Biochemical and Biophysical Research Communications
Association of phospholamban with a cGMP kinase signaling complex☆
Section snippets
Materials and methods
Unless indicated otherwise, all procedures including buffers used for the purification of the membranes and the protein complex were carried out at 0–4 °C. All buffers contained following protease inhibitors: 1 mM benzamidine, leupeptin, and 0.3 mM phenylmethylsulfonyl fluoride.
Preparation of smooth muscle membranes. Bovine tracheal smooth muscle was prepared and phosphorylated as described previously [12]. The preparation of microsomal membranes was modified. EDTA (5 mM) and the above
Characterization of the cGK signaling complex
Several previous studies showed that smooth muscle membranes contain a number of cGKI substrates termed G0, G1, G2, G3, and G4 [10], [12], [17]. Recently, G0, identified as IP3 receptor type I (IP3RI) [18], G1, a new protein termed IRAG (IP3 receptor associated cGMP kinase substrate), and G2, cGMP kinase Iβ, were demonstrated to form a multimeric complex [12]. The nature of the other two substrates remained unclear. MALDI-TOF analysis of the purified putative substrates G3 (65 kDa) and G4 (30
Discussion
This study analyzed the composition of a cGMP kinase signaling complex present in the microsomal membranes of tracheal smooth muscle. The analysis of the cGMP kinase substrates G3 and G4 showed that these proteins were derived proteolytically from the recently identified cGMP kinase substrate IRAG. However, we identified by sequencing, immune decoration, and immune precipitation additional members of the signaling complex. Calponin and α-actin are cytoskeletal proteins. Presumably, they anchor
Acknowledgements
We thank Dr. F. Wuytack for SERCA IIa and SERCA IIb polyclonal antibodies and Dr. M. Aepfelbacher for the Rho A monoclonal antibody. This work was supported by grants from Deutsche Forschungsgemeinschaft, Sanderstiftung, Fond der Chemischen Industrie, and stipend by the TU München to A.K.
References (26)
- et al.
Phospholamban: a protein coming of age
Biochem. Biophys. Res. Commun.
(1997) - et al.
Phospholamban inhibitory function is activated by depolymerization
J. Biol. Chem.
(1997) - et al.
Atrial natriuretic peptide-dependent phosphorylation of smooth muscle cell particulate fraction proteins is mediated by cGMP-dependent protein kinase
J. Biol. Chem.
(1989) - et al.
Molecular determinants of the interaction between the inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate (IRAG) and cGMP kinase Ibeta
J. Biol. Chem.
(2001) - et al.
Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa
Anal. Biochem.
(1987) - et al.
Purification and characterization of 240-kDa cGMP-dependent protein kinase substrate of vascular smooth muscle. Close resemblance to inositol 1,4,5-trisphosphate receptor
J. Biol. Chem.
(1994) - et al.
cGMP-dependent protein kinase phosphorylates and inactivates RhoA
Biochem. Biophys. Res. Commun.
(2001) - et al.
Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle
J. Biol. Chem.
(2000) - et al.
Signaling microdomains define the specificity of receptor-mediated InsP(3) pathways in neurons
Neuron
(2002) - et al.
Role of phospholamban in NO/EDRF-induced relaxation in rat aorta
Life Sci.
(1992)
Phospholamban: protein structure, mechanism of action, and role in cardiac function
Physiol. Rev.
Cyclic GMP-dependent protein kinase phosphorylates phospholamban in isolated sarcoplasmic reticulum from cardiac and smooth muscle
Biochem. J.
Regulation of sarcoplasmic reticulum protein phosphorylation by localized cyclic GMP-dependent protein kinase in vascular smooth muscle cells
Mol. Pharmacol.
Cited by (42)
Endothelium-independent vasodilator effect of 2-nitro-1-phenyl-1-propanol on mesenteric resistance vessels in rats
2017, European Journal of PharmacologyCitation Excerpt :Cellular responses to cGMP involve target proteins, known as cGMP-dependent protein kinases (e.g., protein kinase G [PKG]; Lucas et al., 2000). Once activated, targeted proteins may induce other events that result in lower Ca2+ release from intracellular stores (Xia et al., 2001) or higher Ca2+ sequestration to the sarcoplasmic reticulum (Koller et al., 2003). Vasorelaxant compounds that stimulate guanylate cyclase can also modulate the gating of K+ channels, resulting in pronounced hyperpolarization and the inhibition of force in vascular smooth muscle (Kubo et al., 1994).
A Restricted Repertoire of de Novo Mutations in ITPR1 Cause Gillespie Syndrome with Evidence for Dominant-Negative Effect
2016, American Journal of Human GeneticsCitation Excerpt :ITPR1 and ATCA2 might interact in smooth muscle as components of the cGMP kinase signaling complex.49
Cyclic GMP-Dependent Protein Kinase: Targeting and Control of Expression
2009, Handbook of Cell Signaling, Second EditionNitric Oxide in Cardiac Surgery: A Review Article
2023, BiomedicinesStudy of blood rheological parameters and NO in coronary artery disease patients with and without collaterals
2023, Clinical Hemorheology and Microcirculation
- ☆
Abbreviations: RIα, regulatory subunit Iα of cAMP kinase; NDKB, nucleoside diphosphate kinase B; MALDI, matrix-assisted laser desorption ionisation; ES, nano-electrospray mass spectrometry.