Effect of Phosphomimetic Mutation of Caldesmon on the Migration Activity of Vascular Smooth Muscle Cells

Abstract

Migration of differentiated smooth muscle cells is usually pathogenic. It contributes to diseases such as atherosclerosis. The low molecular weight isoform of caldesmon (l-CaD) binds actin filaments in mammalian cells and modulates the assembly of the actin cytoskeleton. To determine the effect of phosphorylation of l-CaD on the mobility of cultured vascular smooth muscle cells, we have performed Transwell migration assays on A7r5 cells expressing l-CaD variants with mutations at the phosphorylation sites mediated by PAK (Ser452 and Ser482) and/or ERK (Ser497 and Ser527). The chemotactic migration activity of transfected cells compared to the normal, untransfected cells was evaluated. Among all constructs the A1234 mutant (Ser residues at all 4 positions changed to Ala) resulted in most hindered mobility. The relative migration activity for the A1234-transfected cells was about 13% of the vehicle-transfected cells. Transfection with wild-type l-CaD decreased the rate of cell migration to a lesser extent (∼50% of the control cells). Cells transfected with l-CaD mutated only at the PAK sites (A1A2) or the ERK sites (A3A4) also migrated approximately 50% slower than those control cells. Apparently, both ERK and PAK contribute to the mobility of A7r5 cells and the effects are comparable and additive. Phosphorylation was indeed found at the ERK sites of ectopically expressed A1A2 and l-CaD, but not that of A3A4 and A1234. The mobility of cells transfected with D1234 (all 4 Ser changed to Asp) was higher than that of cells transfected with all other CaD variants, but still about 20% lower than the control cells. Taken together, these results suggest l-CaD plays a role in controlling the migration activity of smooth muscle cells, and reversible phosphorylation of l-CaD facilitates this activity. Supported by NIH (HL-92252).