Abstract
LIM-kinases (LIMKs) play crucial roles in various cell activities, including migration, division, and morphogenesis, by phosphorylating and inactivating cofilin. Using a bimolecular fluorescence complementation assay to detect the actin-cofilin interaction, we screened LIMK1 inhibitors and identified two effective inhibitors, damnacanthal (Dam) and MO-26 (a pyrazolopyrimidine derivative). These compounds have already been shown to inhibit Lck, a Src family tyrosine kinase. However, in vitro kinase assays revealed that Dam inhibited LIMK1 more effectively than Lck. Dam suppressed LIMK1-induced cofilin phosphorylation and deceleration of actin retrograde flow in lamellipodia in N1E-115 cells. Dam impaired CXCL12-induced chemotactic migration of Jurkat T lymphocytes and Jurkat-derived, Lck-deficient JCaM1.6 cells and also inhibited serum-induced migration and invasion of MDA-MB-231 breast carcinoma cells. These results suggest that Dam has the potential to suppress cell migration and invasion primarily through the inhibition of LIMK kinase activity. Topical application of Dam also suppressed hapten-induced migration of epidermal Langerhans cells in mouse ears. Dam provides a useful tool for investigating cellular and physiological functions of LIMKs and holds promise for the development of agents against LIMK-related diseases. The bimolecular fluorescence complementation assay system used in this study will provide a useful method to screen for inhibitors of various protein kinases.