LIM kinase 1 modulates opsonized Zymosan-triggered activation of macrophage-like U937 cells: Possible involvement of phosphorylation of cofilin and reorganization of actin cytoskeleton

We have previously reported that cofilin, an actinbinding protein, plays an important role in phagocyte functions, such as respiratory burst, phagocytosis, and chemotaxis. On the other hand, it was recently found that LIM motif-containing kinase (LIMK) phosphorylates cofilin. In this work, we investigated the roles of LIMK in activated phagocytes. The results of immunostaining showed that in dormant phagocytes the endogenous LIMK1 was diffusely distributed in the cytosol of macrophage-like U937 cells, and when activated by opsonized zymosan (OZ), it was translocated to plasma membranes. Green fluorescence protein (GFP)-conjugated LIMK was expressed in the phagocytes, and the GFP-positive cells were isolated by a fluorescence-activated cell sorter. The isolated wild-type LIMK-overexpressing cells produced superoxide at a rate that was 3.2-fold higher than that of only GFP-expressing control cells, whereas the respiratory burst of dominant negative LIMK1 (D460A)-expressing cells decreased to 31% of that of the control cells. Phagocytic activity monitored by using Texas Red-labeled OZ was also decreased in the D460A-expressing cells. By immunoblotting using a specific anti-phosphorylated cofilin antibody, it was revealed that in the OZ-activated wild-type LIMK1-GFP-expressing cells, the phosphorylated cofilin increased by 2.3-fold, and that in the OZ-activated D460A-GFP-expressing cells, the phosphorylated cofilin decreased to 47% of that of only GFP-expressing cells (mock control). Furthermore, in the wild-type LIMK1-expressing cells, OZ-evoked increase in filamentous actin was markedly enhanced, whereas in the dominant negative LIMK1-expressing cells, the total level of F-actin was strongly suppressed. These results suggest that LIMK1 regulates the functions of phagocytes through phosphorylation of cofilin and enhances the formation of filamentous actin.