Differential inhibition by α and εPKC pseudosubstrate sequences: A putative mechanism for preferential εPKC activation in neonatal cardiac myocytes

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Abstract

The aims of the current study were: 1) to determine if the εPKC pseudosubstrate peptide (εφ) (NH2-RKRQGAVRRRVHQVNG-COOH) could be used as an εPKC-selective inhibitor in neonatal cardiac myocytes (NCMs) and 2) to determine if differences in the α and εPKC autoinhibitory pseudosubstrate mechanisms could play roles in α and εPKC-selective functions. Introduction of the εφ into NCMs by transient permeabilization modestly attenuated 3nM 4-β PMA-induced slowing of contraction rate, an εPKC mediated response (Circ Res. 76:654-663; J. Biol. Chem. 271:24962-24966). In contrast, the αPKC pseudosubstrate peptide (αφ) (NH2-RFARKGALRQKNVHEVK-COOH) was 6- to 10-fold more potent at antagonizing the 3 nM 4-β PMA-induced slowing of contraction rate. Addition of purified PKC to the particulate cell fraction of NCMs promoted 32P incorporation into 3 proteins of ∼18, ∼46 and ∼97 kDa. The αφ antagonized these phosphorylations with IC50 values of 1 - 5 μM. These IC50 values were 1.8 - 4.7-fold lower than those observed for the εφ. In in vitro phosphorylation assays with recombinant α or ε PKC isozymes the εφ failed to inhibit the εPKC isozyme as potently as the αφ peptide but both the αφ and the εφ were equally effective inhibitors of the recombinant αPKC isozyme. In addition, in vitro cleavage of the εφ by the protease Arg-C in lysates from NCMs treated with 3 nM 4-β PMA was greatly enhanced when compared to that of the αPKC isozyme. Our studies suggest that the εφ cannot be used as a selective inhibitor of the εPKC isozyme in NCMs and that there are differences in the εPKC and αPKC autoinhibitory pseudosubstrate mechanisms.

Original languageEnglish (US)
Pages (from-to)3153-3172
Number of pages20
JournalLife sciences
Volume74
Issue number25
DOIs
StatePublished - May 7 2004

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Cardiac Myocytes
Isoenzymes
Chemical activation
Phosphorylation
Peptides
Inhibitory Concentration 50
Assays
Proteins
In Vitro Techniques

Keywords

  • Arg-C
  • Cardiac myocyte
  • Pseudosubstrates
  • α and εPKC

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Pharmacology, Toxicology and Pharmaceutics(all)

Cite this

@article{2ebce283cdf648939926068452495b56,
title = "Differential inhibition by α and εPKC pseudosubstrate sequences: A putative mechanism for preferential εPKC activation in neonatal cardiac myocytes",
abstract = "The aims of the current study were: 1) to determine if the εPKC pseudosubstrate peptide (εφ) (NH2-RKRQGAVRRRVHQVNG-COOH) could be used as an εPKC-selective inhibitor in neonatal cardiac myocytes (NCMs) and 2) to determine if differences in the α and εPKC autoinhibitory pseudosubstrate mechanisms could play roles in α and εPKC-selective functions. Introduction of the εφ into NCMs by transient permeabilization modestly attenuated 3nM 4-β PMA-induced slowing of contraction rate, an εPKC mediated response (Circ Res. 76:654-663; J. Biol. Chem. 271:24962-24966). In contrast, the αPKC pseudosubstrate peptide (αφ) (NH2-RFARKGALRQKNVHEVK-COOH) was 6- to 10-fold more potent at antagonizing the 3 nM 4-β PMA-induced slowing of contraction rate. Addition of purified PKC to the particulate cell fraction of NCMs promoted 32P incorporation into 3 proteins of ∼18, ∼46 and ∼97 kDa. The αφ antagonized these phosphorylations with IC50 values of 1 - 5 μM. These IC50 values were 1.8 - 4.7-fold lower than those observed for the εφ. In in vitro phosphorylation assays with recombinant α or ε PKC isozymes the εφ failed to inhibit the εPKC isozyme as potently as the αφ peptide but both the αφ and the εφ were equally effective inhibitors of the recombinant αPKC isozyme. In addition, in vitro cleavage of the εφ by the protease Arg-C in lysates from NCMs treated with 3 nM 4-β PMA was greatly enhanced when compared to that of the αPKC isozyme. Our studies suggest that the εφ cannot be used as a selective inhibitor of the εPKC isozyme in NCMs and that there are differences in the εPKC and αPKC autoinhibitory pseudosubstrate mechanisms.",
keywords = "Arg-C, Cardiac myocyte, Pseudosubstrates, α and εPKC",
author = "Johnson, {John A.}",
year = "2004",
month = "5",
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doi = "10.1016/j.lfs.2003.11.016",
language = "English (US)",
volume = "74",
pages = "3153--3172",
journal = "Life Sciences",
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TY - JOUR

