Structural features of LIM kinase that control effects on the actin cytoskeleton

LIM kinase phosphorylates and inactivates the actin binding/depolymerizing factor cofilin and induces actin cytoskeletal changes. Several unique struc tural features within LIM kinase were investigated for their roles in regul ation of LIM kinase activity. Disruption of the second LIM domain or the PD Z domain or deletion of the entire amino terminus increased activity in viv o measured as increasing aggregation of the actin cytoskeleton. A kinase de leted alternate splice product was identified and characterized. This alter nate splice product and a kinase inactive mutant inhibited LIM kinase in vi vo, indicating that the amino terminus suppresses activity of the kinase do main. Mutation of threonine 508 in the activation loop to valine abolished activity whereas replacement with 2 glutamic acid residues resulted in a fu lly active enzyme. Dephosphorylation of LIM kinase inhibited cofilin phosph orylation. Mutation of the basic insert in the activation loop inhibited ac tivity in vivo, but not in vitro. These results indicate phosphorylation is an essential regulatory feature of LIM kinase and indicate that threonine 508 and the adjacent basic insert sequences of the activation loop are requ ired for this process. A combination of structural features are thus involv ed in receiving upstream signals that regulate LIM kinase-induced actin cyt oskeletal reorganization.