The commonly used cGMP-dependent protein kinase type I (cGKI) inhibitor Rp-8-Br-PET-cGMPS can activate cGKI in vitro and in intact cells

Nadejda Valtcheva; Peter Nestorov; Alexander Beck; Michael Russwurm; Matthias Hillenbrand; Pascal Weinmeister; Robert Feil

doi:10.1074/jbc.M806161200

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The commonly used cGMP-dependent protein kinase type I (cGKI) inhibitor Rp-8-Br-PET-cGMPS can activate cGKI in vitro and in intact cells

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The commonly used cGMP-dependent protein kinase type I (cGKI) inhibitor Rp-8-Br-PET-cGMPS can activate cGKI in vitro and in intact cells

Nadejda Valtcheva, Peter Nestorov, Alexander Beck, Michael Russwurm, Matthias Hillenbrand, Pascal Weinmeister et Robert Feil

The Journal of biological chemistry, Vol.284(1), pp.556-562

02/01/2009

DOI: https://doi.org/10.1074/jbc.M806161200

PMID: 19008225

Résumé

Animals

Cell Adhesion Molecules - chemistry

Cell Adhesion Molecules - metabolism

Cyclic GMP - analogs & derivatives

Cyclic GMP - chemistry

Cyclic GMP - pharmacology

Enzyme Activation - drug effects

Isoenzymes - antagonists & inhibitors

Isoenzymes - chemistry

Isoenzymes - metabolism

Mice

Mice, Knockout

Microfilament Proteins - chemistry

Microfilament Proteins - metabolism

Muscle, Smooth, Vascular - cytology

Muscle, Smooth, Vascular - enzymology

Myocytes, Smooth Muscle - cytology

Myocytes, Smooth Muscle - enzymology

Phosphoproteins - chemistry

Phosphoproteins - metabolism

Phosphorylation - drug effects

Protein Kinase Inhibitors - chemistry

Protein Kinase Inhibitors - pharmacology

Protein Kinases - chemistry

Protein Kinases - metabolism

Small-molecule modulators of cGMP signaling are of interest to basic and clinical research. The cGMP-dependent protein kinase type I (cGKI) is presumably a major mediator of cGMP effects, and the cGMP analogue Rp-8-Br-PET-cGMPS (Rp-PET) (chemical name: beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer) is currently considered one of the most permeable, selective, and potent cGKI inhibitors available for intact cell studies. Here, we have evaluated the properties of Rp-PET using cGKI-expressing and cGKI-deficient primary vascular smooth muscle cells (VSMCs), purified cGKI isozymes, and an engineered cGMP sensor protein. cGKI activity in intact VSMCs was monitored by cGMP/cGKI-stimulated cell growth and phosphorylation of vasodilator-stimulated phosphoprotein. Unexpectedly, Rp-PET (100 microm) did not efficiently antagonize activation of cGKI by the agonist 8-Br-cGMP (100 microm) in intact VSMCs. Moreover, in the absence of 8-Br-cGMP, Rp-PET (100 microm) stimulated cell growth in a cGKIalpha-dependent manner. Kinase assays with purified cGKI isozymes confirmed the previously reported inhibition of the cGMP-stimulated enzyme by Rp-PET in vitro. However, in the absence of the agonist cGMP, Rp-PET partially activated the cGKIalpha isoform. Experiments with a fluorescence resonance energy transfer-based construct harboring the cGMP binding sites of cGKI suggested that binding of Rp-PET induces a conformational change similar to the agonist cGMP. Together, these findings indicate that Rp-PET is a partial cGKIalpha agonist that under certain conditions stimulates rather than inhibits cGKI activity in vitro and in intact cells. Data obtained with Rp-PET as cGKI inhibitor should be interpreted with caution and not be used as sole evidence to dissect the role of cGKI in signaling processes.

