title: PKC-mediated phosphorylation regulates c-FLIP ubiquitylation and stability.
abstract: 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 PKCalpha and PKCbeta. 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.
authors.affiliation: Turku Centre for Biotechnology, University of Turku and Abo Akademi University, FIN-20521, Turku, Finland.
authors.forename: A
authors.initials: A
authors.lastname: Kaunisto
authors.forename: V
authors.initials: V
authors.lastname: Kochin
authors.forename: T
authors.initials: T
authors.lastname: Asaoka
authors.forename: A
authors.initials: A
authors.lastname: Mikhailov
authors.forename: M
authors.initials: M
authors.lastname: Poukkula
authors.forename: A
authors.initials: A
authors.lastname: Meinander
authors.forename: J E
authors.initials: JE
authors.lastname: Eriksson