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P. Russell, X. Zhao, D. Garland; Decreased Levels of Rho-Kinase Alter Levels of Phosphorylated Caldesmon. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2970.
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Rho-kinase is one of the serine/threonine kinases in the intracellular signaling pathway. This kinase influences the phosphorylation of myosin light chain and can affect cytoskeletal functions. We wished to determine other proteins that might be changed when Rho-kinase is silenced in cells.
Rho kinase was stably silenced using a shRNA in human retinal pigment epithelial ARPE-19 cells. Two-dimensional gel electrophoresis was run on the proteins from Rho-kinase silenced cells as well as control cells. The gels were stained to reveal the phosphoproteins, and the Progenesis software was used to reveal those phosphorylated proteins that were increased or decreased with this silencing. Because of its association with the cytoskeleton, one of these proteins was investigated to determine which isoforms were present in the ARPE-19 cells.
Although transient transfection by siRNA could reduce the mRNA level of Rho-kinase by 80%, the stable transfection using shRNA and puromycin resistance selection decreased the mRNA to 49.8% of the level expressed in control cells. After two-dimensional gel electrophoresis, one of the phosphoproteins identified by the Progenesis software that had decreased expression was identified by mass spectroscopy as caldesmon. Because caldesmon has multiple isoforms, real time PCR was used to identify the isoforms present in the ARPE-19 cells. These cells contain 115 times more caldesmon II than caldesmon I. The type of caldesmon in the ARPE-19 cells is similar to the caldesmon found in WI-38 cells rather than the form present in HeLa cells.
Caldesmon is a protein that binds calmodulin, actin and tropomyosin. Phosphorylation of this protein influences its binding to actin and can influence the cytoskeleton of the cell. Reduction of Rho-kinase in a cell has the potential of decreasing focal adhesions and stress fiber assembly. Since phosphorylated caldesmon is thought to destabilize actin, a lowered level of the phosphorylated caldesmon in the silenced ARPE-19 cells may indicate a cellular mechanism for compensating for the decreased Rho-kinase.
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