Studies on the regulatory domain of Ca2+/calmodulin-dependent protein kinase II: Functional analyses of arginine 283 using synthetic inhibitory peptides and site-directed mutagenesis of the α subunit

Yiu Lian Fong, Thomas Soderling

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Abstract

The regulatory role of Arg283 in the autoinhibitory domain of Ca2+/calmodulin-dependent protein kinase II was investigated using substituted inhibitory synthetic peptides and site-directed mutation of the expressed kinase. In the synthetic peptide corresponding to the autoinhibitory domain (residues 281-309) of Ca2+/calmodulin-dependent protein kinase II, substitution of Arg283 by other residues increased the IC50 values of the peptides in the following order: Arg ≪ Lys ≪ Gln ≪ Glu. Site-directed mutations of Arg283 to glutamic acid and glutamine in the kinase α subunit cDNA were transcribed and translated in vitro. The expressed enzymes had the same total kinase activities, determined in the presence of Ca2+/CaM, but the Glu283 mutant had a slightly higher Ca2+-independent kinase activity (5.46 ± 0.88%) compared to the wild-type Arg283 (1.86 ± 0.71%) and the Gln283 mutant (2.15 ± 0.60%). When the expressed kinases were subjected to limited autophosphorylation on ice to monitor generation of the Ca2+-independent activity, the Arg283 kinase attained maximal Ca2+-independent activity (about 20%) within 30 s, whereas the Gln283 and Glu283 mutants attained maximal Ca2+-independence only after about 40 min of autophosphorylation. The results indicate that Arg283 is a very important determinant for the regulatory autophosphorylation of Thr286 that generates the Ca2+-independent activity but is not essential for the other multiple autophosphorylations within Ca2+/calmodulin-dependent protein kinase II, and that Arg283 is only one of several important residues for the inhibitory potency of the autoinhibitory domain.

Original languageEnglish (US)
Pages (from-to)11091-11097
Number of pages7
JournalJournal of Biological Chemistry
Volume265
Issue number19
StatePublished - Jul 5 1990
Externally publishedYes

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Calcium-Calmodulin-Dependent Protein Kinase Type 2
Mutagenesis
Site-Directed Mutagenesis
Arginine
Phosphotransferases
Peptides
Mutation
Ice
Glutamine
Inhibitory Concentration 50
Glutamic Acid
Substitution reactions
Complementary DNA
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{4128a769cf0a491798dd77d11f74713e,
title = "Studies on the regulatory domain of Ca2+/calmodulin-dependent protein kinase II: Functional analyses of arginine 283 using synthetic inhibitory peptides and site-directed mutagenesis of the α subunit",
abstract = "The regulatory role of Arg283 in the autoinhibitory domain of Ca2+/calmodulin-dependent protein kinase II was investigated using substituted inhibitory synthetic peptides and site-directed mutation of the expressed kinase. In the synthetic peptide corresponding to the autoinhibitory domain (residues 281-309) of Ca2+/calmodulin-dependent protein kinase II, substitution of Arg283 by other residues increased the IC50 values of the peptides in the following order: Arg ≪ Lys ≪ Gln ≪ Glu. Site-directed mutations of Arg283 to glutamic acid and glutamine in the kinase α subunit cDNA were transcribed and translated in vitro. The expressed enzymes had the same total kinase activities, determined in the presence of Ca2+/CaM, but the Glu283 mutant had a slightly higher Ca2+-independent kinase activity (5.46 ± 0.88{\%}) compared to the wild-type Arg283 (1.86 ± 0.71{\%}) and the Gln283 mutant (2.15 ± 0.60{\%}). When the expressed kinases were subjected to limited autophosphorylation on ice to monitor generation of the Ca2+-independent activity, the Arg283 kinase attained maximal Ca2+-independent activity (about 20{\%}) within 30 s, whereas the Gln283 and Glu283 mutants attained maximal Ca2+-independence only after about 40 min of autophosphorylation. The results indicate that Arg283 is a very important determinant for the regulatory autophosphorylation of Thr286 that generates the Ca2+-independent activity but is not essential for the other multiple autophosphorylations within Ca2+/calmodulin-dependent protein kinase II, and that Arg283 is only one of several important residues for the inhibitory potency of the autoinhibitory domain.",
author = "Fong, {Yiu Lian} and Thomas Soderling",
year = "1990",
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T1 - Studies on the regulatory domain of Ca2+/calmodulin-dependent protein kinase II

