Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: phosphorylation • cytoskeleton • cell adhesions/cell junctions