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B. K. Tripathi, P. S. Zelenka; Cdk5 Specifically Affects Rho Activation Required for Myosin-Dependent Contraction and Stress Fiber Maintenance in Spreading Lens Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4767.
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© ARVO (1962-2015); The Authors (2016-present)
We previously showed that inhibiting Cdk5 reduced Rho-dependent myosin phosphorylation in lens epithelial cells, leading to changes in stress fiber organization. However, since the actin polymerization required for stress fiber assembly also requires Rho activation, it was not clear whether the loss of myosin phosphorylation by Cdk5 inhibition was due to a specific effect on Rho-dependent myosin contraction. The present study tests whether Cdk5 has a specific effect on the myosin-dependent contraction required for stress fiber maintenance.
Human lens epithelial cells were allowed to spread on 10 µg/ml fibronectin for 2 hr. Cdk5 activity was inhibited by a pharmacological inhibitor (olomoucine, 15 µM)), dominant negative GFP-Cdk5(D144N), or by Cdk5 siRNA. Rho and ROCK activity were blocked by specific inhibitors CT04 (2.0 ug/ml)) and Y-27632 (10 µM), respectively. Relative levels of Cdk5, Cdk5(pY15), MRLC, myosin binding subunit (MBS), MBS(pT853), and Thr18/Ser19 diphosphorylated MRLC (dpMRLC) were measured by immunoblotting. Rho-GTP levels were measured by affinity chromatography with Rhotekin-RBD. Cytoskeletal organization and subcellular localization of specific proteins were determined by confocal immunofluorescence microscopy.
Two hours after plating on fibronectin, 97% of cells showed well-formed stress fibers. Suppressing Cdk5 activity significantly reduced Rho-GTP formation and ROCK activation at this time. Immunofluorescence microscopy demonstrated that Cdk5 and its activator, p35, co-localized with dpMRLC on contracting stress fibers, consistent with a role in myosin regulation. Inhibiting Cdk5 with Cdk5 specific inhibitor olomoucine after stress fibers were fully formed reduced dpMRLC and dissociated pre-formed stress fibers. Bundling of thin radial actin fibrils and transverse arcs into stress fibers was blocked.
Inhibiting Cdk5 activity affects the Rho activation required for MRLC diphosphorylation and stress fiber maintenance, leading to loss of preformed stress fibers. Localization of Cdk5, p35, and Cdk5(pY15) on contracting stress fibers indicates local activation of Rho, in the vicinity of the contracting stress fibers.
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