CDC25A phosphatase promotes cell cycle progression by activating G 1 cyclin-dependent kinases and has been postulated to be an oncogene because of its ability to cooperate with RAS to transform rodent fibroblasts. In this study, we have identified apoptosis signal-regulating kinase 1 (ASK1) as a CDC25A-interacting protein by yeast two-hybrid screening. ASK1 activates the p38 mitogen-activated protein kinase (MAPK) and c-Jun NH 2 -terminal protein kinase-stress-activated protein kinase (JNK/SAPK) pathways upon various cellular stresses. Coimmunoprecipitation studies demonstrated that CDC25A physically associates with ASK1 in mammalian cells, and immunocytochemistry with confocal laser-scanning microscopy showed that these two proteins colocalize in the cytoplasm. The carboxyl terminus of CDC25A binds to a domain of ASK1 adjacent to its kinase domain and inhibits the kinase activity of ASK1, independent of and without effect on the phosphatase activity of CDC25A. This inhibitory action of CDC25A on ASK1 activity involves diminished homo-oligomerization of ASK1. Increased cellular expression of wild-type or phosphatase-inactive CDC25A from inducible transgenes suppresses oxidant-dependent activation of ASK1, p38, and JNK1 and reduces specific sensitivity to cell death triggered by oxidative stress, but not other apoptotic stimuli. Thus, increased expression of CDC25A, frequently observed in human cancers, could contribute to reduced cellular responsiveness to oxidative stress under mitogenic or oncogenic conditions, while it promotes cell cycle progression. These observations propose a mechanism of oncogenic transformation by the dual function of CDC25A on cell cycle progression and stress responses.

The ability of the cytoskeleton to deform and reform is a crucial aspect of many cellular responses (5). This is especially true of motile and dividing cells where the cytoskeleton must deform and reform on demand. Interactions between cells and the extracellular matrix also appear to be important in cell survival (22). Integrin ligation by the extracellular matrix plays a crucial role in organizing the cytoskeleton (25), and the loss of substrate attachment is known to induce apoptosis (anoikis) (14). On the other hand, studies on epithelial cells grown in three-dimensional culture have shown that integrin-extracellular matrix interactions promote the organization of the cytoskeleton and resistance to apoptotic stimuli (42).The organization and stiffness of the cytoskeleton are determined in large part by the forces generated by actin and myosin II (12). The actin-myosin II interaction in smooth muscle and nonmuscle cells is regulated by the phosphorylation of serine 19 of the 20-kDa light chain of myosin II (1,11,37,39,44). This reaction, which is catalyzed by myosin light chain kinase (MLCK), stimulates the actin-activated, Mg 2ϩ -dependent ATPase activity of myosin II (1). Work from many laboratories has shown that MLC 20 phosphorylation and dephosphorylation are required for smooth muscle contraction and relaxation (for reviews, see references 11, 37, and 39). Other experiments have shown that MLC 20 phosphorylation/dephosphorylation plays a central role in cell motility (25,33,43,45), endothelial (41, 46) and epithelial (3,15,19) barrier function, and cell division (13,34,47).Apoptosis is a carefully regulated cellular process that is important in developing and maintaining tissue homeostasis (40). Dysregulation of the apoptotic process underlies pathologies including cancer, autoimmune diseases, and neurodegenerative disorders. Biochemical events associated with apoptosis include caspase activation, mitochondrial disruption, and genome digestion (20,24). Another hallmark of apoptosis is a profound change in cell shape that is apparently mediated by restructuring the cytoskeleton. While actin (4) and actin-binding proteins (26) have been implicated in mediating these cytoskeletal changes, the role of myosin II in apoptosis is poorly understood. Because actin and myosin II work together to stabilize the cytoskeleton and to define cell shape, we investigated how MLCK and the phosphorylation/dephosphorylation of the 20-kDa light chain of myosin II (MLC 20 ) are involved in apoptosis. In the present study we show that MLC 20 is dephosphorylated during apoptosis and that the dephosphorylation of MLC 20 , effected by destabilizing the cytoskeleton or by direct inhibition of MLCK, triggers cell death. We also show that targeted inhibition of MLCK induced apoptosis in vivo. MATERIALS AND METHODSCell culture. Smooth muscle cells (SMC) were isolated from porcine pulmonary artery by enzymatic digestion as described previously (7). Cells were grown in culture dishes in Dulbecco's modified Eagle medium (DMEM; Gibco BRL...

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