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First published online 26 July 2005
doi: 10.1242/jcs.02493

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MARCKS is a major PKC-dependent regulator of calmodulin targeting in smooth muscle

¹ Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472, USA
² Wellesley College, 106 Central Street, Wellesley, MA 02481, USA
³ Department Pharmacology and Pharmaceutical Science, Kitasato University, Tokyo 108-8641, Japan
⁴ Department of Medicine, Harvard Medical School, Boston, MA 02115, USA

^* Author for correspondence (e-mail: morgan{at}bbri.org)

Accepted 13 May 2005

Calmodulin (CaM) is a ubiquitous transducer of intracellular Ca²⁺ signals and plays a key role in the regulation of the function of all cells. The interaction of CaM with a specific target is determined not only by the Ca²⁺-dependent affinity of calmodulin but also by the proximity to that target in the cellular environment. Although a few reports of stimulus-dependent nuclear targeting of CaM have appeared, the mechanisms by which CaM is targeted to non-nuclear sites are less clear. Here, we investigate the hypothesis that MARCKS is a regulator of the spatial distribution of CaM within the cytoplasm of differentiated smooth-muscle cells. In overlay assays with portal-vein homogenates, CaM binds predominantly to the MARCKS-containing band. MARCKS is abundant in portal-vein smooth muscle (16 µM) in comparison to total CaM (40 µM). Confocal images indicate that calmodulin and MARCKS co-distribute in unstimulated freshly dissociated smooth-muscle cells and are co-targeted simultaneously to the cell interior upon depolarization. Protein-kinase-C (PKC) activation triggers a translocation of CaM that precedes that of MARCKS and causes multisite, sequential MARCKS phosphorylation. MARCKS immunoprecipitates with CaM in a stimulus-dependent manner. A synthetic MARCKS effector domain (ED) peptide labelled with a photoaffinity probe cross-links CaM in smooth-muscle tissue in a stimulus-dependent manner. Both cross-linking and immunoprecipitation increase with increased Ca²⁺ concentration, but decrease with PKC activation. Introduction of a nonphosphorylatable MARCKS decoy peptide blocks the PKC-mediated targeting of CaM. These results indicate that MARCKS is a significant, PKC-releasable reservoir of CaM in differentiated smooth muscle and that it contributes to CaM signalling by modulating the intracellular distribution of CaM.

Key words: Calmodulin, PKC, Smooth muscle, MARCKS

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