Association of phospholamban with a cGMP kinase signaling complex

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Abstract

The cGMP kinase signaling complex identified previously in tracheal smooth muscle membranes contains a number of cGMP kinase substrates termed G0 through G4. G0, G1, and G2 were identified as IP3 receptor I (IP3RI), IRAG, and cGMP kinase I. Sequencing of purified G3 and G4 showed that these proteins were proteolytic cleavage products of IRAG. However, the purified cGMP kinase signaling complex contained following additional proteins: α-actin, calponin H1, and phospholamban (PLB) as verified by MALDI-TOF as well as MS/MS sequencing and immune detection. The complex of these six proteins was immune precipitated by antibodies to each protein. The proteins were phosphorylated by the endogenous cGMP kinase I with the exception of α-actin and calponin H1. The complex did not contain the Ca2+-ATPase SERCA II. PLB, IP3RI, and cGMP kinase Iβ were co-immune precipitated after expression in COS-7 cells. These results suggest that PLB may have additional functions to regulate the activity of SERCA II.

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, 0.5μg/μl 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)

  • H.K Simmerman et al.

    Phospholamban: protein structure, mechanism of action, and role in cardiac function

    Physiol. Rev.

    (1998)
  • L Raeymaekers et al.

    Cyclic GMP-dependent protein kinase phosphorylates phospholamban in isolated sarcoplasmic reticulum from cardiac and smooth muscle

    Biochem. J.

    (1988)
  • T.L Cornwell et al.

    Regulation of sarcoplasmic reticulum protein phosphorylation by localized cyclic GMP-dependent protein kinase in vascular smooth muscle cells

    Mol. Pharmacol.

    (1991)
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    Abbreviations: RIα, regulatory subunit Iα of cAMP kinase; NDKB, nucleoside diphosphate kinase B; MALDI, matrix-assisted laser desorption ionisation; ES, nano-electrospray mass spectrometry.

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