Purchase this article with an account.
B. K. Tripathi, C. Y. Gao, F. Qiao, P. Zelenka; Cdk5 Activity Regulates Myosin Phosphorylation in Lens Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5410.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Cyclin dependent kinase 5 (Cdk5) activity promotes adhesion and retards migration of lens epithelial cells. Moreover, the Cdk5 activating proteins p35 and p39 interact specifically with stress fiber components, such as α-actinin and myosin essential light chain (MLC17). Here we test whether effects of Cdk5 on adhesion and migration reflect Cdk5-dependent regulation of myosin regulatory light chain (MRLC) phosphorylation.
Cultured human lens epithelial cells (FHL124) were plated on fibronectin and examined at intervals from 15 to 120min. Relative levels of Cdk5, Cdk5(pY15), MRLC and Thr18/Ser19 diphosphorylated MRLC were measured by immunoblotting. Rho-GTP levels were measured by affinity chromatography with Rhotekin-RBD. Cdk5 activity was inhibited using 15 µM olomoucine and by overexpression of the dominant negative construct, GFP-Cdk5(D144N) in transfected cells. Cdk5 expression was suppressed with siRNA oligonucleotides. Cell morphology was assessed by immunofluorescence of cytoskeletal proteins.
MRLC phosphorylation increased more than 3-fold during the period from 15 min to 120 min after plating. This increase was correlated with a parallel increase in Cdk5 activity, Cdk5(Y15) phosphorylation, and Rho activation. During this time, cells underwent a characteristic change in cell shape from rounded to stellate, consistent with myosin contraction. Inhibiting Cdk5 activity with 15 µM olomoucine reduced the level of Thr18/Ser19 diphosphorylated MRLC (38.0%, p<0.05; N=5) observed at 120 min. Suppression of Cdk5 with siRNA or inhibition of Cdk5 activity by GFP-Cdk5-N144 transfection also significantly inhibited Thr18/Ser19 MRLC diphosphorylation and reduced the percentage of cells that assumed the stellate configuration. Controls transfected with scrambled siRNA oligonucleotides or with wild type GFP-Cdk5 did not show these effects.
Cdk5 may regulate lens epithelial cell adhesion and migration by regulating myosin contractility. Since MRLC lacks a consensus site for phosphorylation by Cdk5, Cdk5 likely impinges on upstream signaling inputs responsible for MRLC phosphorylation.
This PDF is available to Subscribers Only