PKC-mediated phosphorylation regulates c-FLIP ubiquitylation and stability

• Published in: Cell death and differentiation Vol. 16; no. 9; pp. 1215 - 1226
• Main Authors: Kaunisto, A, Kochin, V, Asaoka, T, Mikhailov, A, Poukkula, M, Meinander, A, Eriksson, J E
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.09.2009; Nature Publishing Group
• Subjects: Apoptosis; Biochemistry; Biology; Biomedical and Life Sciences; Cancer research; CASP8 and FADD-Like Apoptosis Regulating Protein - metabolism; Caspase 8 - metabolism; Cell Biology; Cell Cycle Analysis; Cell death; Cell Line, Tumor; Humans; K562 Cells; Kinases; Life Sciences; Ligands; Medical research; Mutation; original-paper; Phosphorylation; Protein Isoforms - metabolism; Protein Kinase C - metabolism; Proteins; Stem Cells; Tetradecanoylphorbol Acetate - pharmacology; TNF-Related Apoptosis-Inducing Ligand - pharmacology; Tumor necrosis factor-TNF; Ubiquitination; Finland; apoptosis; PKC; c-FLIP; phosphorylation; ubiquitylation
• ISSN: 1350-9047; 1476-5403
• DOI: 10.1038/cdd.2009.35

Cellular FLICE-inhibitory protein (c-FLIP) proteins are crucial regulators of the death-inducing signaling complex (DISC) and caspase-8 activation. To date, three c-FLIP isoforms with distinct functions and regulation have been identified. Our previous studies have shown that the stability of c-FLIP proteins is subject to isoform-specific regulation, but the underlying molecular mechanisms have not been known. Here, we identify serine 193 as a novel in vivo phosphorylation site of all c-FLIP proteins and demonstrate that S193 phosphorylation selectively influences the stability of the short c-FLIP isoforms, as S193D mutation inhibits the ubiquitylation and selectively prolongs the half-lives of c-FLIP short (c-FLIP S ) and c-FLIP Raji (c-FLIP R ). S193 phosphorylation also decreases the ubiquitylation of c-FLIP long (c-FLIP L ) but, surprisingly, does not affect its stability, indicating that S193 phosphorylation has a different function in c-FLIP L . The phosphorylation of this residue is operated by the protein kinase C (PKC), as S193 phosphorylation is markedly increased by treatment with 12-O-tetradecanoylphorbol-13-acetate and decreased by inhibition of PKC α and PKC β . S193 mutations do not affect the ability of c-FLIP to bind to the DISC, although S193 phosphorylation is increased by death receptor stimulation. Instead, S193 phosphorylation affects the intracellular level of c-FLIP S , which then determines the sensitivity to death-receptor-mediated apoptosis. These results reveal that the differential stability of c-FLIP proteins is regulated in an isoform-specific manner by PKC-mediated phosphorylation.