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 Cancer Research UK Cambridge Research Institute, The Li Ka Shing Centre, UK 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: chr26@cam.ac.uk *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 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 formation of a complex between MT1-MMP and furin Finally, we report that disruption of the interaction between GRASP55 and furin led to a reduction 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 MT2MMP (uniprotkb:P51511) by two hybrid (MI:0018) l MINT-7897821: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with MT3MMP (uniprotkb:P51512) by two hybrid (MI:0018) l MINT-7897577: GRASP55 (uniprotkb:Q9R064) and MT1-MMP (uniprotkb:P50281) colocalize (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, fluorescenceactivated 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 C Roghi et al Role of GRASP55 in MT1-MMP activation l l l l l l l l l l l l l MINT-7897617, MINT-7897659, MINT-7897681, MINT-7897702, MINT-7897725, MINT7898032, MINT-7898011, MINT-7897907, MINT-7897884: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with MT1-MMP (uniprotkb:P50281) by two hybrid (MI:0018) MINT-7898002: MT1-MMP (uniprotkb:P50281) physically interacts (MI:0914) with Furin (uniprotkb:P09958) by anti bait coimmunoprecipitation (MI:0006) MINT-7897500: MT1-MMP (uniprotkb:P50281) and Giantin (uniprotkb:Q14789) colocalize (MI:0403) by fluorescence microscopy (MI:0416) MINT-7897750, MINT-7897394: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with MT1-MMP (uniprotkb:P50281) by anti tag coimmunoprecipitation (MI:0007) MINT-7897562: MT1-MMP (uniprotkb:P50281) and GRASP55 (uniprotkb:Q9H8Y8) colocalize (MI:0403) by fluorescence microscopy (MI:0416) MINT-7897512: TGN46 (uniprotkb:O43493) and MT1-MMP (uniprotkb:P50281) colocalize (MI:0403) by fluorescence microscopy (MI:0416) MINT-7897921, MINT-7897975: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with Furin (uniprotkb:P09958) by two hybrid (MI:0018) MINT-7898052, MINT-7897410: MT1-MMP (uniprotkb:P50281) physically interacts (MI:0915) with GRASP55 (uniprotkb:Q9R064) by anti bait coimmunoprecipitation (MI:0006) MINT-7897951: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with PC7 (uniprotkb:Q16549) by two hybrid (MI:0018) MINT-7897866: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with MT5MMP (uniprotkb:Q9Y5R2) by two hybrid (MI:0018) MINT-7897633: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with TGFA (uniprotkb:P01135) by two hybrid (MI:0018) MINT-7897551: GRASP55 (uniprotkb:Q9H8Y8) and Giantin (uniprotkb:Q14789) colocalize (MI:0403) by fluorescence microscopy (MI:0416) MINT-7897938: GRASP55 (uniprotkb:Q9R064) physically interacts (MI:0915) with PC5/6B (uniprotkb:Q04592) by two hybrid (MI:0018) Introduction Extracellular matrix (ECM) remodelling is a crucial process occurring during cell migration and invasion in various physiological (i.e embryonic development, ovulation, angiogenesis, wound healing) and pathological processes, including rheumatoid arthritis, tumour growth, invasion and metastasis [1] Of all the different proteolytic systems involved in ECM turnover, the matrix metalloproteinases (MMPs) have been reported to exert a dominant effect [2] MMPs are a large family of structurally and functionally related multi-domain zinc-dependent endopeptidases that collectively are able to degrade virtually all proteins of the ECM MMPs are mainly soluble enzymes released by the cell in the extracellular milieu, although membrane-bound MMPs (membrane type-MMPs or MT-MMPs) have also been identified and are ideally positioned for regulating pericellular proteolysis [3] Membrane-type matrix metalloproteinase (MT1MMP; MMP14; EC 3.4.24.80) is by far the most extensively studied member of the MT-MMP subfamily MT1-MMP is a type transmembrane MMP involved in pericellular ECM turnover [4], as well as in the proteolytic processing of cell surface receptors [4,5] MT1-MMP is also involved in the activation of pro-MMP2 and pro-MMP13, leading to the indirect increase in its repertoire of substrates [6,7] MT1-MMP has a wide spectrum of cellular functions [5,8] Elevated MT1-MMP expression, which is well documented in many tumours, has been correlated with key processes of tumour progression [9,10], including angiogenesis [11], cell migration and invasion [12], cell growth [13], and metastatic spread Inhibition or silencing of the protease has been found to significantly reduce the invasive phenotype of tumour cells, implicating a leading role for MT1-MMP in such processes [12,14] There is mounting evidence that the short intracellular domain (ICD) of MT1-MMP (21 amino acids) plays an important role in multiple MT1-MMP-mediated cellular events [15] MT1-MMP ICD has been involved in cell migration [16] and invasion into reconstituted basement membrane [17,18] The MT1-MMP ICD is also critical for the intracellular trafficking of the enzyme [19–23] and its targeting to invadopodia in invasive cells [24] The ICD of MT1-MMP has been found to modulate multiple signal transduction pathways [16,25–27] and participates in the homophilic interaction between MT1-MMP monomers [28] Recently, the LL572 di-leucine motif has been reported FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS 3159 Role of GRASP55 in MT1-MMP activation C Roghi et al to influence the O-glycosylation pattern of MT1-MMP [29] Post-translational modifications of the MT1MMP ICD have also been reported with the palmitoylation of the cysteine 574 (C574) residue [30] and the phosphorylation of the tyrosine 573 (Y573) [31] and threonine 567 (T567) [32] residues The MT1-MMP ICD has been reported to interact with the multifunctional protein p32/gC1qR [21], a protein with homology to members of the Cupin superfamily (MTCBP-1) [33], as well as with the l2 subunit of the clathrincoated pits adapter protein (AP-2) [18] and phosphocaveolin-1 in src overexpressing cells [34] Golgi reassembly stacking protein 55 (GRASP55) is a peripheral Golgi matrix protein that has been implicated, in vitro, in the post-mitotic stacking of Golgi cisternae [35] Cryo-electron microscopy has shown that GRASP55 is found predominantly in the medial-cisternae of the Golgi complex of HeLa cells [35] GRASP55 interacts with Golgin-45 and the complex is crucial for maintaining Golgi structure [36] In addition to its contribution to the Golgi exoskeleton, GRASP55 has also been reported to be involved in the intracellular transport of pro-transforming growth factor (TGF)-a [37], CD8a or the frizzled receptor Fz4 [38], as well as in the Golgi retention of