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 PKCa and PKCb. 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.

Modeling Reveals That Dynamic Regulation of c-FLIP Levels Determines Cell-to-Cell Distribution of CD95-mediated Apoptosis

“…To fit experimental data, the model incorporates both the long and short c-FLIP isoforms to account for their different reaction mechanisms. The two short c-FLIP isoforms, c-FLIP S and c-FLIP R , have been shown to affect caspase-8 activation and apoptosis sensitivity in a similar way (8,10,27,33), and they have similar half-lives. As the c-FLIP property that is important for this study is its half-life, and as the half-lives of the short forms of c-FLIP, i.e.…”

“…Parameter estimation in population models based on experimental findings will also yield possibilities to estimate rate-limiting steps among the processes that determine the regulation of c-FLIP isoforms and thereby the processing of DISC signals. For example, as mentioned in the Introduction, c-FLIP isoforms are subjected to active isoform-specific phosphorylation (27,28) and ubiquitylation and degradation (9,10). All the post-translational modifications seem to have distinct roles and features not only in regulating the stability of the proteins but also in determining their functions.…”

“…As the c-FLIP property that is important for this study is its half-life, and as the half-lives of the short forms of c-FLIP, i.e. c-FLIP S and c-FLIP R , are the same in all the model cell lines (8,10,27,33), we can assume that c-FLIP S and c-FLIP R will behave in the same way. Therefore, no distinction has been made between the two short isoforms in this study.…”

“…However, the reported high turnover rate of c-FLIP (8,10) implies that c-FLIP bound in the DISC is rapidly replenished with newly synthesized c-FLIP, tending to counteract changes in c-FLIP levels. Moreover, a number of experimental reports (8,10,27) indicate that regulation of the stability and consequent concentration levels of c-FLIP plays an important role in controlling apoptotic sensitivity.…”

“…This feature was employed to validate the modeling results using empirical cell population data. The model can be used to study, apart from ubiquitylation-mediated degradation, other rate-limiting steps in DISC signaling such as the recently defined isoformspecific phosphorylation processes of c-FLIP (27,28) that partly regulate ubiquitylation and partly regulate other aspects.…”