Abstract
The tumor suppressor, death-associated protein kinase (DAPK), is a Ca(2+)/calmodulin-regulated Ser/Thr kinase with an important role in regulating cytoskeletal dynamics. Autophosphorylation within the calmodulin-binding domain at Ser-308 inhibits DAPK catalytic activity. Dephosphorylation of Ser-308 by a previously unknown phosphatase enhances kinase activity and proteasome-mediated degradation of DAPK. In these studies, we identified two holoenzyme forms of protein phosphatase 2A (PP2A), ABalphaC and ABdeltaC, as DAPK-interacting proteins. These phosphatase holoenzymes dephosphorylate DAPK at Ser-308 in vitro and in vivo resulting in enhanced kinase activity of DAPK. The enzymatic activity of PP2A also negatively regulates DAPK levels by enhancing proteasome-mediated degradation of the kinase. Overexpression of wild type DAPK induces cell rounding and detachment in HEK293 cells; however, this effect is not observed following expression of an inactive DAPK S308E mutant. Finally, activation of DAPK by PP2A was found to be required for ceramide-induced anoikis. Together, our results provide a mechanism by which PP2A and DAPK activities control cell adhesion and anoikis.
Highlights
Mediator of apoptosis induced by tumor necrosis factor ␣ [3, 6]
To validate the tandem affinity purification (TAP) results, we examined the ability of death-associated protein kinase (DAPK) to co-immunoprecipitate with B␣ and the highly homologous (90% identity) B␦ subunit of phosphatase 2A (PP2A)
Both the structural (A) and catalytic (C) subunits of PP2A were detected in the FLAG-B subunit eluates, whereas no PP2A subunits were observed in FLAG immune complexes isolated from lysates of control cells
Summary
Mediator of apoptosis induced by tumor necrosis factor ␣ [3, 6]. Forced expression of DAPK results in morphological changes, including cell rounding, shrinking, detachment, and anoikis in multiple cell types [4, 5]. To determine whether enhanced activation of PP2A altered endogenous DAPK protein levels, we treated HeLa cells with increasing amounts of FTY720 in the presence or absence of OA.
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