p24a, a member of the p24 family of cargo receptors [39] In the present study, we report in detail on the interaction between the MT1-MMP ICD and GRASP55 using a mammalian two-hybrid (M2H) system Using this approach, we have identified the GRASP55 binding site in the ICD of MT1-MMP, as well as the GRASP55 domains involved in the interaction with MT1-MMP ICD We also describe the GRASP55 interaction with the furin ICD, and provide evidence that GRASP55 could play an important role in the furin-mediated proteolytic activation of the MT1MMP zymogen Results MT1-MMP co-immunoprecipitates with GRASP55 Although the presence of MT1-MMP and GRASP55 (p59) in the same complex has been suggested by Kuo et al [37], the functional implications of this interaction have yet to be fully investigated In steady-state HT1080 cells, MT1-MMP is mainly present at the cell surface and in the endosomal compartment [22] and virtually no protease can be detected in the Golgi apparatus We therefore transfected these cells with an exogenous wild-type MT1-MMP cDNA (Fig 1A), aiming to detect the protease in the early secretory pathway Cells were then lysed and the extract was 3160 A + – – pCDNA3.1 Zeo+ + MT1-MMP kDa 60 IB: MT1-MMP 50 40 60 IB: GRASP55 50 40 50 40 IB: β-actin B + – IgG – + anti MT1-MMP kDa 60 IB: GRASP55 50 40 C – – + – – + + + MT1/MYC GRASP55F kDa IP: FLAG 60 IB: MYC 60 IB: FLAG Input lysates IB: MYC 60 Fig MT1-MMP co-immunoprecipitates with GRASP55F (A) Protein extracts prepared from HT1080 cells transiently transfected with pCDNA3.1 Zeo+ (lane 1) or full-length MT1-MMP construct were analyzed by IB with antibodies directed against MT1-MMP, GRASP55 and b-actin (B) Protein extract prepared from HT1080 transiently transfected with MT1-MMP were immunoprecipitated with rabbit control IgGs (lane 1) or with the rabbit polyclonal antibody directed against MT1-MMP (lane 2) and analyzed by IB using a monoclonal antibody to GRASP55 The arrow identifies immunoprecipitated MT1-MMP (C) Protein extracts prepared from HT1080 cells transiently transfected with pCDNA3.1 Zeo+ and MT1/MYC (lane 2), pCDNA3.1 Zeo+ and GRASP55F (lane 3) and MT1/MYC and GRASP55F (lane 4) were immunoprecipitated with the FLAG M2 monoclonal antibody and the associated MT1-MMP was detected by IB using the MYC tag monoclonal antibody Expression of the transfected construct was monitored in the input lysates using specific antibodies The black arrowhead indicates IgG (immunoglobulin heavy chain) FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS C Roghi et al Role of GRASP55 in MT1-MMP activation A B C D E F G H I J K L Fig Subcellular localization of GRASP55 and MT1-MMP in bbHT1080 Fixed and permeabilized bbHT1080 cells were incubated with antibodies directed against MT1-MMP (A, D, J), GRASP55 (G, K), TGN46 (E) and giantin (B, H) The co-localization can be observed in yellow in the merged panels (C, F, I, L) Arrowheads depict membranous structures where the proteins co-localize Scale bar = lm FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS 3161 Role of GRASP55 in MT1-MMP activation C Roghi et al Fig Co-localization of MT1-MMP and GRASP55 in live cells Four consecutive frames (4 s apart) of time lapse sequence collected from HT1080 co-transfected cells with EYFP/MT1 and GRASP55-GFP The arrowheads are examples of dynamic vesicles containing both fluorescent proteins MT1/EYFP was pseudo-coloured in red during post-acquisition processing The co-localization between GRASP55-GFP and MT1/EYFP can be observed in yellow in the merged panels Scale bar = 16 lm immunoprecipitated with nonspecific rabbit IgGs or with the rabbit polyclonal antibody directed against MT1-MMP As shown in Fig 1B, immunoprecipitation of MT1-MMP led to the co-precipitation of a small amount of endogenous GRASP55, as detected by immunoblotting (IB) No GRASP55 was detected when the pre-immune IgGs were used Co-precipitation between MT1-MMP and GRASP55 was also observed in HT1080 cells expressing exogenous Myctagged MT1-MMP (MT1/MYC) and FLAG-tagged GRASP55 (GRASP55F) (Fig 1C, lane 4) or HeLa cells (Fig S1) No MT1-MMP was detected in control immunoprecipitations (Fig 1C, lanes 1–3 and Fig S1) MT1-MMP co-localizes with GRASP55 The co-immunoprecipitation of MT1-MMP and GRASP55 prompted us to investigate whether these two proteins co-localized in the same membranous compartment To investigate this, we used HT1080 cells stably expressing wild-type MT1-MMP (bbHT1080) In these cells, MT1-MMP (Fig 2A, 2D) co-localized 3162 extensively with the medial Golgi marker giantin (Fig 2B) and with the trans-Golgi network (TGN) membrane protein marker TGN46 (Fig 2E) Endogenous GRASP55 (Fig 2G, 2K) was also found to co-localize with giantin (Fig 2H) [40] and a clear co-localization with MT1-MMP (Fig 2J) could also be observed (Fig 2L) in these cells The co-localization of MT1-MMP and GRASP55 was next assessed using live video microscopy We generated an enhanced yellow fluorescent protein (EYFP)-tagged MT1-MMP construct (MT1/EYFP), where the EYFP tag replaced the entire MT1-MMP catalytic domain In HT1080, the intracellular trafficking of MT1/EYFP was indistinguishable from that of wild-type MT1-MMP and both constructs were found to accumulate in the Golgi apparatus and the TGN in these cells (C Roghi, unpublished data) HT1080 cells were then transiently co-transfected with MT1/EYFP and the GRASP55-green fluorescent protein (GFP) fusion protein as previously described [35] and the localization of both proteins was studied in live cells Separation of the GFP and EYFP signals was achieved using a Zeiss META confocal microscope (see FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS C Roghi et al Role of GRASP55 in MT1-MMP activation A VP16-MT1 VP16 PGGGFFFRRHGTPRRLLYCQRSLLDKV VP16-MT1 FRR VP16 PGGGFFAAAHGTPRRLLYCQRSLLDKV VP16-MT1 HGT VP16 PGGGFFFRRAAAPRRLLYCQRSLLDKV VP16-MT1 PRR VP16 PGGGFFFRRHGTAAALLYCQRSLLDKV VP16-MT1 LLY VP16 PGGGFFFRRHGTPRRAAACQRSLLDKV VP16-MT1 CQR VP16 PGGGFFFRRHGTPRRLLYAAASLLDKV VP16-MT1 SLL VP16 PGGGFFFRRHGTPRRLLYCQRAAADKV VP16 MT1 VP16-MT1 DKV VP16 PGGGFFFRRHGTPRRLLYCQRSLLAAA VP16-MT1 Y VP16 PGGGFFFRRHGTPRRLLACQRSLLDKV VP16-MT1 LL VP16 PGGGFFFRRHGTPRRAAYCQRSLLDKV VP16-TGF-α VP16 KHCEWCRALICRHEKPSALLKGRTACCHSETVV GAL4 GAL4 GRASP55 GAL4 Fig MT1-MMP interaction with GRASP55 (A) Schematic representation of VP16-TGF-a, VP16-MT1 and the VP16-MT1 mutant constructs The mutated amino acids are shown in bold and the PGGG linker is shown in italics (B) Interaction between full-length GRASP55 (GAL4GRASP55) and MT1-MMP ICD (VP16-MT1) or (C) TGF-a ICD (VP16-TGF-a) using the M2H assay GAL4 GRASP55 500 Luminescence (arbitrary