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The p90 ribosomal S6 kinase (RSK) is a mediator of smooth muscle contractility
Mykhaylo Artamonov1, Ko Momotani, Darkhan Utepbergenov
1Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia, United States of America.
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 [Ca(2+)]. This pathway is referred to as Ca(2+)-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 Ca(2+)- 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 Ca(2+), 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.
Insights
The p90 ribosomal S6 kinase (RSK) plays a novel role in smooth muscle (SM) contraction by directly phosphorylating myosin regulatory light chain (RLC20). RSK inhibition reduces SM contractile force, highlighting its importance beyond gene expression regulation.
Area of Science:
- Physiology
- Molecular Biology
- Biochemistry
Background:
- Smooth muscle (SM) contraction is primarily regulated by myosin light chain kinase (MLCK) and RhoA-dependent ROCK kinase pathways.
- Existing models suggest Ca(2+)-sensitization is key, but other Ser/Thr kinases may also contribute to SM regulation.
- The p90 ribosomal S6 kinase (RSK) has been traditionally linked to gene expression, not contractile mechanisms.
Purpose of the Study:
- To investigate the potential role of p90 ribosomal S6 kinase (RSK) in regulating smooth muscle (SM) contractility.
- To determine if RSK activity influences myosin regulatory light chain (RLC20) and MYPT1 phosphorylation.
- To elucidate the mechanism by which RSK affects SM contraction.
Main Methods:
- Used a selective RSK inhibitor (BI-D1870) to assess its impact on agonist- and Ca(2+)-induced SM contraction.
- Measured phosphorylation levels of RLC20 and MYPT1 following RSK inhibition.
- Employed an irreversible MLCP inhibitor (microcystin-LR) to differentiate between direct and indirect effects of RSK on RLC20 phosphorylation.
- Assessed activation of ERK1/2 and PDK1 upon agonist stimulation to confirm RSK activation pathways.
Main Results:
- RSK inhibition significantly suppressed SM contractile force during both Ca(2+) and agonist (U46619) stimulation.
- Inhibition of RSK led to decreased phosphorylation of both RLC20 and MYPT1.
- RSK inhibition reduced RLC20 phosphorylation independently of changes in phosphatase activity, suggesting direct or indirect phosphorylation by RSK.
- Agonist stimulation activated ERK1/2 and PDK1, consistent with RSK activation.
Conclusions:
- p90 ribosomal S6 kinase (RSK) is a novel regulator of smooth muscle (SM) contractility.
- RSK directly or indirectly phosphorylates RLC20, contributing to SM force generation.
- This finding expands the known functions of RSK beyond gene expression into the realm of muscle contraction regulation.
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