T1 - Differential inhibition by α and εPKC pseudosubstrate sequences

T2 - A putative mechanism for preferential εPKC activation in neonatal cardiac myocytes

AU - Johnson, John A.

PY - 2004/5/7

Y1 - 2004/5/7

N2 - The aims of the current study were: 1) to determine if the εPKC pseudosubstrate peptide (εφ) (NH2-RKRQGAVRRRVHQVNG-COOH) could be used as an εPKC-selective inhibitor in neonatal cardiac myocytes (NCMs) and 2) to determine if differences in the α and εPKC autoinhibitory pseudosubstrate mechanisms could play roles in α and εPKC-selective functions. Introduction of the εφ into NCMs by transient permeabilization modestly attenuated 3nM 4-β PMA-induced slowing of contraction rate, an εPKC mediated response (Circ Res. 76:654-663; J. Biol. Chem. 271:24962-24966). In contrast, the αPKC pseudosubstrate peptide (αφ) (NH2-RFARKGALRQKNVHEVK-COOH) was 6- to 10-fold more potent at antagonizing the 3 nM 4-β PMA-induced slowing of contraction rate. Addition of purified PKC to the particulate cell fraction of NCMs promoted 32P incorporation into 3 proteins of ∼18, ∼46 and ∼97 kDa. The αφ antagonized these phosphorylations with IC50 values of 1 - 5 μM. These IC50 values were 1.8 - 4.7-fold lower than those observed for the εφ. In in vitro phosphorylation assays with recombinant α or ε PKC isozymes the εφ failed to inhibit the εPKC isozyme as potently as the αφ peptide but both the αφ and the εφ were equally effective inhibitors of the recombinant αPKC isozyme. In addition, in vitro cleavage of the εφ by the protease Arg-C in lysates from NCMs treated with 3 nM 4-β PMA was greatly enhanced when compared to that of the αPKC isozyme. Our studies suggest that the εφ cannot be used as a selective inhibitor of the εPKC isozyme in NCMs and that there are differences in the εPKC and αPKC autoinhibitory pseudosubstrate mechanisms.

AB - The aims of the current study were: 1) to determine if the εPKC pseudosubstrate peptide (εφ) (NH2-RKRQGAVRRRVHQVNG-COOH) could be used as an εPKC-selective inhibitor in neonatal cardiac myocytes (NCMs) and 2) to determine if differences in the α and εPKC autoinhibitory pseudosubstrate mechanisms could play roles in α and εPKC-selective functions. Introduction of the εφ into NCMs by transient permeabilization modestly attenuated 3nM 4-β PMA-induced slowing of contraction rate, an εPKC mediated response (Circ Res. 76:654-663; J. Biol. Chem. 271:24962-24966). In contrast, the αPKC pseudosubstrate peptide (αφ) (NH2-RFARKGALRQKNVHEVK-COOH) was 6- to 10-fold more potent at antagonizing the 3 nM 4-β PMA-induced slowing of contraction rate. Addition of purified PKC to the particulate cell fraction of NCMs promoted 32P incorporation into 3 proteins of ∼18, ∼46 and ∼97 kDa. The αφ antagonized these phosphorylations with IC50 values of 1 - 5 μM. These IC50 values were 1.8 - 4.7-fold lower than those observed for the εφ. In in vitro phosphorylation assays with recombinant α or ε PKC isozymes the εφ failed to inhibit the εPKC isozyme as potently as the αφ peptide but both the αφ and the εφ were equally effective inhibitors of the recombinant αPKC isozyme. In addition, in vitro cleavage of the εφ by the protease Arg-C in lysates from NCMs treated with 3 nM 4-β PMA was greatly enhanced when compared to that of the αPKC isozyme. Our studies suggest that the εφ cannot be used as a selective inhibitor of the εPKC isozyme in NCMs and that there are differences in the εPKC and αPKC autoinhibitory pseudosubstrate mechanisms.

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KW - α and εPKC

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