Tài liệu Báo cáo khoa học: Delineation of exoenzyme S residues that mediate the interaction with

Delineation of exoenzyme S residues that mediate the

interaction with 14-3-3 and its biological activity

Lubna Yasmin1,*, Anna L. Jansson1,*, Tooba Panahandeh1

, Ruth H. Palmer3

, Matthew S. Francis2

and Bengt Hallberg1

1 Department of Medical Biosciences ⁄ Pathology, Umea˚ University, Sweden

2 Department of Molecular Biology, Umea˚ University, Sweden

3 Umea˚ Center for Molecular Pathogenesis, Umea˚ University, Sweden

14-3-3 proteins are a group of highly conserved intra￾cellular dimeric molecules, expressed in plants, inverte￾brates and higher eukaryotes, with no intrinsic activity.

14-3-3 proteins play an important role in several signa￾ling pathways and 14-3-3 interacts with proteins in a

phospho-specific manner, using a defined consensus￾binding motif [1–3]. Several of these interacting part￾ners have recognized functions, which include enzymes

in biosynthetic metabolism, ion channels and regula￾tors of growth in plants [4–6]. It has been shown that

many human proteins can also bind directly to 14-3-3

in a phosphorylation-dependent manner, placing

14-3-3 as a central regulatory molecule in several

physiological processes such as biosynthetic metabo￾lism, cell proliferation, and survival in human cells

[3,7,8].

Crystal structure analyses of the 14-3-3 dimer alone

or in complex with peptides or native binding partners

has revealed the presence of a basic cluster in the

amphipathic groove of each monomer which mediates

the interaction of 14-3-3 with the phospho-amino acid

residues in its interaction partners. Therefore it is likely

that each dimer contains two binding pockets and can

interact with a single target or with multiple binding

partners. Further, it has been observed that interaction

between 14-3-3 proteins and its target partner(s) can

Keywords

ADP-ribosylation; coenzyme binding site;

cytotoxicity; NAD-dependent; cystic fibrosis;

Pseudomonas aeruginosa

Correspondence

B. Hallberg, Department of Medical

Biosciences ⁄ Pathology, Building 6M,

2nd floor, Umea˚ University, 901 87 Umea˚,

Sweden

Fax: + 46 90 785 2829

Tel: + 46 90 785 2523

E-mail: [email protected]

*Both authors contributed equally to this

work.

(Received 5 October 2005, revised 7

December 2005, accepted 12 December

2005)

doi:10.1111/j.1742-4658.2005.05100.x

14-3-3 proteins belong to a family of conserved molecules expressed in all

eukaryotic cells, which play an important role in a multitude of signaling

pathways. 14-3-3 proteins bind to phosphoserine ⁄ phosphothreonine motifs

in a sequence-specific manner. More than 200 14-3-3 binding partners have

been found that are involved in cell cycle regulation, apoptosis,

stress responses, cell metabolism and malignant transformation. A phos￾phorylation-independent interaction has been reported to occur between

14-3-3 and a C-terminal domain within exoenzyme S (ExoS), a bacterial

ADP-ribosyltransferase toxin from Pseudomonas aeruginosa. In this study,

we have investigated the effect of amino acid mutations in this C-terminal

domain of ExoS on ADP-ribosyltransferase activity and the 14-3-3 interac￾tion. Our results suggest that leucine-428 of ExoS is the most critical resi￾due for ExoS enzymatic activity, as cytotoxicity analysis reveals that

substitution of this leucine significantly weakens the ability of ExoS to

mediate cell death. Leucine-428 is also required for the ability of ExoS to

modify the eukaryotic endogenous target Ras. Finally, single amino acid

substitutions of positions 426–428 reduce the interaction potential of 14-3-3

with ExoS in vitro.

Abbreviations

ADPRT, ADP-ribosyltransferase; BD, binding domain; ExoS, exoenzyme S; FAS, factor activating exoenzyme S; GAP, GTPase-activating

protein; GEF, guanine exchange factor; GTPase, GTP binding protein; Ras, rat sarcoma.

638 FEBS Journal 273 (2006) 638–646 ª 2006 The Authors Journal compilation ª 2006 FEBS