Abstract
In the canonical model of smooth muscle (SM) contraction, the contractile force is generated by phosphorylation of the myosin regulatory light chain (RLC20) by the myosin light chain kinase (MLCK). Moreover, phosphorylation of the myosin targeting subunit (MYPT1) of the RLC20 phosphatase (MLCP) by the RhoA-dependent ROCK kinase, inhibits the phosphatase activity and consequently inhibits dephosphorylation of RLC20 with concomitant increase in contractile force, at constant intracellular [Ca2+]. This pathway is referred to as Ca2+-sensitization. There is, however, emerging evidence suggesting that additional Ser/Thr kinases may contribute to the regulatory pathways in SM. Here, we report data implicating the p90 ribosomal S6 kinase (RSK) in SM contractility. During both Ca2+- and agonist (U46619) induced SM contraction, RSK inhibition by the highly selective compound BI-D1870 (which has no effect on MLCK or ROCK) resulted in significant suppression of contractile force. Furthermore, phosphorylation levels of RLC20 and MYPT1 were both significantly decreased. Experiments involving the irreversible MLCP inhibitor microcystin-LR, in the absence of Ca2+, revealed that the decrease in phosphorylation levels of RLC20 upon RSK inhibition are not due solely to the increase in the phosphatase activity, but reflect direct or indirect phosphorylation of RLC20 by RSK. Finally, we show that agonist (U46619) stimulation of SM leads to activation of extracellular signal-regulated kinases ERK1/2 and PDK1, consistent with a canonical activation cascade for RSK. Thus, we demonstrate a novel and important physiological function of the p90 ribosomal S6 kinase, which to date has been typically associated with the regulation of gene expression.
Highlights
Contraction and relaxation of smooth muscle (SM), which is found in the walls of blood vessels, lymphatic vessels, bladder, uterus, the gastrointestinal, respiratory and reproductive tracts, as well as other hollow organs, play a critical role in the regulation of blood pressure, pulmonary resistance, gastrointestinal motility, urogenital and erectile function [1]
The release of select agonists, which act on G-protein coupled receptors (GPCRs), up-regulates the cytosolic GTPase RhoA, which in turn activates the Rho-associated protein kinase-ROCK
We show that inhibition of ribosomal S6 kinase (RSK) significantly reduces contractile response in intact SM stimulated by either high [K+] or the thromboxane A2 (TXA2) analogue U46619, which typically induces Ca2+-sensitization cascade through the activation of the TXA2 receptors and the RhoA/ROCK signaling cascade
Summary
Contraction and relaxation of smooth muscle (SM), which is found in the walls of blood vessels, lymphatic vessels, bladder, uterus, the gastrointestinal, respiratory and reproductive tracts, as well as other hollow organs, play a critical role in the regulation of blood pressure, pulmonary resistance, gastrointestinal motility, urogenital and erectile function [1]. In SM, contraction is induced when Ca2+-bound calmodulin binds to and activates the myosin light chain kinase (MLCK), which phosphorylates the regulatory myosin light chain (RLC20) on Ser, with concomitant activation of the ATPase activity of myosin [1]. Over the past two decades, it has been shown that this relatively simple Ca2+/calmodulin-dependent paradigm is modulated in a complex and often tissue-specific way by phenomena which are Ca2+-independent and which amplify the contractile response to Ca2+, leading to ‘Ca2+ sensitization’ [5,6]. Two distinct molecular mechanisms have been reported for this pathway. The first of these invokes Ca2+-independent activation of kinases other than MLCK, capable of direct phosphorylation of RLC20. The second mechanism involves agonist-dependent downregulation of MLCP, leading to increased contractility at constant intracellular Ca2+ concentration ([Ca2+]i). The release of select agonists (e.g. thromboxane A2), which act on G-protein coupled receptors (GPCRs), up-regulates the cytosolic GTPase RhoA, which in turn activates the Rho-associated protein kinase-ROCK
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