T2 - Functional analyses of arginine 283 using synthetic inhibitory peptides and site-directed mutagenesis of the α subunit

AU - Fong, Yiu Lian

AU - Soderling, Thomas

PY - 1990/7/5

Y1 - 1990/7/5

N2 - The regulatory role of Arg283 in the autoinhibitory domain of Ca2+/calmodulin-dependent protein kinase II was investigated using substituted inhibitory synthetic peptides and site-directed mutation of the expressed kinase. In the synthetic peptide corresponding to the autoinhibitory domain (residues 281-309) of Ca2+/calmodulin-dependent protein kinase II, substitution of Arg283 by other residues increased the IC50 values of the peptides in the following order: Arg ≪ Lys ≪ Gln ≪ Glu. Site-directed mutations of Arg283 to glutamic acid and glutamine in the kinase α subunit cDNA were transcribed and translated in vitro. The expressed enzymes had the same total kinase activities, determined in the presence of Ca2+/CaM, but the Glu283 mutant had a slightly higher Ca2+-independent kinase activity (5.46 ± 0.88%) compared to the wild-type Arg283 (1.86 ± 0.71%) and the Gln283 mutant (2.15 ± 0.60%). When the expressed kinases were subjected to limited autophosphorylation on ice to monitor generation of the Ca2+-independent activity, the Arg283 kinase attained maximal Ca2+-independent activity (about 20%) within 30 s, whereas the Gln283 and Glu283 mutants attained maximal Ca2+-independence only after about 40 min of autophosphorylation. The results indicate that Arg283 is a very important determinant for the regulatory autophosphorylation of Thr286 that generates the Ca2+-independent activity but is not essential for the other multiple autophosphorylations within Ca2+/calmodulin-dependent protein kinase II, and that Arg283 is only one of several important residues for the inhibitory potency of the autoinhibitory domain.

AB - The regulatory role of Arg283 in the autoinhibitory domain of Ca2+/calmodulin-dependent protein kinase II was investigated using substituted inhibitory synthetic peptides and site-directed mutation of the expressed kinase. In the synthetic peptide corresponding to the autoinhibitory domain (residues 281-309) of Ca2+/calmodulin-dependent protein kinase II, substitution of Arg283 by other residues increased the IC50 values of the peptides in the following order: Arg ≪ Lys ≪ Gln ≪ Glu. Site-directed mutations of Arg283 to glutamic acid and glutamine in the kinase α subunit cDNA were transcribed and translated in vitro. The expressed enzymes had the same total kinase activities, determined in the presence of Ca2+/CaM, but the Glu283 mutant had a slightly higher Ca2+-independent kinase activity (5.46 ± 0.88%) compared to the wild-type Arg283 (1.86 ± 0.71%) and the Gln283 mutant (2.15 ± 0.60%). When the expressed kinases were subjected to limited autophosphorylation on ice to monitor generation of the Ca2+-independent activity, the Arg283 kinase attained maximal Ca2+-independent activity (about 20%) within 30 s, whereas the Gln283 and Glu283 mutants attained maximal Ca2+-independence only after about 40 min of autophosphorylation. The results indicate that Arg283 is a very important determinant for the regulatory autophosphorylation of Thr286 that generates the Ca2+-independent activity but is not essential for the other multiple autophosphorylations within Ca2+/calmodulin-dependent protein kinase II, and that Arg283 is only one of several important residues for the inhibitory potency of the autoinhibitory domain.

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