units) 100 200 300 400 500 Luminescence (arbitrary units) 100 200 300 400 500 + VP16 + VP16 + VP16 + VP16 MT1 MT1 C GAL4 + VP16 GAL4 GRASP55 + VP16 GAL4 + VP16 TGF-α GAL4 GRASP55 + VP16 TGF-α A GAL4 + VP16 MT1 GAL4 GRASP55 + VP16 MT1 GAL4 GRASP55 + VP16 MT1 FRR GAL4 GRASP55 + VP16 MT1 HGT GAL4 GRASP55 + VP16 MT1 PRR GAL4 GRASP55 + VP16 MT1 LLY GAL4 GRASP55 + VP16 MT1 CQR GAL4 GRASP55 + VP16 MT1 SLL GAL4 GRASP55 + VP16 MT1 DKV – + + * – + – ** ** – – + * + – – B *** Fig The LLY motif in the MT1-MMP ICD is crucial for the interaction with GRASP55 (A) Interaction between GAL4-GRASP55 and VP16-MT1 or MT1-MMP ICD triple mutants (B) Cell lysates prepared from HT1080 cells transfected with pCDNA3.1 Zeo+ and MT1/ MYC (lane 1), pCDNA3.1 Zeo+ and GRASP55F (lane 2), pCDNA3.1 Zeo+ and MT1 LLY/MYC (lane 3), GRASP55F and MT1 LLY/MYC (lane 4) and GRASP55F and MT1/MYC (lane 5) were immunoprecipitated with the FLAG M2 antibody MT1-MMP present in the immunoprecipitate was detected by IB using the MYC tag antibody Levels of transfected proteins were monitored in input lysates using specific antibodies Luminescence (arbitrary units) 100 200 300 400 B + – + MT1/MYC MT1 LLY/MYC GRASP55F IP: FLAG IB: MYC 50 WB: MYC 60 Input lysates WB: FLAG 60 FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS 3163 Role of GRASP55 in MT1-MMP activation C Roghi et al Materials and methods) As previously described in fixed bbHT1080 (Fig 2), we clearly observed, in live HT1080 cells, the presence of both tagged proteins in the same membrane compartment (Fig 3, merged), thus confirming the results that were observed previously in fixed bbHT1080 cells (Fig 2) Interestingly, we also noted that MT1/EYFP and GRASP55-GFP also co-localized in very dynamic unidentified cytoplasmic membranous structures (Fig 3, arrowheads) MT1-MMP intracellular domain is involved in the interaction with GRASP55 If the co-immunoprecipitation between MT1-MMP and GRASP55 is functionally relevant, there should be an interaction between the peripheral scaffolding protein and the ICD of MT1-MMP To investigate this, we used an M2H system MT1-MMP ICD flanked by an N-terminal PGGG linker was fused to the VP16 activation domain (VP16-MT1; Fig 4A) and full-length GRASP55 was fused to the GAL4 DNA binding domain (GAL4-GRASP55) Both constructs were transiently co-transfected in HT1080 cells together with the reporter plasmid pG5luc, which contains the firefly luciferase gene under the control of five GAL4 binding sites After 24 h of transfection, the firefly luciferase activity was measured, as described in the Materials and methods, and the values obtained were normalized according to transfection efficiency using the Renilla reniformis luciferase expressed by the pBIND vector Co-expression of VP16-MT1 and GAL4-GRASP55 fusion proteins (Fig 4B, lane 4) in HT1080 resulted in the production of significantly higher firefly luciferase luminescence compared to the controls (Fig 4B, lanes 1–3), demonstrating an interaction between the MT1-MMP ICD and GRASP55 in the M2H system Using this assay, we also observed an interaction between the VP16-TGF-a ICD (Fig 4A) and GAL4-GRASP55 (Fig 4C, lane 4), therefore confirming the interaction of these two proteins previously observed biochemically [37] or using a yeast two-hybrid assay [39] Interestingly, in the M2H system, the interaction between GRASP55 and TGF-a ICD did not require the oligomerization of the TGF-a ICD as previously observed using a yeast two-hybrid assay [39] The MT1-MMP LLY motif is important for the interaction with GRASP55 We next sought to define the nature of the GRASP55 binding site in the MT1-MMP ICD pACT plasmids driving the expression of MT1-MMP ICDs containing single-, double- and triple-point mutations were generated (Fig 4A) and used in the M2H system Systematic analysis of the interaction between the triple MT1-MMP ICD mutants and GAL4-GRASP55 revealed that, apart from the VP16-MT1 FFR (Fig 5A, lane 3) and VP16-MT1 DKV mutants (Fig 5A, lane 9), all the other triple mutants (Fig 5A, lanes 4–8) displayed a marked and significant reduction of luciferase activity compared to the wild-type VP16-MT1 construct (Fig 5A, lane 2) In particular, the mutation of the LLY573 motif (LLY571-573AAA) (Fig 5A, lane 6) resulted in a complete inhibition of % homology with MT1-MMP ICD A VP16-MT1 VP16 PGGGFFFRRHGTPRRLLYCQRSLLDKV VP16-MT2 VP16 PGGGVQMQRKGAPRVLLYCKRSLQEWV 57.1% VP16-MT3 VP16 PGGGFQFKRKGTPRHILYCKRSMQEWV 57.1% VP16-MT5 VP16 PGGGFQFKNKTGPQPVTYYKRPVQEWV 23.8% B GAL4 + VP16 MT3 GAL4 GRASP55 + VP16 MT3 GAL4 GRASP55 + GAL4 GAL4 GRASP55 + VP16 + VP16 GAL4 GRASP55 + VP16 MT5 VTY 3164 VP16 MT3 ILY MT5 MT5 Luminescence (arbitrary units) 100 200 300 400 500 ** + VP16 MT2 GAL4 GAL4 GRASP55 + VP16 MT2 GAL4 GRASP55 + VP16 MT2 LLY - Fig The MT2-MMP LLY motif is involved in the interaction with GRASP55 (A) Schematic representation of the VP16-MT1, VP16-MT2, VP16-MT3 and VP16-MT5 constructs Amino acids conserved between MT1-MMP ICD and either MT2-, MT3- or MT5-MMP are shown in bold (B) Interactions between GAL4-GRASP55 and VP16-MT2, VP16-MT2 LLY, VP16-MT3, VP16-MT3 ILY, VP16-MT5 and VP16-MT5 VTY were tested using the M2H system **P < 0.001 FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS C Roghi et al Role of GRASP55 in MT1-MMP activation ting that the disruption of the interaction between MT1-MMP and GRASP55 could affect the activation of the protease Taken together, and having been obtained using different experimental approaches, our data demonstrate that the LLY573 motif in MT1-MMP ICD plays an important role in the interaction between MT1-MMP with GRASP55 Interestingly, we were unable to co-immunoprecipitate MT1-MMP and the soluble G2A GRASP55F mutant (Fig S3) [35,37,41], suggesting that the Golgi localization of GRASP55 is crucial for its interaction with MT1-MMP the interaction between MT1-MMP ICD and GAL4-GRASP55 IB analysis revealed that all triple mutants were expressed to a similar level (data not shown), indicating that the differences in interaction observed were not a result of impaired protein production or stability Our data therefore suggest that most of the MT1-MMP ICD is implicated in the interaction with GRASP55, with the LLY573 motif playing a critical role in the interaction between the two proteins A reduction of luciferase activity was also observed using the VP16-MT1 Y (MT1Y573A) (Fig S2, lane 5) and VP16-MT1 LL (LL570-572AA) mutants (Fig S2, lane 4), although not to the level observed with the LLY570-573AAA triple mutant (Fig S2, lane 3), demonstrating that the mutation of the whole LLY573 motif is needed to abolish the interaction of