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

Abstract

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.

Cytoskeletal remodeling in differentiated vascular smooth muscle is actin isoform dependent and stimulus dependent

“…Ratio measurements for phalloidin-to-DNase I ratios followed a modification of previously published methods (17). Phalloidin has been shown to be highly specific for F-actin, and DNase I was used to identify G-actin and discriminate it from F-actin (34).…”

“…The tissues, after peptide loading and extensive washing, are visually inspected, before proceeding with the experiment, for the yellowish-green color, confirming the successful loading of the FITC-labeled peptide. Details of this method are published in past studies from our laboratory using the same tissue and successfully loading TAT-peptides and peptides with similar TAT-like sequences (17,24,37).…”

Extracellular growth factors and mitogens cooperate to drive mitochondrial biogenesis

“…As shown in supplementary material Fig. S4A, it had no effect on the increased expression of mitochondria-related genes induced by NRG+IGF but it did block the phosphorylation of MARCKS (supplementary material Fig. Journal of Cell Science 122 (24) S4B), a target of both novel and conventional PKCs (Disatnik et al, 2002;Gallant et al, 2005). To investigate the role of the atypical PKCs that are not blocked by BIM, we used an inhibitor specific for PKC and PKC, with similar results (supplementary material Fig.…”

Focal adhesion signaling is required for myometrial ERK activation and contractile phenotype switch before labor

“…2B see arrows in insets). Vinculin staining shows the characteristic pattern of adhesion plaque staining in differentiated smooth muscle cells [Moore et al, 2004;Gallant et al, 2005;Marganski et al, 2005]. The antibody used recognizes only vinculin isoforms in a myometrial homogenate (Fig.…”