Introduction The regulation of actomyosin contractility plays a key role in various cellular functions, such as smooth-muscle contraction (Tan et al., 1992; Fukata et al., 2001), stress fiber and focal adhesion formation (Tan et al., 1992; Chrzanowska-Wodnicka and Burridge, 1996), neuronal morphogenesis (Lin et al., 1996; Amano et al., 1998; Wylie et al., 1998), cytokinesis (De Lozanne and Spudich, 1987; Komatsu et al., 2000) and apoptotic membrane blebbing (Mills et al., 1998). In both smooth muscle and nonmuscle cells, actomyosin contractility is primarily regulated by phosphorylation of the regulatory light chain of myosin II (MLC) (Tan et al., 1992). Phosphorylation of MLC at Thr18 and Ser19 enhances the actin-activated myosin motor activity (Kamm and Stull, 1989), whereas phosphorylation at Ser1/2 and Thr9 by protein kinase C (PKC) decreases its affinity for actin (Bengur et al., 1987). Phosphorylation of MLC at Thr18 and Ser19 is regulated by the MLC kinases and phosphatases. Myosin- light-chain kinase (MLCK) is responsible for calcium- dependent MLC phosphorylation at the stimulatory sites (Kamm and Stull, 1989). Rho kinase (ROCK) can both inactivate MLC phosphatase by phosphorylating its myosin- binding subunit (Kimura et al., 1996; Feng et al., 1999; Kawano et al., 1999) and directly phosphorylate MLC at MLCK site (Amano et al., 1996; Ueda et al., 2002). The p21- activated kinase was also reported to increase MLC phosphorylation in certain cell types (Kiosses et al., 1999; Sells et al., 1999). Through alterations of the MLC phosphorylation, these kinases and phosphatases regulate actomyosin contractile forces, profoundly influencing cell motility and morphology (Wilson et al., 1991; Klemke et al., 1997; Dong et al., 1998; Fukata et al., 1999). Contractility of nonmuscle cells is intimately associated with adhesion and adhesion-dependent signaling. Focal adhesions are sites of contact between the cell surface and the extracellular matrix, where the associated stress fibers terminate. They consist of both structural proteins that link integrins to the actin cytoskeleton and signaling proteins that transduce a range of adhesion-dependent signals (Sastry and Burridge, 2000; Zamir and Geiger, 2001). The assembly and stabilization of focal adhesions is greatly influenced by myosin-II-driven contractility. Chemicals that inhibit either MLC phosphorylation or actin-dependent myosin ATPase activity cause a rapid loss of both stress fibers and focal adhesions (Volberg et al., 1994; Chrzanowska-Wodnicka and Burridge, 1996). Similar effects are observed by dominant- negative inhibition of myosin IIA heavy chain (Wei and Adelstein, 2000) or overexpression of caldesmon (Helfman et al., 1999), a protein that restrains the interactions of myosin heads with actin filaments. Conversely, activation of the Rho/ROCK pathway stimulates the formation of both stress fibers and their associated focal adhesions in a contractility-dependent manner (Amano et al., 1997; Uehata et al., 1997). MLC phosphorylation is also involved in apoptosis. Death-associated protein kinase (DAP kinase) is a 4777 Death-associated protein kinase (DAP kinase) is a pro- apoptotic, calcium/calmodulin-dependent serine/threonine kinase. Here, we report that DAP kinase phosphorylates the regulatory light chain of myosin II (MLC) both in vitro and in vivo, and that this phosphorylation occurs preferentially at residue Ser19. In quiescent fibroblasts, DAP kinase stabilizes stress fibers through phosphorylation of MLC, but it is dispensable for the formation of peripheral microfilament bundles. This cytoskeletal effect of DAP kinase occurs before the onset of apoptosis and does not require an intact death domain. In addition, DAP kinase is required for serum-induced stress-fiber formation, which is associated with the upregulation of its catalytic activity. Despite being both sufficient and necessary for the assembly or maintenance of stress fibers, DAP kinase is incapable of stimulating the formation of focal adhesions in quiescent cells. Moreover, it promotes the disassembly of focal adhesions but not stress fibers in cells receiving serum factors. Together, our results identify a novel and unique function of DAP kinase in the uncoupling of stress fibers and focal adhesions. Such uncoupling would lead to a perturbation of the balance between contractile and adhesion forces and subsequent cell detachment, which might contribute to its pro-apoptotic activity. Key words: DAP kinase, Myosin phosphorylation, Stress fibers, Focal adhesions, Apoptosis Summary Uncoordinated regulation of stress fibers and focal adhesions by DAP kinase Jean-Cheng Kuo 1 , Jia-Ren Lin 1 , James M. Staddon 2 , Hiroshi Hosoya 3 and Ruey-Hwa Chen 1, * 1 Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan 2 Eisai London Research Laboratories, University College London, London, UK 3 Department of Biological Science, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan *Author for correspondence (e-mail: rhchen@ha.mc.ntu.edu.tw) Accepted 22 July 2003 Journal of Cell Science 116, 4777-4790 © 2003 The Company of Biologists Ltd doi:10.1242/jcs.00794 Research Article