PKC-mediated phosphorylation regulates c-FLIP ubiquitylation and stability

Area of Science:

• Cellular and Molecular Biology
• Apoptosis Regulation
• Signal Transduction Pathways

Background:

• Cellular FLICE-inhibitory protein (c-FLIP) isoforms regulate apoptosis by modulating the death-inducing signaling complex (DISC) and caspase-8 activation.
• Isoform-specific regulation of c-FLIP protein stability is known, but the underlying mechanisms remain unclear.

Purpose of the Study:

• To identify the molecular mechanisms governing the isoform-specific stability of c-FLIP proteins.
• To investigate the role of serine 193 phosphorylation in c-FLIP regulation and its impact on apoptosis.

Main Methods:

• Identification of serine 193 as a novel in vivo phosphorylation site for all c-FLIP isoforms.
• Site-directed mutagenesis (S193D) to assess the functional consequences of phosphorylation.
• Analysis of ubiquitylation and protein half-life of c-FLIP isoforms.
• Pharmacological inhibition and activation of protein kinase C (PKC) pathways.
• Assessment of c-FLIP binding to the DISC and sensitivity to death-receptor-mediated apoptosis.

Main Results:

• Serine 193 phosphorylation selectively prolongs the half-lives of short c-FLIP isoforms (c-FLIP(S) and c-FLIP(R)) by inhibiting their ubiquitylation.
• Phosphorylation at S193 decreases ubiquitylation of c-FLIP long (c-FLIP(L)) but does not affect its stability.
• Protein kinase C (PKC) alpha and beta are identified as the kinases responsible for S193 phosphorylation.
• S193 phosphorylation increases upon death receptor stimulation but does not alter c-FLIP binding to the DISC.
• S193 phosphorylation influences intracellular c-FLIP(S) levels, thereby affecting sensitivity to apoptosis.

Conclusions:

• Differential stability of c-FLIP isoforms is regulated by PKC-mediated phosphorylation at serine 193 in an isoform-specific manner.
• Serine 193 phosphorylation represents a key regulatory node controlling c-FLIP levels and apoptosis sensitivity.