Rac is a small GTPase of the Rho family that mediates stimulus-induced actin cytoskeletal reorganization to generate lamellipodia. Little is known about the signalling pathways that link Rac activation to changes in actin filament dynamics. Cofilin is known to be a potent regulator of actin filament dynamics, and its ability to bind and depolymerize actin is abolished by phosphorylation of serine residue at 3; however, the kinases responsible for this phosphorylation have not been identified. Here we show that LIM-kinase 1 (LIMK-1), a serine/threonine kinase containing LIM and PDZ domains, phosphorylates cofilin at Ser 3, both in vitro and in vivo. When expressed in cultured cells, LIMK-1 induces actin reorganization and reverses cofilin-induced actin depolymerization. Expression of an inactive form of LIMK-1 suppresses lamellipodium formation induced by Rac or insulin. Furthermore, insulin and an active form of Rac increase the activity of LIMK-1. Taken together, our results indicate that LIMK-1 participates in Rac-mediated actin cytoskeletal reorganization, probably by phosphorylating cofilin.

LIM-kinase 1 (LIMK1) phosphorylates cofilin, an actin-depolymerizing factor, and regulates actin cytoskeletal reorganization. LIMK1 is activated by the small GTPase Rho and its downstream protein kinase ROCK. We now report the site of phosphorylation of LIMK1 by ROCK. In vitro kinase reaction revealed that the active forms of ROCK phosphorylated LIMK1 on the threonine residue and markedly increased its cofilin-phosphorylating activity. A LIMK1 mutant (T508A) with replacement of Thr-508 within the activation loop of the kinase domain by alanine was neither phosphorylated nor activated by ROCK. Replacement of Thr-508 by serine changed the ROCK-catalyzed phosphorylation residue from threonine to serine. A LIMK1 mutant with replacement of Thr-508 by two glutamates increased the kinase activity about 2-fold but was not further activated by ROCK. In addition, wild-type LIMK1, but not its T508A mutant, was activated by co-expression with ROCK in cultured cells. These results suggest that ROCK activates LIMK1 in vitro and in vivo by phosphorylation at Thr-508. Together with the recent finding that PAK1, a downstream effector of Rac, also activates LIMK1 by phosphorylation at Thr-508, these results suggest that activation of LIMK1 is one of the common targets for Rho and Rac to reorganize the actin cytoskeleton.Actin cytoskeletal reorganization is essential for various cell activities, including motility, morphological change, adhesion, and cytokinesis (1). Various extracellular stimuli induce changes in actin organization, but little is known about signaling pathways which link the external stimuli to the machinery controlling actin polymerization and organization. Cofilin can bind to and depolymerize actin filaments and is considered to be a potent regulator of actin filament dynamics (2, 3). The actin-binding and -depolymerizing activity of cofilin is inhibited by phosphorylation at Ser-3 (4). We and other investigators recently provided evidence that LIM-kinase 1 (LIMK1) 1 and LIM-kinase 2 (LIMK2) phosphorylate cofilin in vitro and in vivo specifically at Ser-3 and are involved in the actin-filament dynamics by phosphorylating and inactivating cofilin (5, 6). LIMK1 and LIMK2 are members of a novel subfamily of protein serine/threonine kinases with characteristic structural features composed of two LIM domains and a PDZ domain at the N terminus and a protein kinase domain at the C terminus (7-11). We and other workers also showed that LIM-kinases are activated in cultured cells by Rho family small GTPases, Rac and Rho, albeit the activation being indirect (5, 6, 12).The Rho family small GTPases, Rho, Rac, and Cdc42, play a central role in regulating actin reorganization through downstream effectors, the activities of which are controlled by interactions with active GTP-bound forms of the Rho family (13, 14). Rho-associated kinases, termed ROCK, ROK, or Rho-kinase, are serine/threonine kinases implicated in Rho-mediated actin reorganization, such as formation of stress fibers and focal adhesions and smooth muscle contr...

Axl, Sky, and Mer, members of an Axl/Sky receptor tyrosine kinase subfamily, are typified by the cell adhesion molecule-related extracellular domain. The product of growth arrest-specific gene 6 (Gas6), structurally homologous to the anticoagulant protein S, was recently identified as the ligand for Axl and Sky, but the ligand for Mer remained unknown. We have now obtained evidence that Gas6 can also function as a ligand for Mer. Co-precipitation analysis, using soluble receptors of Axl, Sky, and Mer (Axl-Fc, Sky-Fc, and Mer-Fc) composed of the extracellular domain of receptors fused to the Fc domain of immunoglobulin G1, clearly showed that Gas6, but not protein S, specifically bound to Axl-Fc, Sky-Fc, and Mer-Fc fusion proteins. Quantitative kinetic analyses using a BIAcore biosensor instrument revealed dissociation constants (Kd) of the binding of rat Gas6 to Axl-Fc, Sky-Fc, and Mer-Fc are 0.4, 2.7, and 29 nM, respectively. We also found that Gas6 stimulated tyrosine phosphorylation of Axl, Sky, and Mer receptors ectopically expressed in Chinese hamster ovary cells. Taken together, these findings suggest that Gas6 is a common ligand for Axl, Sky, and Mer, all known members of an Axl/Sky receptor subfamily.

Gas6, a product of a growth arrest-specific gene 6, potentiates proliferation of vascular smooth muscle cells and prevents cell death of vascular smooth muscle cells. It has been also demonstrated that Gas6 is a ligand of receptor tyrosine kinases Axl, Sky, and Mer. Gas6 contains ␥-carboxyglutamic acid residues, which are found in some blood coagulation factors and mediate the interaction of the coagulation factors with negatively charged phospholipid. In this study, we clarified that Gas6 specifically bound to phosphatidylserine and the binding was dependent on Ca 2؉ and ␥-carboxyglutamic acid residues. Furthermore, we found that U937 cells, which express Gas6 receptor on their surfaces, adhered to phosphatidylserine-coated enzyme-linked immunosorbent assay (ELISA) plate only in the presence of Gas6 and Ca 2؉ . U937 cells also bound to ELISA plate coated with phosphatidylinositol, but the binding was independent of Gas6 and Ca 2؉ . On the other hand, U937 cells did not adhere to phosphatidylcholine-or phosphatidylethanolamine-coated ELISA plate even in the presence of Gas6 and Ca 2؉ . These findings suggest that Gas6 may play a role in recognition of cells exposing phosphatidylserine on their surfaces by phagocytic cells, which is supposed to be one of the mechanisms for clearing dying cells.

Sky (also calledRse

No abstract

Gas6 is a ligand for an Axl/Sky receptor tyrosine kinase subfamily and has a structure composed of a Gla domain, four EGF-like domains and a C-terminal sex hormone-binding globulin (SHBG)-like domain. When examining the role of each domain in receptor-binding and biological activities of Gas6, we found that receptor-binding and mitogenic activities were markedly reduced by inhibiting γ-carboxylation of the Gla domain, while a Gas6 mutant composed of only an SHBG-like domain retained both of these activities. Thus, the SHBG-like domain is apparently an entity indispensable for Gas6 activities, and γ-carboxylation of the Gla domain has a regulatory role in retaining the activity of native Gas6.

No abstract