Nitric oxide synthase regulatory sites: Phosphorylation by cyclic AMP-dependent protein kinase, protein kinase C, and calcium/calmodulin protein kinase; identification of flavin and calmodulin binding sites

David S. Bredt, Christopher D. Ferris, Solomon H Snyder

Research output: Contribution to journalArticle

Abstract

Nitric oxide (NO) is an important molecular messenger accounting for endothelial-derived relaxing activity in blood vessels, mediating cytotoxic actions of macrophages, and functioning as a neurotransmitter in the brain and periphery. NO synthase (NOS) from brain has been purified to homogeneity and molecularly cloned. We now report that NOS is stoichiometrically phosphorylated by cAMP dependent protein kinase, protein kinase C, and calcium/calmodulin-dependent protein kinase, with each kinase phosphorylating a different serine site on NOS. Activation of PKC in transfected cells reduces NOS enzyme activity by ≈77% in intact cells and by 50% in protein homogenates from these cells. Utilizing fluorescence spectroscopy we find that purified monomer NOS contains 1 molar equivalent of both FMN and FAD. This stoichiometry is supported by enzymatic digestion of the flavins with phosphodiesterase, and titration of the FMN with a specific FMN binding protein. We demonstrate that purified NOS is labeled by a photoaffinity derivative of calmodulin. These recognition sites on NOS provide multiple means for regulation of NO levels and "cross-talk" between second messenger systems.

Original languageEnglish (US)
Pages (from-to)10976-10981
Number of pages6
JournalJournal of Biological Chemistry
Volume267
Issue number16
StatePublished - Jun 5 1992

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Calcium-Calmodulin-Dependent Protein Kinases
Phosphorylation
Calmodulin
Cyclic AMP-Dependent Protein Kinases
Nitric Oxide Synthase
Protein Kinases
Protein Kinase C
Binding Sites
Calcium
Flavin Mononucleotide
Brain
Nitric Oxide
Calcium-Calmodulin-Dependent Protein Kinase Kinase
Flavins
Flavin-Adenine Dinucleotide
Macrophages
Fluorescence Spectrometry
Phosphoric Diester Hydrolases
Blood vessels
Fluorescence spectroscopy

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Nitric oxide synthase regulatory sites: Phosphorylation by cyclic AMP-dependent protein kinase, protein kinase C, and calcium/calmodulin protein kinase; identification of flavin and calmodulin binding sites",
abstract = "Nitric oxide (NO) is an important molecular messenger accounting for endothelial-derived relaxing activity in blood vessels, mediating cytotoxic actions of macrophages, and functioning as a neurotransmitter in the brain and periphery. NO synthase (NOS) from brain has been purified to homogeneity and molecularly cloned. We now report that NOS is stoichiometrically phosphorylated by cAMP dependent protein kinase, protein kinase C, and calcium/calmodulin-dependent protein kinase, with each kinase phosphorylating a different serine site on NOS. Activation of PKC in transfected cells reduces NOS enzyme activity by ≈77{\%} in intact cells and by 50{\%} in protein homogenates from these cells. Utilizing fluorescence spectroscopy we find that purified monomer NOS contains 1 molar equivalent of both FMN and FAD. This stoichiometry is supported by enzymatic digestion of the flavins with phosphodiesterase, and titration of the FMN with a specific FMN binding protein. We demonstrate that purified NOS is labeled by a photoaffinity derivative of calmodulin. These recognition sites on NOS provide multiple means for regulation of NO levels and {"}cross-talk{"} between second messenger systems.",
author = "Bredt, {David S.} and Ferris, {Christopher D.} and Snyder, {Solomon H}",
year = "1992",
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T2 - Phosphorylation by cyclic AMP-dependent protein kinase, protein kinase C, and calcium/calmodulin protein kinase; identification of flavin and calmodulin binding sites

AU - Bredt, David S.

AU - Ferris, Christopher D.

AU - Snyder, Solomon H

PY - 1992/6/5

Y1 - 1992/6/5

N2 - Nitric oxide (NO) is an important molecular messenger accounting for endothelial-derived relaxing activity in blood vessels, mediating cytotoxic actions of macrophages, and functioning as a neurotransmitter in the brain and periphery. NO synthase (NOS) from brain has been purified to homogeneity and molecularly cloned. We now report that NOS is stoichiometrically phosphorylated by cAMP dependent protein kinase, protein kinase C, and calcium/calmodulin-dependent protein kinase, with each kinase phosphorylating a different serine site on NOS. Activation of PKC in transfected cells reduces NOS enzyme activity by ≈77% in intact cells and by 50% in protein homogenates from these cells. Utilizing fluorescence spectroscopy we find that purified monomer NOS contains 1 molar equivalent of both FMN and FAD. This stoichiometry is supported by enzymatic digestion of the flavins with phosphodiesterase, and titration of the FMN with a specific FMN binding protein. We demonstrate that purified NOS is labeled by a photoaffinity derivative of calmodulin. These recognition sites on NOS provide multiple means for regulation of NO levels and "cross-talk" between second messenger systems.

AB - Nitric oxide (NO) is an important molecular messenger accounting for endothelial-derived relaxing activity in blood vessels, mediating cytotoxic actions of macrophages, and functioning as a neurotransmitter in the brain and periphery. NO synthase (NOS) from brain has been purified to homogeneity and molecularly cloned. We now report that NOS is stoichiometrically phosphorylated by cAMP dependent protein kinase, protein kinase C, and calcium/calmodulin-dependent protein kinase, with each kinase phosphorylating a different serine site on NOS. Activation of PKC in transfected cells reduces NOS enzyme activity by ≈77% in intact cells and by 50% in protein homogenates from these cells. Utilizing fluorescence spectroscopy we find that purified monomer NOS contains 1 molar equivalent of both FMN and FAD. This stoichiometry is supported by enzymatic digestion of the flavins with phosphodiesterase, and titration of the FMN with a specific FMN binding protein. We demonstrate that purified NOS is labeled by a photoaffinity derivative of calmodulin. These recognition sites on NOS provide multiple means for regulation of NO levels and "cross-talk" between second messenger systems.

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