Sequential phosphorylation of adjacent serine residues on the N‐terminal region of cardiac troponin‐I: structure‐activity implications of ordered phosphorylation

Quirk, Philip G.; Patchell, Valerie B.; Gao, Yuan; Levine, Barry A.; Perry, S. V.

doi:10.1016/0014-5793(95)00812-n

Cited by 28 publications

(32 citation statements)

References 11 publications

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“…Here we found strong evidence for constitutive phosphorylation of Ser 22 rather than for Ser 23 in human cTnI. This apparent disagreement of our results with the literature may be explained by our analysis of the basal phosphorylation state of intact human cTnI phosphorylated in vivo in contrast to those previous in vitro studies (61)(62)(63)(64)(65). In vivo, Ser 22 may be phosphorylated by kinases in addition to PKA, such as PKC (12,66,67), cyclic GMP-dependent protein kinase (10), and protein kinase D (14).…”

Section: Discussioncontrasting

confidence: 57%

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Unraveling Molecular Complexity of Phosphorylated Human Cardiac Troponin I by Top Down Electron Capture Dissociation/Electron Transfer Dissociation Mass Spectrometry

Zabrouskov

Schwartz

et al. 2008

Molecular & Cellular Proteomics

106

163

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Cardiac troponin I (cTnI), the inhibitory subunit of the thin filament troponin-tropomyosin regulatory complex, is required for heart muscle relaxation during the cardiac cycle. Expressed only in cardiac muscle, cTnI is widely used in the clinic as a serum biomarker of cardiac injury. In vivo function of cTnI is influenced by phosphorylation and proteolysis; therefore analysis of post-translational modifications of the intact protein should greatly facilitate the understanding of cardiac regulatory mechanisms and may improve cTnI as a disease biomarker. cTnI (24 kDa, pI ϳ 9.5) contains twelve serine, eight threonine, and three tyrosine residues, which presents a challenge for unequivocal identification of phosphorylation sites and quantification of positional isomers. In this study, we used top down electron capture dissociation and electron transfer dissociation MS to unravel the molecular complexity of cTnI purified from human heart tissue. High resolution MS spectra of human cTnI revealed a high degree of heterogeneity, corresponding to phosphorylation, acetylation, oxidation, and C-terminal proteolysis. Thirty-six molecular ions of cTnI were detected in a single ESI/FTMS spectrum despite running as a single sharp band on SDS-PAGE. Electron capture dissociation of monophosphorylated cTnI localized two major basal phosphorylation sites: a well known site at Ser 22 and a novel site at Ser

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Section: Discussioncontrasting

confidence: 57%

“…Sequential (or ordered) phosphorylation of these serine residues on cTnI has been suggested based upon phosphorylation of synthetic peptides by PKA in vitro (61)(62)(63)(64). In those studies, Ser 23 was phosphorylated first followed by Ser 22 at a ϳ10-fold slower rate.…”

Section: Discussionmentioning

confidence: 99%

Unraveling Molecular Complexity of Phosphorylated Human Cardiac Troponin I by Top Down Electron Capture Dissociation/Electron Transfer Dissociation Mass Spectrometry

Zabrouskov

Schwartz

et al. 2008

Molecular & Cellular Proteomics

106

163

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“…In some, but not all, proteins regulated by phosphorylation, the substitution of a phosphoacceptor serine or threonine with an acidic residue can mimic the effect of phosphorylation at that locus (22,23). However, for Thr 189 of the Na-K-Cl cotransporter, replacement with aspartate did not restore activity.…”

Section: Resultsmentioning

confidence: 98%

A Regulatory Locus of Phosphorylation in the N Terminus of the Na-K-Cl Cotransporter, NKCC1