MT1MMP ICD with GRASP55 The importance of the LLY573 motif in the MT1MMP ICD observed in the M2H assay was next confirmed by co-immunoprecipitation HT1080 cells were transiently co-transfected with GRASP55F together with MT1/MYC or MT1 LLY/MYC (LLY570-573 AAA) triple mutant and total cell lysates were subjected to immunoprecipitation using the FLAG tag monoclonal antibody As previously observed, we detected a clear interaction between GRASP55F and the wild-type MYC-tagged MT1-MMP (Fig 5B, lane 5) when both proteins were expressed in HT1080 cells Mutation of the LLY573 motif to AAA573 in MT1MMP ICD led to a marked reduction in the amount of MT1-MMP present in the immunoprecipitated material (Fig 5B, lane 4), thus confirming the important role of the LLY573 motif in the interaction between the protease and GRASP55 Interestingly, mutation of the LLY573 motif led to the detection of pro-MT1-MMP in the immunoprecipitate, sugges- GRASP55 interacts with MT2-, MT3- and MT5-MMP The sequences of cytoplasmic domains of the four MT-MMPs are conserved (Fig 6A) Interestingly, the LLY573 motif in MT1-MMP ICD was completely conserved in MT2-MMP (LLY660), whereas ILY598 and VTY636 sequences were found in MT3-MMP and MT5-MMP ICDs, respectively (Fig 6A) To test whether MT2-, MT3- and MT5-MMP ICDs could also interact with GRASP55, we generated VP16-MT2, -MT3 and -MT5 chimeras (Fig 6A) As shown in Fig 6B, all three ICDs (Fig 6B, lanes 2, and 8) showed a clear interaction with GRASP55 We also tested whether the LLY660 motif in MT2-MMP, the ILY598 motif in MT3-MMP or the VTY636 motif in MT5-MMP could also be involved in the interaction with GRASP55 Accordingly, VP16-MT2 LLY, VP16MT3 ILY and VP16-MT5 VTI triple mutants were generated and used in the M2H assay As previously observed for MT1-MMP, mutation of the MT2-MMP LLY660 motif to AAA660 significantly decreased the interaction with GRASP55 (Fig 6B, lane 3) By GAL4 + VP16 GAL4 GRASP55 + VP16 GAL4 GRASP55 + VP16 MT1 + VP16 MT1 MT1 MT1 GAL4 P2 Fig GRASP55 PDZ2 and region are important for the interaction with the MT1-MMP ICD (A) Interactions between VP16-MT1 and GAL4-GRASP55, GRASP55 PDZ1 (GAL4-P1), GRASP55 PDZ2 (GAL4-P2) or GRASP55 region (GAL4-R3) and (B) between VP16-TFG a and GAL4GRASP55 or GAL4-P1 were tested using the M2H system 500 + VP16 Luminescence (arbitrary units) 100 200 300 400 + VP16 500 GAL4 P1 Luminescence (arbitrary units) 100 200 300 400 A GAL4 R3 MT1 B GAL4 + VP16 TGF-α GAL4 GRASP55 + VP16 TGF-α GAL4 P1 + VP16 TGF-α FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS 3165 Role of GRASP55 in MT1-MMP activation C Roghi et al + VP16 Furin + VP16 Furin GAL4 + VP16 PC5/6B GAL4 GRASP55 + VP16 PC5/6B Luminescence (arbitrary units) 100 200 300 400 GAL4 GRASP55 500 * GAL4 Luminescence (arbitrary units) 100 200 300 400 A PC7 B + VP16 + VP16 GAL4 GRASP55 + VP16 Furin Furin Furin Furin + VP16 Furin 500 + VP16 + VP16 * PC7 * + VP16 GAL4 GRASP55 + VP16 GAL4 GAL4 GAL4 GRASP55 GAL4 P1 GAL4 P2 GAL4 R3 Fig GRASP55 interacts with furin, PC5/ 6B and PC7 (A) Interactions between GAL4-GRASP55 and furin, PC5/6B or PC7 ICDs were tested using the M2H system (B) Interaction between VP16-furin and GAL4-GRASP55, GRASP55 PDZ1 (GAL4P1), GRASP55 PDZ2 (GAL4-P2) or GRASP55 Region (GAL4-R3) (C) Furin co-localized with GRASP55 bbHT1080 cells, transfected with a full-length furin cDNA, were permeabilized and stained with polyclonal antibodies against furin and GRASP55 Arrows show examples of membrane compartment containing GRASP55 and furin Scale bar = 10 lm C GRASP55 Furin contrast, mutation of MT3-MMP ILY598 (Fig 6B, lane 6) and MT5-MMP VTY636 to AAA (Fig 6B, lane 9) had no effect on GRASP55 binding MT1-MMP ICD binds to PDZ2 domain and region of GRASP55 GRASP55 contains two non-overlapping and structurally independent PSD-95/SAP90 Drosophila septate junction protein discs-large and epithelial tight junction ZO-1 (PDZ) domains in its N-terminal half, followed by a third region of approximately 250 amino acids without known structural motif (region 3) We next aimed to identify the region(s) of GRASP55 that interacts with MT1-MMP ICD GRASP55 PDZ1 (amino acids 1–107), GRASP55 PDZ2 (amino acids 84–172) and GRASP55 region (amino acids 173–454) were each fused to the GAL4 DNA binding domain and used together with VP16-MT1 in the M2H system MT1-MMP ICD was found to interact with full-length GRASP55 (Fig 7A, lane 3), as well as with GRASP55 PDZ2 (P2; Fig 7A, lane 5) and GRASP55 region (R3; Fig 7A, lane 6) However, no interaction was 3166 found between VP16-MT1 and GAL4-GRASP55 PDZ1 (P1; Fig 7A, lane 4), despite the expression of the GAL4-GRASP55 PDZ1 chimera in HT1080 (data not shown) TGF-a was previously reported to coimmunoprecipitate with a very small amount of flagged tagged GRASP55 PDZ1 domain [37] In our hands, no interaction between TGF-a ICD and GRASP55 PDZ1 could be observed in the M2H assay (Fig 7B, lane 3) The lack of interaction could result from a mis-folding of GRASP55 PDZ1 subsequent to its fusion to the GAL4 DNA binding domain We therefore cannot rule out an interaction between MT1-MMP ICD and the GRASP55 PDZ1 domain GRASP55 binds to furin, PC5/6B and PC7 intracellular domains The pro-convertase furin has previously been implicated in the activation of pro-MT1-MMP [42] Because MT1-MMP activation occurs during the intracellular traffic of the protease, we tested whether furin could interact, via its ICD, with GRASP55 Accordingly, we generated a VP16-furin construct FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS C Roghi et al Role of GRASP55 in MT1-MMP activation C A – + – + – – + + kDa Furin GRASP55F – + EGFP-furin ICD 64 pro-MT1-MMP active-MT1-MMP kDa IP: MT1-MMP * 98 64 IB: Furin 50 IB: Furin 98 IB: MT1-MMP 50 IB: β-actin 64 IB: FLAG 50 Input lysate 36 64 IB: MT1-MMP 50 – + + + MT1/MYC – + + + GRASP55F – B – 05 0.5 10 1.0 EGFP furin EGFP-furin ICD (μg) D *** 20 kDa IP: FLAG 50 IB: MYC 60 MMP2 (Arbritary units × 105) 60 IB: GFP 15 ** *** n.s 10 50 IB: FLAG 60 Input lysate 60 IB: MYC 50 Fig GRASP55 is important for MT1-MMP–furin complex formation and activation of pro-MT1-MMP (A) Lysates of bbHT1080 cells transfected with pCDNA3.1 Zeo+ vector control (lane 1), pCDNA3.1 Zeo+ and furin (lane 2), pCDNA3.1 Zeo+ and GRASP55 (lane 3) or with furin and GRASP55F (lane 4) were immunoprecipitated using the affinity-purified anti-MT1-MMP IgGs and the associated furin was detected by IB Levels of transfected proteins were monitored in input lysates using specific antibodies Asterisks marks endogenous furin immunoprecipitated by MT1-MMP in bbHT1080 (B) Expression of EGFP-furin ICD disrupted the formation of the