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

Abstract-Subcellular targeting of kinases controls their activation and access to substrates. Although Ca 2ϩ /calmodulindependent 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 ␥ isoform of CaMKII that contains a potential targeting sequence in its association domain . Antisense knockdown of CaMKII␥ G-2 inhibited extracellular signal-related kinase (ERK) activation, myosin phosphorylation, and contractile force in dSMCs. Confocal colocalization analysis revealed that in unstimulated dSMCs CaMKII␥ G-2 is bound to a cytoskeletal scaffold consisting of interconnected vimentin intermediate filaments and cytosolic dense bodies. On activation with a depolarizing stimulus, CaMKII␥ G-2 is released into the cytosol and subsequently targeted to cortical dense plaques. Comparison of phosphorylation and translocation time courses indicates that, after CaMKII␥ G-2 activation, and before CaMKII␥ 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 CaMKII␥ G-2 inhibited targeting. Furthermore, prevention of CaMKII␥ 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. Key Words: CaMKII Ⅲ smooth muscle Ⅲ contractility Ⅲ targeting Ⅲ extracellular signal-regulated kinase C a 2ϩ /calmodulin-dependent protein kinase II (CaMKII) is a Ser/Thr kinase that is expressed as 4 different isoforms (␣, ␤, ␥, and ␦). Each member of this kinase family possesses 3 major domains ( Figure 1A): an N-terminal catalytic/regulatory domain containing a Ser/Thr kinase region and overlapping autoinhibitory and Ca 2ϩ /calmodulin binding regions, a central-linker domain containing variable regions, and a C-terminal association domain that promotes oligomerization. 1 CaMKII is not active until Ca 2ϩ /calmodulin binds to its regulatory domain. This binding allows CaMKII to undergo autophosphorylation at Thr287 (numbering according to the ␥ isoform), which, in turn, increases its affinity for Ca 2ϩ /calmodulin and allows kinase activity even in the absence of Ca 2ϩ /calmodulin. The ␣ and ␤ isoforms of CaMKII are predominantly expressed in neural tissue, 2 where they have been demonstrated to be involved in synaptic plasticity, memory, and learning. 3,4 In contrast, differentiated smooth muscle cells (dSMCs) express mainly the ␥ isoform of CaMKII, 5 which has been linked to contractile activity by the use of antisense and phar...

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

“…All procedures were performed according to protocols approved by the Institutional Care and Use Committee. As previously described (37), ferrets (Marshall Farms, North Rose, NY) were euthanized by an overdose of chloroform, and the aorta was quickly removed from animals and dissected in oxygenated (95% O2-5% CO2) physiological salt solution (PSS) consisting of (in mM) 120 NaCl, 5.9 KCl, 1.2 NaH 2PO4, 25 NaHCO3, 11.5 dextrose, 1 CaCl2, and 1.4 MgCl2 (pH 7.4). The aorta was carefully dissected to remove all connective tissue and the endothelium under a dissecting microscope.…”

“…Quick-frozen tissue samples were homogenized and analyzed via immunoblotting as previously published (37). Briefly, the homogenized samples were boiled in Laemmli sample buffer.…”

“…Cells were freshly enzymatically dissociated from aorta tissue by a previously published method (37).…”

The non-excitable smooth muscle: Calcium signaling and phenotypic switching during vascular disease

“…In these cells, CaMKII γ isoforms have been implicated in the control of the differentiated contractile function [71,88,120]. However, cultured VSMC predominantly express the δ 2 (also referred to as δ C ) isoform [124].…”

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.…”