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Titre: The commonly used cGMP-dependent protein kinase type I (cGKI) inhibitor Rp-8-Br-PET-cGMPS can activate cGKI in vitro and in intact cells
Créateurs - sans rôle: Nadejda Valtcheva - Interfakulta¨res Institut fu¨r Biochemie, Universita¨t Tu¨bingen, 72076 Tu¨bingen, the Zentrum fu¨r Klinische Massenspektrometrie GmbH, 74076 Heilbronn, the Institut fu¨r Pharmakologie und Toxikologie, Universita¨t Bochum, 44780 Bochum, and the Institut fu¨r Pharmakologie und Toxikologie, Technische Universita¨t Mu¨nchen, 80802 Mu¨nchen Germany
Peter Nestorov - Interfakulta¨res Institut fu¨r Biochemie, Universita¨t Tu¨bingen, 72076 Tu¨bingen, the Zentrum fu¨r Klinische Massenspektrometrie GmbH, 74076 Heilbronn, the Institut fu¨r Pharmakologie und Toxikologie, Universita¨t Bochum, 44780 Bochum, and the Institut fu¨r Pharmakologie und Toxikologie, Technische Universita¨t Mu¨nchen, 80802 Mu¨nchen Germany
Alexander Beck - Interfakulta¨res Institut fu¨r Biochemie, Universita¨t Tu¨bingen, 72076 Tu¨bingen, the Zentrum fu¨r Klinische Massenspektrometrie GmbH, 74076 Heilbronn, the Institut fu¨r Pharmakologie und Toxikologie, Universita¨t Bochum, 44780 Bochum, and the Institut fu¨r Pharmakologie und Toxikologie, Technische Universita¨t Mu¨nchen, 80802 Mu¨nchen Germany
Michael Russwurm - Interfakulta¨res Institut fu¨r Biochemie, Universita¨t Tu¨bingen, 72076 Tu¨bingen, the Zentrum fu¨r Klinische Massenspektrometrie GmbH, 74076 Heilbronn, the Institut fu¨r Pharmakologie und Toxikologie, Universita¨t Bochum, 44780 Bochum, and the Institut fu¨r Pharmakologie und Toxikologie, Technische Universita¨t Mu¨nchen, 80802 Mu¨nchen Germany
Matthias Hillenbrand - Interfakulta¨res Institut fu¨r Biochemie, Universita¨t Tu¨bingen, 72076 Tu¨bingen, the Zentrum fu¨r Klinische Massenspektrometrie GmbH, 74076 Heilbronn, the Institut fu¨r Pharmakologie und Toxikologie, Universita¨t Bochum, 44780 Bochum, and the Institut fu¨r Pharmakologie und Toxikologie, Technische Universita¨t Mu¨nchen, 80802 Mu¨nchen Germany
Pascal Weinmeister - Interfakulta¨res Institut fu¨r Biochemie, Universita¨t Tu¨bingen, 72076 Tu¨bingen, the Zentrum fu¨r Klinische Massenspektrometrie GmbH, 74076 Heilbronn, the Institut fu¨r Pharmakologie und Toxikologie, Universita¨t Bochum, 44780 Bochum, and the Institut fu¨r Pharmakologie und Toxikologie, Technische Universita¨t Mu¨nchen, 80802 Mu¨nchen Germany
Robert Feil - Interfakulta¨res Institut fu¨r Biochemie, Universita¨t Tu¨bingen, 72076 Tu¨bingen, the Zentrum fu¨r Klinische Massenspektrometrie GmbH, 74076 Heilbronn, the Institut fu¨r Pharmakologie und Toxikologie, Universita¨t Bochum, 44780 Bochum, and the Institut fu¨r Pharmakologie und Toxikologie, Technische Universita¨t Mu¨nchen, 80802 Mu¨nchen Germany. Electronic address: robert.feil@uni-tuebingen.de
Détails de publication: The Journal of biological chemistry, Vol.284(1), pp.556-562
Identifiants: 99154040009311
Unité académique: Institut de Génétique Moléculaire de Montpellier - IGMM
Langue: English
Type de ressource: Journal article