complex between MT1-MMP and GRASP55 Lysates of HT1080 cells transfected with pCDNA3.1 Zeo+ vector control (lane 1), MT1/MYC and GRASP55F (lane 2), MT1/MYC and GRASP55F and 0.5 lg EGFP-furin ICD (lane 3) or MT1/MYC and GRASP55F and 1.0 lg of EGFP-furin ICD (lane 4) were immunoprecipitated with the FLAG antibody and the associated MT1-MMP was detected by IB using the MYC tag antibody Top black arrowheads indicate IgGs The bottom black arrowhead indicates a crossreaction (C) IB analysis of protein extracts prepared from the EGFP-negative (lane 1) and -positive (lane 2) cell population sorted in Fig S4 Equal amounts of total protein (6 lg) were loaded and the expression of MT1-MMP (pro and active) was analyzed by IB Protein loading was controlled using the b-actin polyclonal antibody (D) Expression of furin decrease MT1-MMP cell surface activity HT1080 cells were transiently transfected with empty vector pCDNA3.1 Zeo+, furin, GRASP55, furin + GRASP55, MT1-MMP, MT1-MMP + furin, MT1-MMP + GRASP55 and MT1-MMP + GRASP55 + furin 4b-Phorbol 12-myristate 13-acetate was used at 50 ngỈlL)1 After 24 h, supernatants were collected and analyzed by zymography Data represent the mean ± SEM of two independent experiments where the furin ICD was fused to VP16 We also generated the VP16-PC5/6B and VP16-PC7 chimeras where the ICDs of PC5/6B and PC7 type I transmembrane pro-convertases were also fused to VP16 Comparable to furin, PC5/6B and PC7 have previously been reported to process tetrabasic cleavage sites [43] and their roles in pro-MT1-MMP activation have been suggested both in vitro [44] and in vivo [42] As shown in Fig 8A, a clear interaction between furin ICD and GRASP55 could be observed (Fig 8A, lane 2) compared to the control (Fig 8A, lane 1) We also observed a significant interaction between GRASP55 and PC5/6B (Fig 8A, lane 4) or PC7 (Fig 8A, lane 6) ICDs We next tested the interaction between furin ICD and the GRASP55 domain constructs described previously As shown in Fig 8B, we detected an interaction between furin ICD and PDZ2 (Fig 8B, lane 5) and region (Fig 8B, lane 6) of GRASP55 No interaction was detected between GRASP55 PDZ1 (Fig 8B, lane 4) and furin ICD, FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS 3167 Role of GRASP55 in MT1-MMP activation C Roghi et al possibly as a result of the same reasons previously reported for MT1-MMP and TGF-a The interaction observed between the furin ICD and GRASP55 lead us to test whether both proteins could be detected in the same membrane compartment We were unable to detecte the endogenous level of furin in HT1080 cells This is in agreement with previous observations [45] To localize the proprotein-convertase in HT1080 cells, we transfected the cells with a low amount of the furin cDNA (250 ng) In furin overexpressing HT1080 cells, we could clearly detect the pro-convertase in cytoplasmic vesicles scattered throughout the cell cytoplasm, as well as in the TGN (Fig 8C, furin) Staining of GRASP55 (Fig 8C, GRASP55) revealed a limited co-localization between the two proteins Interestingly, in fixed samples, we also observed co-localization between furin and GRASP55 in cytoplasmic vesicular structures (Fig 8C, arrows), as previously observed for MT1-MMP and GRASP55 in HT1080 cells by video microscopy (Fig 3) GRASP55 expression induces formation of a MT1-MMP–furin complex The interaction on the one hand between MT1-MMP and GRASP55 and on the other hand between GRASP55 and furin led us to hypothesize that GRASP55 could potentially play a role in the formation of a complex between MT1-MMP and furin To test this hypothesis, bbHT1080 cells, which stably overexpress MT1-MMP, were transfected with furin, GRASP55F or with both furin and GRASP55F Total cell lysates were subjected to immunoprecipitation using the MT1-MMP antibody and immunocomplexes were then probed with the furin polyclonal antibody (Fig 9A) No furin was immunoprecipitated with MT1-MMP in bbHT1080 cells transfected with pCDNA 3.1 Zeo+ alone (Fig 9A, lane 1) or expressing only GRASP55 (Fig 9A, lane 3) When furin was expressed in bbHT1080 cells, a small amount of the proprotein-convertase was found in the same complex as MT1-MMP (Fig 9A, lane 2, asterisk) Expression of GRASP55F together with furin resulted in a significant increase in the amount of furin co-immunoprecipitated with MT1-MMP (Fig 9A, lane 4), therefore suggesting that GRASP55 could enhance the formation of a complex between MT1-MMP and furin Disruption of the GRASP55–furin complex reduces processing of pro-MT1-MMP GRASP55 appears to play an important role in the formation of the MT1-MMP–furin complex; therefore, 3168 disruption of the interaction between GRASP55 and furin should perturb the activation of pro-MT1-MMP and reduce MT1-MMP activity at the cell surface To test this hypothesis, we generated an enhanced green fluorescent protein (EGFP)-furin ICD construct in which the extracellular domain of the proprotein-convertase was replaced by EGFP Expression of the EGFP-furin ICD construct (1 lg) in HT1080 expressing MT1/MYC and GRASP55F led to a disruption of the complex between these two proteins (Fig 9B) We next tested whether expression of the EGFP-furin chimera could exert a dominant negative effect on proMT1-MMP processing by disrupting the complex between GRASP55 and MT1-MMP To test this, bbHT1080 cells were transfected for 24 h with EGFP-furin ICD Transfected cells were then sorted by fluorescence-activated cell sorting (FACS) into EGFPpositive and EGFP-negative populations (Fig S4) and the expression of pro- and active MT1-MMP was assessed by IB (Fig 9C) Using this approach, we found that high expression of the EGFP-furin ICD construct in bbHT1080 cells (Fig 9C, top panel, lane 2) led to an increased amount of pro-MT1-MMP being detected compared to EGFP-negative cells (Fig 9C, top panel, lane 1) This observation prompted us to test whether the reduction in pro-MT1-MMP activation observed following furin expression would result in a decrease of MT1-MMP activity at the cell surface HT1080 cells were transiently transfected with empty vector pCDNA3.1 Zeo+ (and treated without or with 50 ngỈlL)1 4b-phorbol 12-myristate 13-acetate), furin, GRASP55, furin + GRASP55, MT1-MMP, MT1MMP + furin, MT1-MMP + GRASP55, and MT1MMP + GRASP55 + furin After 24 h, the activation of pro-MMP2 was analyzed by zymography in the cell supernatants As shown in Figs 9D and S5, expression of MT1-MMP, but not furin, GRASP55 or GRASP55 + furin, significantly increased endogenous pro-MMP2 activation Co-expression of furin with MT1-MMP led to a significant reduction in the levels of MMP2 generated compared