Tài liệu Báo cáo khoa học: Golgi reassembly stacking protein 55 interacts with membrane-type (MT)

Golgi reassembly stacking protein 55 interacts with

membrane-type (MT) 1-matrix metalloprotease (MMP) and

furin and plays a role in the activation of the MT1-MMP

zymogen

Christian Roghi1,2, Louise Jones2

*, Matthew Gratian2

, William R. English1,2 and Gillian Murphy1,2

1 Cancer Research UK Cambridge Research Institute, The Li Ka Shing Centre, UK

2 Cambridge Institute for Medical Research, UK

Keywords

furin; GRASP55; intracellular traffic;

MT1-MMP; protease

Correspondence

C. Roghi, Cancer Research UK Cambridge

Research Institute, The Li Ka Shing Centre,

Robinson Way, Cambridge CB2 0RE, UK

Fax: +44 (0)1223 404573

Tel: +44 (0)1223 404472

E-mail: [email protected]

*Present address

KuDOS Pharmaceuticals Ltd, Cambridge

Science Park, UK

(Received 25 March 2010, revised 14 May

2010, accepted 28 May 2010)

doi:10.1111/j.1742-4658.2010.07723.x

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a proteinase

involved in the remodelling of extracellular matrix and the cleavage of a

number of substrates. MT1-MMP is synthesized as a zymogen that requires

intracellular post-translational cleavage to gain biological activity. Furin,

a member of the pro-protein convertase family, has been implicated in the

proteolytic removal of the MT1-MMP prodomain sequence. In the present

study, we demonstrate a role for the peripheral Golgi matrix protein

GRASP55 in the furin-dependent activation of MT1-MMP. MT1-MMP

and furin were found to co-localize with Golgi reassembly stacking protein

55 (GRASP55). Further analysis revealed that GRASP55 associated with

the cytoplasmic domain of both proteases and that the LLY573 motif in the

MT1-MMP intracellular domain was crucial for the interaction with

GRASP55. Overexpression of GRASP55 was found to enhance the forma￾tion of a complex between MT1-MMP and furin. Finally, we report that

disruption of the interaction between GRASP55 and furin led to a reduc￾tion in pro-MT1-MMP activation. Taken together, these data suggest that

GRASP55 may function as an adaptor protein coupling MT1-MMP with

furin, thus leading to the activation of the zymogen.

Structured digital abstract

l MINT-7897990: Furin (uniprotkb:P09958) and GRASP55 (uniprotkb:Q9H8Y8) colocalize

(MI:0403) by fluorescence microscopy (MI:0416)

l MINT-7897801: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with MT2-

MMP (uniprotkb:P51511) by two hybrid (MI:0018)

l MINT-7897821: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with MT3-

MMP (uniprotkb:P51512) by two hybrid (MI:0018)

l MINT-7897577: GRASP55 (uniprotkb:Q9R064) and MT1-MMP (uniprotkb:P50281) coloca￾lize (MI:0403) by fluorescence microscopy (MI:0416)

l MINT-7897366: MT1-MMP (uniprotkb:P50281) physically interacts (MI:0915) with

GRASP55 (uniprotkb:Q9H8Y8) by anti bait coimmunoprecipitation (MI:0006)

Abbreviations

ECM, extracellular matrix; EGFP, enhanced green fluorescent protein; EYFP, enhanced yellow fluorescent protein; FACS, fluorescence￾activated cell sorting; GFP, green fluorescent protein; GRASP, Golgi reassembly stacking protein; GRASP55F, FLAG-tagged GRASP55;

IB, immunoblotting; ICD, intracellular domain; M2H, mammalian two-hybrid; MMP, matrix metalloprotease; MT1/EYFP, EYFP-tagged

MT1-MMP; MT1/MYC, Myc-tagged MT1-MMP; MT-MMP, membrane-type MMP; PDZ, PSD-95/SAP90 Drosophila septate junction protein

discs-large and epithelial tight junction ZO-1; TGF, transforming growth factor; TGN, trans-Golgi network.

3158 FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS