MARCKS is a major PKC-dependent regulator of calmodulin targeting in smooth muscle.
Calmodulin (CaM) is a ubiquitous transducer of intracellular Ca(2+) 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 Ca(2+)-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 ( approximately 16 microM) in comparison to total CaM ( approximately 40 microM). 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 Ca(2+) 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.
Gallant
Cynthia
C
Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472, USA.
You
Jae Young
JY
Sasaki
Yasuharu
Y
Grabarek
Zenon
Z
Morgan
Kathleen G
KG
D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002147:Calmodulin / Q000378:metabolism*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005289:Ferrets; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism*; D009119:Muscle Contraction / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010455:Peptides / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology
D002147:Calmodulin; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010455:Peptides; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C; D002118:Calcium