Slingshot (SSH) phosphatases and LIM kinases (LIMK)regulate actin dynamics via a reversible phosphorylation (inactivation) of serine 3 in actin-depolymerizing factor (ADF) and cofilin. Here we demonstrate that a multi-protein complex consisting of SSH-1L, LIMK1, actin, and the scaffolding protein, 14-3-3f, is involved, along with the kinase, PAK4, in the regulation of ADF/cofilin activity. Endogenous LIMK1 and SSH-1L interact in vitro and co-localize in vivo, and this interaction results in dephosphorylation and downregulation of LIMK1 activity. We also show that the phosphatase activity of purified SSH-1L is F-actin dependent and is negatively regulated via phosphorylation by PAK4. 14-3-3f binds to phosphorylated slingshot, decreases the amount of slingshot that co-sediments with F-actin, but does not alter slingshot activity. Here we define a novel ADF/cofilin phosphoregulatory complex and suggest a new mechanism for the regulation of ADF/cofilin activity in mediating changes to the actin cytoskeleton.

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S U M M A R Y The LIM kinase family includes two proteins: LIMK1 and LIMK2. These proteins have identical genomic structure and overall amino acid identity of 50%. Both proteins regulate actin polymerization via phosphorylation and inactivation of the actin depolymerizing factors ADF/cofilin. Although the function of endogenous LIMK1 is well established, little is known about the function of the endogenous LIMK2 protein. To understand the specific role of endogenous LIMK2 protein, we examined its expression in embryonic and adult mice using a rat monoclonal antibody, which recognizes specifically the PDZ domain of LIMK2 but not that of LIMK1. Immunoblotting and immunoprecipitation analyses of mouse tissues and human and mouse cell lines revealed widespread expression of the 75-kDa LIMK2 protein. Immunofluorescence analysis demonstrated that the cellular localization of LIMK2 is different from that of LIMK1. LIMK2 protein is found in the cytoplasm localized to punctae and is not enriched within focal adhesions like LIMK1. Immunohistochemical studies revealed that LIMK2 is widely expressed in embryonic and adult mouse tissues and that its expression pattern is similar to that of LIMK1 except in the testes. We have also demonstrated that endogenous LIMK1 and LIMK2 form heterodimers, and that LIMK2 does not always interact with the same proteins as LIMK1. (J Histochem Cytochem 54:487-501, 2006)