Darman

Forbush

2002

Journal of Biological Chemistry

219

217

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The secretory Na-K-Cl cotransporter NKCC1 is activated by secretagogues through a phosphorylationdependent mechanism. We found a phosphorylation stoichiometry of 3.0 ؎ 0.4 phosphorylated residues/ NKCC1 protein harvested from shark rectal gland tubules maximally stimulated with forskolin and calyculin A, showing that at least three sites on the cotransporter are phosphorylated upon stimulation. Three phosphoacceptor sites were identified in the N-terminal domain of the protein (at Thr 184 , Thr 189 , and Thr 202 ) using high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry to analyze tryptic fragments of the radiolabeled cotransporter. None of these residues occurs in the context of strong consensus sites for known Ser/Thr kinases. The threonines and the surrounding amino acids are highly conserved between NKCC1 and NKCC2, and similarities are also present in the Na-Cl cotransporter NCC (or TSC). This strongly suggests that the phosphoregulatory mechanism is conserved among isoforms. The secretory Na-K-Cl cotransporter NKCC1 is a major pathway for net influx of Cl Ϫ in many cells (1, 2). Along with the absorptive Na-K-Cl cotransporter NKCC2 and the thiazidesensitive Na-Cl transporter NCC (or TSC), NKCC1 belongs to the sodium-coupled branch of the cation chloride cotransporter family; this family also includes the K-Cl transporters (KCCs) (3) and two other major branches of putative transporters, including human CIP (4) and SLC12-8 (GenBank TM /EBI accession number AAK94307), whose transport functions are unknown. These proteins are all predicted to have 12 transmembrane-spanning domains that are responsible for ion transport properties (5) and large cytoplasmic N and C termini that are candidates for regulatory domains. NKCC1 is expressed in many cell types and is involved in the regulation of both cell volume and intracellular Cl Ϫ concentration (2, 6). This isoform is also a major component of the basolateral membrane of secretory cells, mediating Cl Ϫ influx, the first step in the transepithelial movement of Cl Ϫ . In contrast, the NKCC2 isoform is limited to the apical membranes of absorptive epithelia such as the thick ascending limb of Henle's loop, where it is a major determinant of NaCl reabsorption from the tubular fluid.In order that the processes of regulatory volume increase, secretion, and absorption may be tightly controlled, the Na-K-Cl cotransporter is subject to strict regulation. The cotransporter is inactive in the basolateral membranes of secretory cells until the application of secretagogues or cell shrinkage causes a phosphorylation-dependent activation of the cotransporter (7-10). Studies on shark rectal gland secretory epithelia have shown an increase in phosphorylation at serines and threonines in response to forskolin or hypertonic stress, and the N terminus of NKCC1 has been shown to be the locus of at least some of the phosphoregulatory sites (7, 11). Although, in many cells, PKA 1 agonists effect an increase in Na-K-Cl co...

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“…It has previously been reported that the PKA-dependent reduction in myofilament Ca 2ϩ sensitivity requires the phosphorylation of both N-terminal phosphorylation sites on troponin I. 34,35 The addition of the second phosphate group to serine 23 in the N-terminus is considerably slower than the phosphorylation of serine 24. 34,35 As such, a PKA-dependent increase in troponin I phosphorylation might precede any functional changes if the phosphorylation of serine 23 was significantly slower.…”

Section: Discussionmentioning

confidence: 99%

Actin Capping Protein

Pyle

Hart

Cooper

et al. 2002

Circulation Research

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Abstract-Actin capping protein (CapZ) binds the barbed ends of actin at sarcomeric Z-lines. In addition to anchoring actin, Z-discs bind protein kinase C (PKC). Although CapZ is crucial for myofibrillogenesis, its role in muscle function and intracellular signaling is unknown. We hypothesized that CapZ downregulation would impair myocardial function and disrupt PKC-myofilament signaling by impairing PKC-Z-disc interaction. To test these hypotheses, we examined transgenic (TG) mice in which cardiac CapZ protein is reduced. Fiber bundles were dissected from papillary muscles and detergent extracted. Some fiber bundles were treated with PKC activators phenylephrine (PHE) or endothelin (ET) before detergent extraction. We simultaneously measured Ca 2ϩ -dependent tension and actomyosin MgATPase activity. CapZ downregulation increased myofilament Ca 2ϩ sensitivity without affecting maximum tension or actomyosin MgATPase activity. Maximum tension and actomyosin MgATPase activity were decreased after PHE or ET treatment of wild-type (WT) muscle. Fiber bundles from TG hearts did not respond to PHE or ET. Immunoblot analysis revealed an increase in myofilament-associated PKC-⑀ after PHE or ET exposure of WT preparations. In contrast, myofilamentassociated PKC-⑀ was decreased after PHE or ET treatment in TG myocardium. Protein levels of myofilamentassociated PKC-␤ were decreased in TG ventricle. C-protein and troponin I phosphorylation was increased after PHE or ET treatment in WT and TG hearts. Basal phosphorylation levels of C-protein and troponin I were higher in TG myocardium. These results indicate that downregulation of CapZ, or other changes associated with CapZ downregulation, increases cardiac myofilament Ca 2ϩ sensitivity, inhibits PKC-mediated control of myofilament activation, and decreases myofilament-associated PKC-␤.

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Sequential phosphorylation of adjacent serine residues on the N‐terminal region of cardiac troponin‐I: structure‐activity implications of ordered phosphorylation

Cited by 28 publications

References 11 publications

Unraveling Molecular Complexity of Phosphorylated Human Cardiac Troponin I by Top Down Electron Capture Dissociation/Electron Transfer Dissociation Mass Spectrometry

Unraveling Molecular Complexity of Phosphorylated Human Cardiac Troponin I by Top Down Electron Capture Dissociation/Electron Transfer Dissociation Mass Spectrometry

A Regulatory Locus of Phosphorylation in the N Terminus of the Na-K-Cl Cotransporter, NKCC1

Actin Capping Protein

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