title: Targeting of a novel Ca+2/calmodulin-dependent protein kinase II is essential for extracellular signal-regulated kinase-mediated signaling in differentiated smooth muscle cells.
abstract: Subcellular targeting of kinases controls their activation and access to substrates. Although Ca2+/calmodulin-dependent protein kinase II (CaMKII) is known to regulate differentiated smooth muscle cell (dSMC) contractility, the importance of targeting in this regulation is not clear. The present study investigated the function in dSMCs of a novel variant of the gamma isoform of CaMKII that contains a potential targeting sequence in its association domain (CaMKIIgamma G-2). Antisense knockdown of CaMKIIgamma G-2 inhibited extracellular signal-related kinase (ERK) activation, myosin phosphorylation, and contractile force in dSMCs. Confocal colocalization analysis revealed that in unstimulated dSMCs CaMKIIgamma G-2 is bound to a cytoskeletal scaffold consisting of interconnected vimentin intermediate filaments and cytosolic dense bodies. On activation with a depolarizing stimulus, CaMKIIgamma G-2 is released into the cytosol and subsequently targeted to cortical dense plaques. Comparison of phosphorylation and translocation time courses indicates that, after CaMKIIgamma G-2 activation, and before CaMKIIgamma G-2 translocation, vimentin is phosphorylated at a CaMKII-specific site. Differential centrifugation demonstrated that phosphorylation of vimentin in dSMCs is not sufficient to cause its disassembly, in contrast to results in cultured cells. Loading dSMCs with a decoy peptide containing the polyproline sequence within the association domain of CaMKIIgamma G-2 inhibited targeting. Furthermore, prevention of CaMKIIgamma G-2 targeting led to significant inhibition of ERK activation as well as contractility. Thus, for the first time, this study demonstrates the importance of CaMKII targeting in dSMC signaling and identifies a novel targeting function for the association domain in addition to its known role in oligomerization.
authors.affiliation: Boston Biomedical Research Institute, Watertown, MA 02472, USA.
authors.forename: William A
authors.initials: WA
authors.lastname: Marganski
authors.forename: Samudra S
authors.initials: SS
authors.lastname: Gangopadhyay
authors.forename: Hyun-Dong
authors.initials: HD
authors.lastname: Je
authors.forename: Cynthia
authors.initials: C
authors.lastname: Gallant
authors.forename: Kathleen G
authors.initials: KG
authors.lastname: Morgan
mesh_terms: D000185:Actinin / Q000032:analysis / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000032:analysis / Q000737:chemistry / Q000502:physiology*; D002454:Cell Differentiation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology*; D005289:Ferrets; D048369:MAP Kinase Kinase 1 / Q000032:analysis; D048370:MAP Kinase Kinase 2 / Q000032:analysis; D020935:MAP Kinase Signaling System / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000502:physiology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D014661:Vasoconstriction; D014746:Vimentin / Q000032:analysis / Q000378:metabolism
chemical_list: D016376:Oligonucleotides, Antisense; D014746:Vimentin; D000185:Actinin; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2