Indiana University-Purdue University Indianapolis (IUPUI) / Modulation of sphingolipid-induced apoptosis is a potential mechanism to enhance the effectiveness of chemotherapeutic drugs. Ceramide is a pleiotropic, sphingolipid produced by cells in response to inflammatory cytokines, chemotherapeutic drugs and ionizing radiation. Ceramide is a potent activator of protein phosphatases, including protein phosphatase 2A (PP2A) leading to dephosphorylation of substrates important in regulating mitochondrial dysfunction and apoptosis. Previous studies demonstrated that death associated protein kinase (DAPK) plays a role in ceramide-induced apoptosis via an unknown mechanism. The tumor suppressor DAPK is a calcium/calmodulin regulated serine/threonine kinase with an important role in regulating cytoskeletal dynamics. Auto-phosphorylation within the calmodulin-binding domain at serine308 inhibits DAPK catalytic activity. Dephosphorylation of serine308 by a hitherto unknown phosphatase enhances kinase activity and proteasomal mediated degradation of DAPK. In these studies, using a tandem affinity purification procedure coupled to LC-MS/MS, we have identified two holoenzyme forms of PP2A as DAPK interacting proteins. These phosphatase holoenzymes dephosphorylate DAPK at Serine308 in vitro and in vivo resulting in enhanced kinase activity of DAPK. The enzymatic activity of PP2A also negatively regulates DAPK protein levels by enhancing proteasomal-mediated degradation of the kinase, as a means to attenuate prolonged kinase activation. These studies also demonstrate that ceramide causes a caspase-independent cell detachment in HeLa cells, a human cervical carcinoma cell line. Subsequent to detachment, these cells underwent caspase-dependent apoptosis due to lack of adhesion, termed anoikis. Overexpression of wild type DAPK induced cell rounding and detachment similar to cells treated with ceramide; however, this effect was not observed following expression of a phosphorylation mutant, S308E DAPK. Finally, the endogenous interaction of DAPK and PP2A was determined to be required for ceramide-induced cell detachment and anoikis. Together these studies have provided exciting and essential new data regarding the mechanisms of cell adhesion and anoikis. These results define a novel cellular pathway initiated by ceramide-mediated activation of PP2A and DAPK to regulate inside-out signaling and promote anoikis.