Abstract
In this study, two alternatively spliced forms of the mouse death-associated protein kinase (DAPK) have been identified and their roles in apoptosis examined. The mouse DAPK-alpha sequence is 95% identical to the previously described human DAPK, and it has a kinase domain and calmodulin-binding region closely related to the 130-150 kDa myosin light chain kinases. A 12-residue extension of the carboxyl terminus of DAPK-beta distinguishes it from the human and mouse DAPK-alpha. DAPK phosphorylates at least one substrate in vitro and in vivo, the myosin II regulatory light chain. This phosphorylation occurs preferentially at Ser-19 and is stimulated by calcium and calmodulin. The mRNA encoding DAPK is widely distributed and detected in mouse embryos and most adult tissues, although the expression of the encoded 160-kDa DAPK protein is more restricted. Overexpression of DAPK-alpha, the mouse homolog of human DAPK has a negligible effect on tumor necrosis factor (TNF)-induced apoptosis. Overexpression of DAPK-beta has a strong cytoprotective effect on TNF-treated cells. Biochemical analysis of TNF-treated cell lines expressing mouse DAPK-beta suggests that the cytoprotective effect of DAPK is mediated through both intrinsic and extrinsic apoptotic signaling pathways and results in the inhibition of cytochrome c release from the mitochondria as well as inhibition of caspase-3 and caspase-9 activity. These results suggest that the mouse DAPK-beta is a negative regulator of TNF-induced apoptosis.
Highlights
Apoptosis is a carefully regulated cellular event with important roles in a number of processes that occur during development and contribute to tissue homeostasis
Biochemical analysis of tumor necrosis factor (TNF)-treated cell lines expressing mouse death-associated protein kinase (DAPK)- suggests that the cytoprotective effect of DAPK is mediated through both intrinsic and extrinsic apoptotic signaling pathways and results in the inhibition of cytochrome c release from the mitochondria as well as inhibition of caspase-3 and caspase-9 activity. These results suggest that the mouse DAPK- is a negative regulator of TNF-induced apoptosis
A recent study has shown that myosin II motor activities activated by the conventional Ca2ϩ/CaM-dependent myosin light chain kinase (MLCK) has an important role in regulating the translocation of at least one death receptor, TNFR-1, to the plasma membrane [8], suggesting an additional role in regulation of the apoptotic response in cells
Summary
Apoptosis is a carefully regulated cellular event with important roles in a number of processes that occur during development and contribute to tissue homeostasis. It is known that other protein kinases in addition to the conventional calcium/calmodulin (Ca2ϩ/CaM)-dependent MLCKs can phosphorylate myosin II regulatory light chain (RLC). These kinases include p21-activated kinase (PAK), rho-activated kinase (RHOK), and death-associated protein kinase (DAPK) [5,6,7]. This kinase family includes ZIP/DLK, DAPK-related apoptosisinducing kinases 1 and 2, DAPK2, and dystrophin-related protein-1 [12,13,14,15,16] These kinases have been shown in vitro to phosphorylate RLC isolated from skeletal muscle [10, 13, 15, 16] or smooth muscle myosin [17], but to date no in vivo substrates have been identified.
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