Tài liệu Báo cáo Y học: Intracellular localization and transcriptional regulation of tumor necrosis

Intracellular localization and transcriptional regulation of tumor

necrosis factor (TNF) receptor-associated factor 4 (TRAF4)

Heike Glauner1

, Daniela Siegmund1

, Hassan Motejadded2

, Peter Scheurich1

, Frank Henkler2

,

Ottmar Janssen3 and Harald Wajant1

1

Institute of Cell Biology and Immunology and 2

Institute of Industrial Genetics, University of Stuttgart, Germany; 3

Institute of

Immunology, Christian-Albrechts-University of Kiel, Germany

To gain insight in the subcellular localization of tumor

necrosis factor receptor-associated factor (TRAF4) we

analyzed GFP chimeras of full-length TRAF4 and various

deletion mutants derived thereof. While TRAF4–GFP (T4–

GFP) was clearlylocalized in the cytoplasm, the N-terminal

deletion mutant, T4(259–470), comprising the TRAF

domain of the molecule, and a C-terminal deletion mutant

consisting mainlyof the RING and zinc finger domains of

TRAF4 were both localized predominantlyto the nucleus.

Passive nuclear localization of T4(259–470) can be ruled out

as the TRAF domain of TRAF4 was sufficient to form high

molecular weight complexes. T4(259–470) recruited full￾length TRAF4 into the nucleus whereas TRAF4 was unable

to change the nuclear localization of T4(259–470). Thus, it

seems that individual T4(259–470) mutant molecules are

sufficient to direct the respective TRAF4–T4(259–470)

heteromeric complexes into the nucleus. In cells forming

cell–cell contacts, TRAF4 was recruited to the sites of con￾tact via its C-TRAF domain. The expression of some TRAF

proteins is regulated bythe NF-jB pathway. Thus, we

investigated whether this pathwayis also involved in the

regulation of the TRAF4 gene. Indeed, in primaryT-cells

and Jurkat cells stimulated with the NF-jB inducers TNF or

phorbol 12-myristate 13-acetate (PMA), TRAF4-mRNA

was rapidlyup-regulated. In Jurkat T-cells deficient for I-jB

kinase c (IKKc, also known as NEMO), an essential com￾ponent of the NF-jB-inducing–IKK complex, induction of

TRAF4 was completelyinhibited. In cells deficient for RIP

(receptor interactive protein), an essential signaling inter￾mediate of TNF-dependent NF-jB activation, TNF-, but

not PMA-induced up-regulation of TRAF4 was blocked.

These data suggest that activation of the NF-jB pathway is

involved in up-regulation of TRAF4 in T-cells.

Keywords: IKKc; NF-jB; T-cells; localization; TRAF4.

The tumor necrosis factor (TNF) receptor-associated factor

(TRAF) familycomprises a group of adaptor proteins that

are involved in signal transduction bymembers of the TNF

receptor and IL1/Toll-receptor family[1,2]. The TRAF

proteins are characterized bya C-terminal homology

domain of about 200 amino acids, called the TRAF

domain. The TRAF domain mediates homo- and hetero￾merization of TRAF proteins and is also responsible for the

majorityof protein–protein interactions that have been

described for these molecules [1,2]. The TRAF domain can

be subdivided into the highlyconserved carboxy-terminal

TRAF-C subdomain, consisting of an eight-stranded anti￾parallel b-sandwich structure and a less conserved amino￾terminal part, the TRAF-N domain, which is organized as a

coiled-coil [1,2]. The TRAF domains form trimeric trefoil￾like structures, with the three TRAF-C domains as leaves

and the trimerized TRAF-N domains as the stalk [3–5]. In

mammalians six different TRAF proteins, designated as

TRAF1–TRAF6, have been described. With respect to the

architecture of the N-terminal domain, TRAF1 is clearly

distinct from all other TRAFs. While the N-terminus of

TRAF2–TRAF6 contains a highlyconserved RING

domain followed bya regularlyspaced cluster of five or

seven zinc fingers, the TRAF1 N-terminus onlycontains a

single zinc finger and no obvious additional structural

elements [1,2]. While TRAF1–TRAF5 have been implicated

mainlyin signaling bymembers of the TNF receptor family,

TRAF6 primarilytransduces signals initiated byIL1/Toll

receptors. In particular, TRAF4 has been shown to interact

with the lymphotoxin-b receptor and the p75 nerve growth

factor receptor in in vitro binding assays [6,7] but the

physiological relevance of these interactions remains to be

elucidated. While there is ample experimental evidence,

including the analyses of knockout mice, for an important

role of TRAF2, TRAF5 and TRAF6 in TNF receptor and

IL1/Toll-receptor induced activation of NF-jB and JNK

(c-Jun N-terminal kinase) [1,2], the role of TRAF1 and

TRAF3 for signal transduction byTNF receptors is poorly

understood. In fact, B-cells from mice deficient in TRAF3

have a defect in immunoglobulin isotype switching in

response to thymus-dependent antigens [8] and TRAF1

knockout mice show an increased TNF-R2-dependent

Correspondence to H. Wajant, Institute of Cell Biologyand

Immunology, University of Stuttgart, Allmandring 31,

70569 Stuttgart, Germany.

Fax: + 49 711 685 7484, Tel.: + 49 711 685 7446,

E-mail: [email protected]

Abbreviations: CHX, cycloheximide; FLIP, fluorescence loss in

photobleaching; IKK, I-jB kinase; NF-jB, nuclear factor jB;

PBMNC, mononuclear cells; PMA, phorbol 12-myristate 13-acetate;

RPA, RNAse protection assay; TNF, tumor necrosis factor;

TRAF, TNF receptor-associated factor.

(Received 24 April 2002, revised 10 July2002,

accepted 13 August 2002)

Eur. J. Biochem. 269, 4819–4829 (2002)
FEBS 2002 doi:10.1046/j.1432-1033.2002.03180.x