either perfused hearts (17, 18) or isolated proteins (14 -16) to determine the m...

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“…To dephosphorylate heart muscle in laboratory animals a different method may be used. For instance, mice can be treated with Propranolol (8 mg/kg) to block β1-adrenoreceptors and deactivate PKA to reduce phosphorylation levels of PKA substrates including Tn and MyBP-C (Bailin, 1979; Wang et al, 2011; Vikhorev et al, 2013). …”

Section: Methodsmentioning

confidence: 99%

“…It was established, soon after the discovery of troponin I (TnI) phosphorylation, that phosphorylation of troponin (Tn) modulates Ca 2+ -regulation by Tn by reducing the Ca 2+ -sensitivity and increasing the force or crossbridge turnover rate at maximally activating Ca 2+ concentrations (Ray and England, 1976; Bailin, 1979; Mope et al, 1980). The magnitude of the Ca 2+ -sensitivity shift has been consistently been measured in the 2–3-fold range.…”

Section: Normal Relationship Between Tni Phosphorylation and Ca2+-regmentioning

confidence: 99%

Investigating the role of uncoupling of troponin I phosphorylation from changes in myofibrillar Ca2+-sensitivity in the pathogenesis of cardiomyopathy

Messer

Marston

2014

Front. Physiol.

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Contraction in the mammalian heart is controlled by the intracellular Ca2+ concentration as it is in all striated muscle, but the heart has an additional signaling system that comes into play to increase heart rate and cardiac output during exercise or stress. β-adrenergic stimulation of heart muscle cells leads to release of cyclic-AMP and the activation of protein kinase A which phosphorylates key proteins in the sarcolemma, sarcoplasmic reticulum and contractile apparatus. Troponin I (TnI) and Myosin Binding Protein C (MyBP-C) are the prime targets in the myofilaments. TnI phosphorylation lowers myofibrillar Ca2+-sensitivity and increases the speed of Ca2+-dissociation and relaxation (lusitropic effect). Recent studies have shown that this relationship between Ca2+-sensitivity and TnI phosphorylation may be unstable. In familial cardiomyopathies, both dilated and hypertrophic (DCM and HCM), a mutation in one of the proteins of the thin filament often results in the loss of the relationship (uncoupling) and blunting of the lusitropic response. For familial dilated cardiomyopathy in thin filament proteins it has been proposed that this uncoupling is causative of the phenotype. Uncoupling has also been found in human heart tissue from patients with hypertrophic obstructive cardiomyopathy as a secondary effect. Recently, it has been found that Ca2+-sensitizing drugs can promote uncoupling, whilst one Ca2+-desensitizing drug Epigallocatechin 3-Gallate (EGCG) can reverse uncoupling. We will discuss recent findings about the role of uncoupling in the development of cardiomyopathies and the molecular mechanism of the process.

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Phosphorylation of a bovine cardiac actin complex

Cited by 29 publications

References 23 publications

Neisseria meningitidis Opc Invasin Binds to the Sulphated Tyrosines of Activated Vitronectin to Attach to and Invade Human Brain Endothelial Cells

Neisseria meningitidis Opc Invasin Binds to the Sulphated Tyrosines of Activated Vitronectin to Attach to and Invade Human Brain Endothelial Cells

Phosphorylation of Both Serine Residues in Cardiac Troponin I Is Required to Decrease the Ca2+ Affinity of Cardiac Troponin C

Investigating the role of uncoupling of troponin I phosphorylation from changes in myofibrillar Ca2+-sensitivity in the pathogenesis of cardiomyopathy

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