Roles of Tyrosine Phosphorylation and Cleavage of Protein Kinase Cδ in Its Protective Effect Against Tumor Necrosis Factor-related Apoptosis Inducing Ligand-induced Apoptosis

Abstract

Protein kinase C␦ (PKC␦) regulates cell apoptosis in a cell-and stimulus-specific manner. Here, we studied the role of PKC␦ in the apoptotic effect of TRAIL in glioma cells. We found that transfection of the cells with a PKC␦ kinase-dead mutant (K376R) or with a small interfering RNA targeting the PKC␦ mRNA increased the apoptotic effect of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), whereas overexpression of PKC␦ decreased it. PKC␦ acted downstream of caspase 8 and upstream of cytochrome c release from the mitochondria. TRAIL induced cleavage of PKC␦ within 2-3 h of treatment, which was abolished by caspase 3, 8, and 9 inhibitors. The cleavage of PKC␦ was essential for its protective effect because overexpression of a caspaseresistant mutant (PKC␦D327A) did not protect glioma cells from TRAIL-induced apoptosis but rather increased it. TRAIL induced translocation of PKC␦ to the perinuclear region and the endoplasmic reticulum and phosphorylation of PKC␦ on tyrosine 155. Using a PKC␦Y155F mutant, we found that the phosphorylation of PKC␦ on tyrosine 155 was essential for the cleavage of PKC␦ in response to TRAIL and for its translocation to the endoplasmic reticulum. In addition, phosphorylation of PKC␦ on tyrosine 155 was necessary for the activation of AKT in response to TRAIL. Our results indicate that PKC␦ protects glioma cells from the apoptosis induced by TRAIL and implicate the phosphorylation of PKC␦ on tyrosine 155 and its cleavage as essential factors in the anti-apoptotic effect of PKC␦.

Regulation of Protein Kinase C function by phosphorylation on conserved and non-conserved sites

“…While PKC plays an important role in apoptosis, its role in this process is dictated by cell type, stimulus and intracellular localisation [10,11,111]. This is probably best exemplified in glioma cells, where PKC positively influences etoposide-mediated apoptosis, whereas it plays a protective role in TRAIL-induced apoptosis in the same cells [112]. In many cases, the generation of a kinase domain fragment of PKC via caspase-3-mediated cleavage at the hinge domain is necessary for PKC to modulate the apoptotic cascade and it has been demonstrated that phosphorylation of different residues on the enzyme regulates this process.…”

“…For example, the apoptotic stimulus TRAIL induces phosphorylation of PKC at Tyr-155 in glioma cells and phosphorylation at this site is necessary for translocation of the enzyme to the endoplasmic recticulum and for the cleavage of PKC to the kinase domain fragment. Phosphorylation at Tyr-155 is therefore an important step in regulating the ability of this enzyme to act as an antiapoptotic kinase in this pathway [112]. PKC is also phosphorylated on Tyr-313 and Tyr-334 in the hinge domain in response to apoptotic stimuli such as TRAIL or cisplatin [113].…”

The Localization of Protein Kinase Cδ in Different Subcellular Sites Affects Its Proapoptotic and Antiapoptotic Functions and the Activation of Distinct Downstream Signaling Pathways

“…Thus, PKCy protects macrophages from apoptosis induced by nitric oxide (23) and exerts antiapoptotic effects on glioma cells treated with tumor necrosis factor -related apoptosis-inducing ligand (TRAIL; ref. 24) or infected with a virulent strain of Sindbis virus (25). Similarly, PKCy promotes survival and chemotherapeutic drug resistance of non -small cell lung cancer cells (26).…”

“…Translocation of PKCy to the Golgi, mitochondria, and nucleus in response to these apoptotic stimuli has been associated with proapoptotic effects of this isoform (5,9,28). In addition, PKCy has been shown to translocate to the endoplasmic reticulum (ER) in cells infected with Sindbis virus and in glioma cells treated with TRAIL, where PKCy exerts antiapoptotic effects (24,25). Although these studies suggest different functions of PKCy in the various subcellular sites, the role of the translocation of PKCy in its proapoptotic and antiapoptotic effects and in the activation of downstream signaling pathways is largely not defined.…”

Downregulation of Bid is associated with PKCɛ-mediated TRAIL resistance

“…The mechanism of TRAIL resistance may vary depending on the PKC isozyme. 14,18,19,24,31 PKCe has been implicated in inhibiting TRAIL-induced apoptosis in glioma and melanoma cells. 18,19,23 Increased levels and activity of PKCe were associated with TRAIL resistance, 19 and overexpression of PKCe inhibited TRAILinduced apoptosis in these cells.…”