to cells expressing only the protease A significant decrease was also observed when furin was co-expressed with MT1-MMP and GRASP55 Taken together, our observations suggest that furin-mediated disruption of the MT1-MMP GRASP55 complex can lead to a reduction of the MT1-MMP, and a consequent decrease of protease activity at the cell surface Our data also revealed that intracellular furin levels are critical for the efficient activation of MT1-MMP The results obtained suggest that GRASP55 might act as a molecular bridge between MT1-MMP and furin and is involved in the furin-mediated activation of pro-MT1-MMP FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS C Roghi et al Discussion In the present study, we have investigated the interaction between GRASP55, a Golgi matrix protein, and MT1-MMP Using the M2H system and a series of MT1-MMP ICD triple mutants, we discovered that mutation of the LLY573 motif to AAA573 completely inhibited the interaction between MT1-MMP ICD and GRASP55, suggesting that this motif is important for the interaction of MT1-MMP ICD with the PDZ2 domain of GRASP55 The interaction of PDZ domains with ICD internal motifs has previously been reported For example, the interaction of the ErbB2 ICD with the Erbin PDZ domain was found to be dependent on a tyrosine at position )7 (the C-terminal residue is referred to as the P0 residue and subsequent residues towards the N-terminal are termed P)1, P)2, etc.) [46] Similarly, a reduced interaction was observed between GRASP55 and TGF-a D152 (lacking the last eight amino acids of the ICD) compared to the TGF-a D158 construct lacking only the last two valine residues, suggesting a role for an internal motif in the interaction between TGF-a ICD and the GRASP55 PDZ1 domain [37] Phosphorylation has been found to modulate interactions between PDZ domains and their ligands [47–49] The affinity of the Erbin–ErbB2 interaction has also been found to decrease by 2.5-fold when P)7 tyrosine is phosphorylated [50] In MT1MMP, tyrosine 573 (P)9) was recently reported to be phosphorylated [31] This opens the possibility that binding of MT1-MMP to GRASP55 could be regulated via tyrosine phosphorylation Surprisingly, MT1-MMP and furin ICDs were also found to interact with the C-terminal region (region 3) of GRASP55 Protein interactions with the region located outside the PDZ domain have previously been observed Sox-4 and eIF5A [51,52] were reported to interact only with the N-terminal region of syntenin-1, thus allowing for the potential binding of different proteins through the PDZ domain(s) and the N-terminal region [53] Our observation suggests that GRASP55 could potentially interact with two MT1MMP molecules, therefore allowing for the homophilic complex formation of the protease, as previously reported [28,54] Forcing the MT1-MMP ICD to dimerize by fusing it to the coil-coiled region of GM130 [39] was found to significantly enhance the interaction between MT1-MMP and GRASP55 (Fig S6) Furthermore, PDZ-containing proteins have previously been reported to self-associate, generating macromolecular complexes This multimerization can involve either of the PDZ domains, as for example in glutamate receptor interacting protein (GRIP1) [55] Role of GRASP55 in MT1-MMP activation or inactivation no afterpotential D (INAD) protein [56] However, it can also be a PDZ-independent mechanism, as for example in the case of the PSD-95 protein, where dimer formation is mediated by the Nterminal region of the protein [57] GRASP65, which is structurally related to GRASP55, has been found to form dimers that can organize into higher-order oligomers in interphase cells [58] GRASP65 dimerization involved the N-terminal GRASP domain (amino acids 1–201), which has been found to be highly conserved between both GRASPs [35] It is therefore possible that, similar to GRASP65, GRASP55 dimerizes or even oligomerizes into multimeric structures and could therefore be involved in the oligomerization of MT1MMP [28,54] MT1-MMP oligomerization, which has been found to facilitate pro-MMP2 activation [54], could therefore provide an explanation for the previously observed intracellular activation of pro-MMP2 [59] Mutation of the LLY573 motif to AAA573 in the MT1-MMP ICD has been reported to significantly decrease the internalization of the protease from the cell surface [18] Because the expression of this mutant at the cell surface is significantly lower than the wildtype MT1-MMP [18], it is reasonable to suggest that this motif also has a role in the exocytosis of protease We have shown that the LLY573 motif is important for the interaction between MT1-MMP ICD and GRASP55 and therefore its mutation to AAA573 could explain the reduction of the level of cell surface expression of this mutated version of MT1-MMP It is important to note that perturbation of the GRASP55 interaction with TGF-a [37], as well as p24 proteins [39], has been reported to affect the normal trafficking of these proteins The expression of the MT1-MMP AAA573 mutant at the cell surface [18] could result from the intracellular traffic of MT1-MMP via an alternative pathway, as previously described [60] Alternatively, GRASP65, which is structurally related to GRASP55, has previously been reported to interact with transmembrane TGF-a, p24a, protein CD8a or the frizzled receptor Fz4 [37–39] and therefore could be involved in the intracellular traffic of MT1-MMP AAA573 to the cell surface It would be interesting to assess the role of GRASP65 with respect to MT1MMP trafficking to the cell surface MT1-MMP, similar to all the other MMPs, is synthesized as a latent zymogen (pro-MT1-MMP) that is activated by endoproteolytic cleavage of its N-terminal inhibitory pro-domain peptide [6,61] Furin has been widely reported to be an activator of pro-MT1-MMP and is considered to be physiologically relevant [42] In the present study, we have reported that both FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS 3169 Role of GRASP55 in MT1-MMP activation C Roghi et al MT1-MMP and furin can interact with GRASP55 Overexpression of GRASP55 has been found to increase the amount of complex containing MT1MMP and furin, and the expression of a catalytically inactive dominant negative furin construct affected the processing of pro-MT1-MMP Taken together, these data reveal a new cellular function for GRASP55 as a molecular bridge between MT1-MMP and furin Disruption of the MT1-MMP interaction with GRASP55 only led to a small inhibition of pro-MT1MMP activation, suggesting that only a small proportion of the MT1-MMP zymogen is activated by a GRASP55-dependent furin-dependent