2 research outputs found
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Phosphorylation at in the ATP-Binding Site of /Calmodulin-Dependent Kinase II as a Mechanism for Switching off the Kinase Activity
CaMKII /calmodulin-dependent kinase II) is a serine/threonine phosphotransferase that is capable of long-term retention of activity due to autophosphorylation at a specific threonine residue within each subunit of its oligomeric structure. The isoform of CaMKII is a significant regulator of vascular contractility. Here, we show that phosphorylation of CaMKII at , a residue located within the ATP-binding site, terminates the sustained activity of the enzyme. To test the physiological importance of phosphorylation at , we generated a phosphospecific antibody and demonstrated an increase in phosphorylation upon depolarization and contraction of blood vessels. To determine if the phosphorylation of affects the kinase activity, we mutated to alanine or aspartic acid. The S26D mutation mimicking the phosphorylated state of CaMKII causes a dramatic decrease in autophosphorylation levels and greatly reduces the catalytic activity towards an exogenous substrate (autocamtide-3), whereas the S26A mutation has no effect. These data combined with molecular modelling indicate that a negative charge at of CaMKII inhibits the catalytic activity of the enzyme towards its autophosphorylation site at most probably by blocking ATP binding. We propose that phosphorylation constitutes an important mechanism for switching off CaMKII activity
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JunB Mediates Basal- and TGFβ1-Induced Smooth Muscle Cell Contractility
Smooth muscle contraction is a dynamic process driven by acto-myosin interactions that are controlled by multiple regulatory proteins. Our studies have shown that members of the AP-1 transcription factor family control discrete behaviors of smooth muscle cells (SMC) such as growth, migration and fibrosis. However, the role of AP-1 in regulation of smooth muscle contractility is incompletely understood. In this study we show that the AP-1 family member JunB regulates contractility in visceral SMC by altering actin polymerization and myosin light chain phosphorylation. JunB levels are robustly upregulated downstream of transforming growth factor beta-1 (TGFβ1), a known inducer of SMC contractility. RNAi-mediated silencing of JunB in primary human bladder SMC (pBSMC) inhibited cell contractility under both basal and TGFβ1-stimulated conditions, as determined using gel contraction and traction force microscopy assays. JunB knockdown did not alter expression of the contractile proteins α-SMA, calponin or SM22α. However, JunB silencing decreased levels of Rho kinase (ROCK) and myosin light chain (MLC20). Moreover, JunB silencing attenuated phosphorylation of the MLC20 regulatory phosphatase subunit MYPT1 and the actin severing protein cofilin. Consistent with these changes, cells in which JunB was knocked down showed a reduction in the F:G actin ratio in response to TGFβ1. Together these findings demonstrate a novel function for JunB in regulating visceral smooth muscle cell contractility through effects on both myosin and the actin cytoskeleton