Tài liệu Báo cáo khoa học: The ubiquitin ligase Itch mediates the antiapoptotic activity of

The ubiquitin ligase Itch mediates the antiapoptotic

activity of epidermal growth factor by promoting the

ubiquitylation and degradation of the truncated C-terminal

portion of Bid

Bilal A. Azakir, Guillaume Desrochers and Annie Angers

De´partement de sciences biologiques, Universite´ de Montre´ al, Que´bec, Canada

Introduction

Itch is a HECT domain ubiquitin ligase of the Nedd4

family, characterized by an N-terminal C2 domain

responsible for guiding intracellular localization to

internal membranes, four WW domains involved in

substrate recognition and a C-terminal catalytic

domain [1]. Itch is best known for its role in immune

system development through regulation of the level of

its target substrates, c-jun and junB [2,3]. However,

other substrates have been identified, and Itch action is

not limited to the immune system [4–10].

Epidermal growth factor (EGF) is well known for

its ability to promote cell growth [11]. It is also a key

regulator of cell survival [12]. Maintaining the balance

between cell survival and apoptosis is critical in the

maintenance of a healthy organism, and tipping the

equilibrium in one or another direction results in either

Keywords

apoptosis; Bid; c-Jun N-terminal kinase;

epidermal growth factor; HECT domain;

ubiquitin

Correspondence

A. Angers, De´partement de sciences

biologiques, Universite´ de Montre´ al, P.O.

Box 6128, station ‘Centre-Ville’, Montre´ al,

Que´bec H3C 3J7, Canada

Fax: +1 514 343 2293

Tel: +1 514 343 7012

E-mail: [email protected]

(Received 2 November 2009, revised 21

December 2009, accepted 24 December

2009)

doi:10.1111/j.1742-4658.2010.07562.x

The truncated C-terminal portion of Bid (tBid) is an important intermedi￾ate in ligand-induced apoptosis. tBid has been shown to be sensitive to pro￾teasomal inhibitors and downregulated by activation of the epidermal

growth factor (EGF) pathway. Here, we provide evidence that tBid is a

substrate of the ubiquitin ligase Itch, which can specifically interact with

and ubiquitinate tBid, but not intact Bid. Consistently, overexpression of

Itch increases cell survival and inhibits caspase 3 activity, whereas downre￾gulation of Itch by RNA interference has the opposite effect, increasing cell

death and apoptosis. Treatment with EGF increases Itch phosphorylation and

activity, and Itch expression is important for the ability of EGF to increase cell

survival after tumour necrosis factor-related apoptosis-inducing ligand treat￾ment. Our findings identify Itch as a key molecule between EGF signalling and

resistance to apoptosis through downregulation of tBid, providing further

details on how EGF receptor and proteasome inhibitors can contribute to the

induction of apoptosis and the treatment of cancer.

Structural digital abstract

l MINT-7542954: ITCH (uniprotkb:Q96J02) physically interacts (MI:0915) with tBid

(uniprotkb:P70444) by anti tag coimmunoprecipitation (MI:0007)

l MINT-7542970: tBid (uniprotkb:P70444) physically interacts (MI:0915) with Ubiquitin

(uniprotkb:P62988) by anti tag coimmunoprecipitation (MI:0007)

l MINT-7542986: ITCH (uniprotkb:Q96J02) physically interacts (MI:0915) with tBid

(uniprotkb:P70444) by bioluminescence resonance energy transfer (MI:0012)

Abbreviations

ATC, anaplastic thyroid carcinoma; BH3, Bcl-2-homology domain-3; BRET, bioluminescent resonance energy transfer; EGF, epidermal growth

factor; JNK, c-Jun N-terminal kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; rLuc, Renilla luciferase; tBid,

truncated C-terminal portion of Bid; TRAIL, tumour necrosis factor-related apoptosis-inducing ligand.

FEBS Journal 277 (2010) 1319–1330 ª 2010 The Authors Journal compilation ª 2010 FEBS 1319