mechanism, with the bulk of activation being GRASP55-independent The interaction observed between GRASP55 and PC5/ 6B or PC7 together with the potential role of these proprotein-convertases in MT1-MMP activation [42,44,62] would support our observation This would suggest that different pro-MT1-MMP activation pathways may co-exist within a cell Finally, MT2-, MT3- and MT5-MMP were also found to interact with GRASP55 Our data led us to hypothesize a role for GRASP55 in the furin-mediated activation of pro-MT2-MMP, pro-MT3-MMP and pro-MT5-MMP [63–65] because all these MT-MMPs harbour a tetrabasic motif sandwiched between the pro- and catalytic domains For MT2-MMP and MT3-MMP, the LLY660 and ILY598 motifs, respectively, were found to be important for the interaction of these MT-MMPs with GRASP55 By contrast, mutation of the VTY636 motif to AAA636 in MT5MMP did not affect the binding of the protease with GRASP55 Previous studies [66,67] have identified the EWV614 motif in MT5-MMP ICD as comprising an important sequence for the interaction with the PDZ proteins: Mint-3, AMPA binding protein and glutamate receptor interacting protein (GRIP) It would therefore be interesting to determine whether this motif is also involved in the interaction between MT5-MMP and GRASP55 GRASP55 could potentially be considered as a molecular bridge involved in connecting furin with various substrates So far, it is unknown whether this mechanism is specific for the type I transmembrane MT-MMPs or whether it comprises a more general mechanism for furin-mediated activation of transmembrane substrates The activation of ADAM17 by furin [68] and our observation of an interaction between GRASP55 and the ICD of ADAM17 (C Roghi and L Jones, unpublished results) would suggest a much wider activation mechanism A role of GRASP55 in bridging MT1-MMP with other transmembrane substrates could also be considered 3170 Materials and methods Antibodies FLAG mouse monoclonal antibody (IB: lgỈmL)1) was obtained from Sigma-Aldrich Company Ltd (Poole, UK) GRASP55 sheep polyclonal antibody (FBA34) [immunofluorescence (IF): lgỈmL)1] was obtained from F Barr (University of Liverpool, UK) [35] GRASP55 mouse monoclonal antibody (B01P, IB: 0.48 lgỈmL)1) was obtained from Abnova (Taipei, Taiwan) Anti-human MT1-MMP sheep heavy chain IgG (N175/6) (IF: lgỈmL)1, IB: 1.5 lgỈmL)1) was described previously [69] MT1-MMP rabbit polyclonal antibody and control rabbit IgGs were obtained from Insight Biotechnology Ltd (Middlesex, UK) MYC (4A6) (IB:1.5 lgỈmL)1) and MT1MMP (LEM-2/15.8) (IF: 10 lgỈmL)1) mouse monoclonal antibody were obtained from Millipore (UK) Ltd (Watford, UK) Polyclonal anti-TGN46 sheep (IF: lgỈmL)1) serum was obtained from Serotec Ltd (Oxford, UK) b-actin (IB: 0.1 ngỈmL)1) and anti-furin (IF: 8.6 lgỈmL)1, IB: 8.6 lgỈmL)1) rabbit Polyclonal antibody were from Abcam plc (Cambridge, UK) Giantin mouse monoclonal antibody was obtained from H.-P Hauri (University of Basel, Switzerland) [70] All secondary antibodies were obtained from Jackson ImmunoResearch Europe Ltd (Soham, UK) and used in accordance with the manufacturer’s instructions Cell culture conditions and transfections All cell culture reagents were obtained from Invitrogen Ltd (Paisley, UK), unless otherwise indicated HT1080 Human fibrosarcoma cells (from Cancer Research UK Research Services, London, UK) were maintained in DMEM containing 10% (v/v) fetal bovine serum (Hyclone Laboratories Inc., UT, USA), mm l-glutamine, 100 mL)1 penicillin and 100 lgỈmL)1 streptomycin at 37 °C in 5% CO2 HT1080 stably expressing wild-type MT1-MMP (bbHT1080 clone #2) were obtained from J Clements (British Biotech plc, Oxford, UK) [71] Transient transfections were performed using FuGENE (Roche Diagnostics Ltd, Lewes, UK) Total DNA transfected was kept constant DNA constructs Full-length wild-type human MT1-MMP cDNA was cloned in pCDNA3.1 Zeo+ vector (Invitrogen Ltd) VP16-MT1 was generated by cloning MT1-MMP intracellular domain (ICD; amino acids 562–582) in frame with the activation domain of herpes simplex virus type (VP16) in the pACT Checkmate Mammalian Two-Hybrid System vector (Promega, Southampton, UK) VP16-MT2, VP16-MT3 and FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS C Roghi et al VP16-MT5 were generated using MT2-MMP (amino acids 647–669), MT3-MMP (amino acids 585–607) and MT5MMP (amino acids 623–645) ICDs MT1-MMP (VP16MT1 Y, VP16-MT1 LL, VP16-MT1 FRR, VP16-MT1 HGT, VP16-MT1 PRR, VP16-MT1 LLY, VP16-MT1 CQR, VP16-MT1 SLL, VP16-MT1 DKV), MT2-MMP (VP16-MT2 LLY), MT3-MMP (VP16-MT3 ILY) and MT5-MMP (VP16-MT5 VTY) mutants were generated by PCR using mutagenic primers All these constructs have a Pro-Gly-Gly-Gly linker (PGGG) between VP16 and the ICD TGF-a ICD (amino acids 126–160) was generated by annealing overlapping oligonucleotides and ‘filling up’ the single strand regions with KOD DNA polymerase (2.5 units; Merck Biosciences Ltd, Nottingham, UK) The resulting double-stranded DNA was cloned in frame with VP16 MT1/MYC has a MYC tag between P312 and T313 in the hinge domain of MT1-MMP LLY570-573AAA MT1/MYC and G2A GRASP55F mutants were generated using the QuikChange II site-directed mutagenesis kit (Stratagene, Amsterdam, the Netherlands) Full-length rat GRASP55 cDNA and N-terminally EGFP-tagged rat GRASP55 (GRASP55-GFP) were obtained from F Barr and colleagues [35] GRASP55F in pCDNA3.1 Zeo+ contained a C-terminal FLAG tag Full-length GRASP55, GRASP55 PDZ1 (amino acids 1–107), GRASP55 PDZ2 (amino acids 84–172) and GRASP55 region (amino acids 173–454) were fused to GAL4 DNA binding domain in the pBIND Checkmate Mammalian Two-Hybrid System vector (Promega) To generate the MT1/EYFP, oligonucleotides coding for MT1-MMP signal sequence (amino acids 1–30) were annealed and inserted upstream of EYFP in pEYFP-N1 (Clontech-Takara Bio Europe, Saint-Germain-en-Laye, France) DNA coding for MT1-MMP hinge, hemopexin, stalk, transmembrane and cytoplasmic domains (amino acids 283–582) was amplified by PCR and cloned downstream of EYFP VP16-PC5/6B, VP16-PC7 and VP16-furin were generated by fusing VP16 to ICDs of mouse PC5/6B (amino acids 1461–1548), human PC7 (amino acids 684– 785) or furin (amino acids 738–794) To create EGFP-furin ICD, the EGFP (pEGFP-N1; Clontech-Takara Bio Europe) stop codon was replaced by PCR with the Pro-Gly-Gly linker The TIMP-3 signal peptide (amino acids 1–23, from D Edwards, UEA, Norwich, UK) was then inserted upstream of EGFP-PGG The furin stalk, transmembrane and intracellular domains (amino acids 708–794) were amplified by PCR and inserted downstream of EGFPPGG This construct lacks the intramolecular cleavage site for