Rho-kinase-mediated Ca²⁺-independent contraction in rat embryo fibroblasts

Abstract

(20,21,23,33,64), neurite outgrowth (1, 8, 57), endothelial cell contraction (23,69,70), and resistance to mechanical perturbations (8,11,31). These myosin II-based cellular events have been considered to be regulated solely by an increase in cytosolic Ca 2ϩ according to the classic smooth muscle model of Ca 2ϩ -coupled contraction. However, within the last 10 years myosin II motor activity has been shown to be activated by Ca 2ϩ -independent pathways (39,41,63,68) in addition to Ca 2ϩ -dependent pathways (7,23,32,70). Ca 2ϩ -dependent cell contraction results from an influx of Ca 2ϩ from the extracellular space or the release of Ca 2ϩ from sequestered internal stores. Ca 2ϩ binds to calmodulin (CaM); the Ca 2ϩ /CaM complex in turn binds to and activates the serine/threonine protein kinase, myosin light chain kinase (MLCK). The active MLCK complex catalyzes myosin II regulatory light chain (RLC) phosphorylation at two sites, Ser-19 and Thr-18. Phosphorylation at these sites is required for myosin II filament formation (6), myosin II interaction with F-actin (23), and an increase in myosin II ATPase activity (7). These phosphorylation driven events are essential for initiation and maintenance of myosin II-based contraction. MLCK, the enzyme responsible for initiating Ca 2ϩ -dependent contraction, exists in several isoforms, resulting from splice variants with distinct intracellular localizations and tissue distributions (3,5,32,52). To date, all MLCK isoforms have been shown biochemically to be strictly dependent on Ca 2ϩ /CaM for activation and are known to catalyze phosphorylation of a single substrate, myosin II RLC.The discovery of the Rho family of proteins initiated study of possible Ca 2ϩ -independent regulation of myosin II motor activity and contraction. A range of protein kinases have now been shown to activate myosin II motor activity by catalyzing phosphorylation of myosin II RLC in vitro or in vivo, including Rho-kinase (2), p21-associated protein kinase (PAK) (9, 73), and integrin-linked kinase (ILK) (10). The most intensively studied of the Ca 2ϩ -independent activators of myosin II motor activity and contraction are those regulated by the Rho family of proteins. Rho is a member of the Ras superfamily of small GTPases, which functions as a molecular switch cycling between the active GTP-bound and inactive GDP-bound state. In the active GTP state, RhoA interacts with its effector molecules to initiate downstream responses. Of several possible Rho effector proteins (4, 17) that bind to GTP-bound Rho, the serine/threonine kinase, Rho-kinase, is the one that has been implicated in the regulation of myosin II and contraction.Rho-kinase exists in two isoforms, ROK␣/ROCK-II (43, 46) and ROK␤/ROCK-I/p160 ROCK (29, 42); both are ubiquitously expressed in various tissues and cells. Rho-kinase isoforms range in mass from 150 to 160 kDa and contain a kinase domain in their NH 2 -terminal domains, a central coil-coiled domain, and a pleckstrin homology domain in the COOH-terminal domains. The coil-coiled domai...

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Myofibroblast communication is controlled by intercellular mechanical coupling

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Horse Chestnut Extract Induces Contraction Force Generation in Fibroblasts through Activation of Rho/Rho Kinase

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