Calcium/Calmodulin-dependent Protein Kinase II Regulation of c-FLIP Expression and Phosphorylation in Modulation of Fas-mediated Signaling in Malignant Glioma Cells

Abstract

(5, 6). FADD has a carboxylterminal DD and an amino-terminal death effector domain (DED). Through its DED, FADD recruits the DED-containing apoptosis-initiating proteases caspase-8 (7, 8) and caspase-10 (9 -11) to the Fas receptor to assemble a DISC (12). In the DISC, caspase-8 is cleaved through autoproteolysis of caspase-8 molecules in close proximity (13). Active caspase-8 subunits are released into the cytoplasm to cleave downstream effector caspases such as caspase-3 (14), which subsequently cleaves its substrates such as DNA fragmentation factor 45 (DFF45) (15), to execute programmed cell death.Recently, the recruitment of other DED-containing proteins to the Fas-mediated DISC, which has been described, modulates DISC functions. These include a family of virus-encoded proteins referred to as v-FLIP (16, 17). v-FLIP contains two DEDs that can bind to the Fas/FADD complex to inhibit Fasmediated apoptosis by interfering with the recruitment of caspase-8 to the DISC. A mammalian cellular homolog of v-FLIP is termed c-FLIP (18), CASH (19), CASPER (20), CLARP (21), FLAME1 (22), I-FLICE (23), MRIT (24), and Usurpin (25). These studies, however, have generated controversy as to the functions of c-FLIP in apoptosis. Some groups have described it as pro-apoptotic (19 -21, 24), whereas others as anti-apoptotic (18,22,25). Recent analysis of Fas-mediated DISC in c-FLIPtransfected BJAB cells has shown that c-FLIP proteins are recruited to the Fas-mediated DISC to inhibit caspase-8 cleavage (26, 27), which supports the role of c-FLIP as an antiapoptotic molecule.The c-FLIP gene is composed of 13 exons that are clustered within ϳ200 kilobases within the caspase-8 and caspase-10 genes on human chromosome 2q33 to 34 (25, 28). c-FLIP is expressed as four main mRNA splice variants but only two forms of protein in human tissues (18,20). The short form protein (c-FLIP S , M r ϳ28) contains two DEDs and is structurally related to v-FLIP; the longer form (c-FLIP L , M r ϳ55) is structurally similar to caspase-8 and contains two DEDs and a caspase-like domain that lacks catalytic activity (18). c-FLIP L is expressed in many tissues, but c-FLIP S is found mainly in lymphatic tissue (18). Expression of c-FLIP mRNA and proteins is regulated by mitogen-activated protein kinase kinase in T lymphocytes (29)

Cellular FLICE-Like Inhibitory Protein (C-FLIP): A Novel Target for Cancer Therapy

“…Treating resistant cells with the CaMK II inhibitor KN-93 inhibited CaMK II activity, reduced c-FLIP expression, inhibited c-FLIP phosphorylation, and rescued Fas agonistic antibody (CH-11) sensitivity [19,71]. Targeting this pathway may provide novel therapeutic strategies in treating cancers with upregulated CaMK II.…”

“…Targeting this pathway may provide novel therapeutic strategies in treating cancers with upregulated CaMK II. Interestingly, phosphorylation of c-FLIP variants by CaMK II appears to promote c-FLIP L recruitment to the DISC and inhibit TRAIL-induced apoptosis [19,71], but phosphorylation of c-FLIP L by protein kinase C or the bile acid glycochenodeoxycholate results in decreased c-FLIP L recruitment to the DISC and increased sensitivity of hepatocellular carcinoma cells to TRAIL-triggered apoptosis [72]. Thus, the particular site of phosphorylation in c-FLIP L appears to influence the functional outcome of this protein on apoptosis.…”

Inositol 1,3,4-Trisphosphate 5/6-Kinase Inhibits Tumor Necrosis Factor-induced Apoptosis

“…Phosphorylation of FADD can enhance apoptosis induced by etoposide (37). Also, PKC II may regulate cellular FLIP expression and phosphorylation in response to Fas (38). It is conceivable that 5/6-kinase could selectively phosphorylate a component within DISC and alter its function specific to TNF␣ signaling.…”

PKC-mediated phosphorylation regulates c-FLIP ubiquitylation and stability

“…Although others did not identify individual phosphorylation sites, they suggested that CaMKII phosphorylates c-FLIP L on threonine, [20][21][22] and that phosphorylation is involved in the detachment of c-FLIP from the TRAIL DISC. 32 However, because CaMKII inhibition did not affect S193 phosphorylation (data not shown), CaMKII is unlikely to be involved in S193 phosphorylation.…”

“…Although we showed earlier that the levels of c-FLIP are determined by ubiquitylation, others have reported that c-FLIP is modulated by phosphorylation. [19][20][21][22] Hence, we studied how these posttranslational modifications regulate c-FLIP in concert.…”