Association of phospholamban with a cGMP kinase signaling complex q Angela Koller, a, * Jens Schlossmann, a Keith Ashman, b Sandrine Uttenweiler-Joseph, c Peter Ruth, d and Franz Hofmann a a Institut f€ ur Pharmakologie und Toxikologie, TU M€ unchen, Biedersteiner Straße 29, 80802 Munich, Germany b Samuel Lunenfeld Institute, 600 University Avenue, Toronto, Ont., Canada M5G 1X5 c IPBS, CNRS, 205, route de Narbonne, 31077 Toulouse Cedex, France d Pharmakologie und Toxikologie, Pharmazeutisches Institut, Universit€ atT€ ubingen, Auf der Morgenstelle 8, 72076 T€ ubingen, Germany Received 18 November 2002 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 IP 3 receptor I (IP 3 RI), 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: a-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 a-actin and calponin H1. The complex did not contain the Ca 2þ -ATPase SERCA II. PLB, IP 3 RI, and cGMP kinase Ib 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. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Phospholamban; cGMP kinase I; IRAG; IP 3 receptor I; a-Actin; Calponin H1; SERCA II; Co-immune precipitation; Signaling complex; Smooth muscle Phospholamban (PLB) is a 5-kDa membrane protein that forms a pentamer and associates with the sarco- plasmatic Ca 2þ -ATPase II (SERCA II) in heart, smooth muscle, and other tissues [1,2]. PLB is phosphorylated by cAMP kinase at Ser 16 , by protein kinase C at Ser 10 , and by Ca 2þ /calmodulin kinase at Thr 17 . Phosphoryla- tion results in dissociation of the protein from the Ca 2þ - ATPase [3] relieving inhibition of Ca 2þ transport from the cytosol to the sarcoplasmatic reticulum. Phosphor- ylation of PLB has been detected in vitro by cGMP kinase I (cGKI) [4] and after stimulation with atrial natriuretic peptide (ANP) [5] in smooth muscle tissue. In intact aortic smooth muscle cells, PLB is phosphory- lated after treatment with sodium nitroprussid or ANP [6]. Deletion of PLB in mice increased vascular tone, but decreased the sensitivity of several smooth muscle preparations against potassium chloride, phenylephrine, and carbachol [7,8]. It was even reported that phos- phorylation of PLB played only a minor role, if any, in cyclic nucleotide-mediated relaxation [9]. Cassnellie and Greengard [10] identified a number of cGMP kinase substrates in membrane fractions from smooth muscle, termed G0 to G4. Several of the sub- strates have been identified. G0 is the IP 3 receptor I, G1 is IRAG, and G2 is cGMP kinase Ib (see [11] for ref- erences). These three proteins are associated in a tight complex and form a cGMP kinase signaling complex in bovine tracheal smooth muscle membranes [12]. Anal- ysis of this complex revealed that it contained additional unknown proteins termed G3 and G4 that were phos- phorylated in the presence of cGMP. The purpose of this study was to identify the nature of G3 and G4. Sequencing of the purified G3 and G4 proteins identified Biochemical and Biophysical Research Communications 300 (2003) 155–160 www.elsevier.com/locate/ybbrc BBRC q Abbreviations: RIa, regulatory subunit Ia of cAMP kinase; NDKB, nucleoside diphosphate kinase B; MALDI, matrix-assisted laser desorption ionisation; ES, nano-electrospray mass spectrometry. * Corresponding author. Fax: +49-89-4140-3261. E-mail address: koller@ipt.med.tu-muenchen.de (A. Koller). 0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S0006-291X(02)02799-7