C-FLIP regulates autophagy by interacting with Beclin-1 and influencing its stability

Luana Tomaipitinca,Antonio Filippini,Federico Giulitti,Claudia Giampietri,Amit Dubey,Elio Ziparo,Angelo Facchiano,Pasquale D’Acunzo,Simonetta Petrungaro,Salvatore Rizza,Francesco Cecconi,Eugenio Gaudio

doi:10.1038/s41419-021-03957-5

Abstract

c-FLIP (cellular FLICE-like inhibitory protein) protein is mostly known as an apoptosis modulator. However, increasing data underline that c-FLIP plays multiple roles in cellular homoeostasis, influencing differently the same pathways depending on its expression level and isoform predominance. Few and controversial data are available regarding c-FLIP function in autophagy. Here we show that autophagic flux is less effective in c-FLIP−/− than in WT MEFs (mouse embryonic fibroblasts). Indeed, we show that the absence of c-FLIP compromises the expression levels of pivotal factors in the generation of autophagosomes. In line with the role of c-FLIP as a scaffold protein, we found that c-FLIPL interacts with Beclin-1 (BECN1: coiled-coil, moesin-like BCL2-interacting protein), which is required for autophagosome nucleation. By a combination of bioinformatics tools and biochemistry assays, we demonstrate that c-FLIPL interaction with Beclin-1 is important to prevent Beclin-1 ubiquitination and degradation through the proteasomal pathway. Taken together, our data describe a novel molecular mechanism through which c-FLIPL positively regulates autophagy, by enhancing Beclin-1 protein stability.

Highlights

FLIP (FLICE-like inhibitory protein) was described for the first time as a viral factor utilized by pathogens to escape apoptotic signalling in host cells [1]
We evaluated by western blotting the levels of Beclin-1, We previously demonstrated that the absence of cellular FLIP (c-FLIP) compro- Vps34 and the phosphorylation status of Atg14 after starvation in mises MEFs response to endoplasmic reticulum (ER) stress-dependent cell death induced a 3 h timeframe
As ER stress response is strictly connected to associates to the phosphatidylinositol-3-kinase Vps34 regulating autophagy activation, we wondered whether autophagic flux was its cognate partners binding

Summary

Introduction

FLIP (FLICE-like inhibitory protein) was described for the first time as a viral factor utilized by pathogens to escape apoptotic signalling in host cells [1]. FLIP homologues were identified in human cells and referred to as cellular FLIP (c-FLIP) [2] Since their characterization as anti-apoptotic factors, it became clear that c-FLIP family members exert multifunctional roles in several cellular processes, as they are involved in embryonic development, cardiac hypertrophy, skeletal muscle homoeostasis, T-cell proliferation and activation of nuclear factor-κ-light-chain-enhancer of activated B cells and extracellular signal-regulated kinase pathways [3,4,5,6,7]. Thirteen distinct splice variants originate from the human CFLAR gene, but only three of them are translated into proteins: the long isoform c-FLIPL (55 kDa) and two short isoforms, namely c-FLIPR (25 kDa) and c-FLIPS (27 kDa) [2]. Numerous lines of evidence show that c-FLIPL can support apoptosis activation [10]

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