Regulation of FLIP Long by the SCF(Skp2) ubiquitin ligase complex


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Jamie Roberts1,Caitriona Holohan1,Joel Riley2,Joanna Majkut1,Patrick Johnston1,Paul Moynagh3,Daniel Longley4
1Queen's University Belfast,2University of Glasgow,3Univerisity of Maynooth,4Queen's Univeristy Belfast

Abstract

Background

Extrinsic apoptosis occurs when death receptors (such as CD95/Fas, TRAIL-R1 and TRAIL-R2) bind their cognate ligands expressed by immune effector cells, stimulating the formation of the Death Inducing Signalling Complex (DISC). FLIP is as an anti-apoptotic protein frequently overexpressed in cancer that inhibits procaspase-8 activation at the DISC. The overexpression of FLIP not only stops apoptosis from occurring in cells that have been exposed to stimuli of the extrinsic apoptotic pathway such as CD95L/FasL and TRAIL, but is also a mediator of drug resistance. Identifying E3 ligases that regulate FLIP’s stability in cancer cells could lead to a novel method of targeting its expression.

Results

Treatment with MLN4924, an indirect inhibitor of the Skp1-Cul1-F-box (SCF) complex, leads to a reduction in ubiquitination of the long FLIP splice form (FLIPL) and increased protein stability, but has no effect on the short FLIP splice form (FLIPS). These effects on FLIPL were also observed when Cullin-1 (Cul1), Skp1 and Rbx1 were silenced using siRNA. Co-immunoprecipitation revealed an interaction between Myc-tagged Cul1 and FLAG-tagged FLIPL but not FLIPS. Multiple truncations of FLIPL were created, and the binding site was identified in the N-terminal pseudo-caspase domain of FLIPL, which is not expressed in FLIPS. Further studies demonstrated that Skp2 also interacts at this region, and its co-overexpression with Cul1 increased FLIPL ubiquitination, indicating that Skp2 may be the F-box protein required for interaction with FLIPL. In further support of this conclusion, the ubiquitination of FLIPL induced by the overexpression of Skp2/Cul1 was blocked by treatment with MLN924. These results provide strong evidence that the SCFSkp2 complex acts as an E3 ligase for FLIPL.

Conclusion

We have for the first time identified the SCFSkp2 ubiquitin ligase complex as an important constitutive regulator of FLIPL ubiquitination and degradation.