furin shedding [72] All constructs were confirmed by sequencing SDS/PAGE and IB All reagents were purchased from Bio-Rad (Hemel Hempstead, UK) SDS/PAGE (10%) and IB were carried out as Role of GRASP55 in MT1-MMP activation previously described [69] Membranes were then stripped using the ReblotÔ Plus kit [Millipore (UK) Ltd] M2H system HT1080 cells (105) were seeded per well of a six-well plates for 24 h prior to transfection (1 lg of pG5luc DNA, lg of DNA for the construct in pBIND and lg of DNA for the construct in pACT) After 16 h at 37 °C, cells were washed in ice-cold NaCl/Pi (137 mm NaCl, 4.3 mm Na2HPO4, 2.7 mm KCl, 1.47 mm KH2PO4) and lysed in passive lysis buffer (200 lL; Promega) for 15 at room temperature After centrifugation (13 000 g for at room temperature), Firefly and Renilla reniformis luciferase activities were measured in 20 lL of lysate using the Dual-Luciferase Reporter Assay System (Promega) and a SpectraFluor Plus plate reader (Tecan UK, Reading, UK) Firefly luciferase activity was normalized to Renilla luciferase activity and is presented as relative luminescence units Each transfection was performed in duplicate and two independent measurements were read per sample All numerical values are shown as the mean ± SEM The graphs presented are representative of at least three experiments Immunoprecipitation HT1080 cells (2 · 105 per well in a six-well plate) were transfected (1–2 lg per construct) for 16 h Cells were then washed twice in ice-cold NaCl/Pi and lysed for in 600 lL of lysis buffer per well [10 mm Tris–HCl, pH 7.4, 150 mm NaCl, 1% (v/v) Triton X-100, 0.5% (v/v) Nonidet P-40, mm EDTA, mm EGTA, mm sodium vanadate] containing protease inhibitor (Roche Diagnostics Ltd) For immunoprecipitation of endogenous GRASP55, cells were lysed in 1% Brij96, mm CaCl2, mm MgCl2, 10 mm Tris–HCl, pH 7.4, 150 mm NaCl containing protease inhibitor (Roche Diagnostics Ltd) After centrifugation (13 000 g for 15 at °C), the supernatant volume was adjusted to 800 lL and 150 lL (input) was kept for IB analysis Antibodies or control IgG (3 lg) were bound to Dynabeads protein G (Invitrogen) for 16 h at °C in NaCl/Pi containing mgỈmL)1 BSA Beads were washed with lysis buffer and mixed with the protein extracts for h at °C under constant rotation Beads were washed three times for 15 with lysis buffer and resuspended in · Laemmli sample buffer (30 lL) Denatured samples were then resolved by SDS/PAGE Indirect immunofluorescence microscopy Cells (1 · 105) seeded on 13 mm round glass coverslips (Agar Scientific, Stansted, UK) were transfected for 16 h Cells were then processed for immunofluorescence microscopy, as described previously [69] FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS 3171 Role of GRASP55 in MT1-MMP activation C Roghi et al Live cell time-lapse microscopy Statistical analysis Transfected HT1080 cells (500 ng of each construct) were imaged with a confocal Zeiss LSM510 META microscope (Zeiss Axiovert 200M platform, · 63/1.4 NA Oil Plan-Apochromat lens; Carl Zeiss Ltd, Welwyn Garden City, UK) The Argon Ion laser (Lasos Lasertechnik GmbH, Jena, Germany) output was restricted to 3% of maximum (< 0.3 mW at focal plane) to minimize phototoxicity and bleaching Coverslips (PeCon GmbH, Erbach, Germany) were placed in a POC-R imaging chamber (PeCon GmbH) containing phenol-red free Hepes-buffered medium and kept at 37 °C using a Heating Insert-P (PeCon GmbH) Time series images (optical section thickness of 1.7 lm) were collected in a lambda acquisition mode at one frame every s for 10–20 Reference EGFP and EYFP emission spectra were captured from singly transfected cells EGFP/EYFP signal unmixing was carried out using Zeiss lsm510 software, version 3.2SP2 Confocal images were post-processed with volocity software (Improvision Systems, Coventry, UK) Movies are presented at ten frames per second, representing a speed increase of ·40 Statistical analysis was performed using the graphpad prism, version (GraphPad Software, Inc., San Diego, CA, USA) Statistical significance was calculated using Student’s t-test Statistical significance was defined as P < 0.05 (*), P < 0.001 (**) and P < 0.0001 (***) FACS bbHT1080, transfected for 24 h with the EGFP-furin ICD construct, were washed with NaCl/Pi and detached from the plastic using NaCl/Pi containing mm EDTA (5 at 37 °C) After centrifugation (500 g for min), cells were washed in NaCl/Pi, resuspended DMEM containing 0.5% (v/v) fetal bovine serum and kept on ice EGFP-negative cells were sorted from the EGFP-positive cells using an Aria SORP flow cytometer (Becton Dickenson, San Jose, CA, USA) Sorting efficiency was controlled by a post-sort analysis Cells were lysed with lysis buffer Protein concentration was determined using a BCA protein assay kit (Perbio Science UK Ltd, Cramlington, UK) The western blot presented is a representative of three independent experiments Pro-MMP2 activation and zymography HT1080 cells (1 · 105 cells) in DMEM and 10% fetal bovine serum were transfected in suspension using 0.5 lg of each DNA construct and 1.5 lL of FuGENEÔ transfection reagent (Roche Diagnostics Ltd) in accordance with the manufacturer’s instructions in 24-well culture dishes The total amount of DNA per transfection was kept constant After 24 h at 37 °C, the medium was removed and replaced with 300 lL per well of serum-free DMEM containing insulin, transferrin and selenium supplements (Sigma-Aldrich) and the cells were incubated at 37 °C for a further 24 h Cell supernatants were harvested and analyzed by gelatin zymography, as described previously [73] Quantification of MMP2 was performed using imagequant tl software, version 7.0 (GE Heathcare, Little Chalfont, UK) 3172 Acknowledgements We would like to thank Drs F Barr, J Creemers, K Shennan, D Edwards and H.-P Hauri for providing reagents used in the present study We thank Greg Veltri, Therese Martin and Michele Bones (CRI Flow Cytometry core unit) and Jane Gray (CRI equipment park) for their technical assistance We thank Neil Taylor, Sue Atkinson, Patricia Eisenach and Helen Gillingham for their helpful discussions and for critically reading the manuscript We would like to acknowledge the support of Cancer Research UK and 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EGFPfurin by FACS Fig S5 Furin expression impairs activation of pro-MMP2 Fig S6 Fusion of GM130 coiled-coil region to MT1-MMP ICD increased its binding to GRASP55 This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 277 (2010) 3158–3175 ª 2010 The Authors Journal compilation ª 2010 FEBS 3175 ... et al MT1-MMP and furin can interact with GRASP55 Overexpression of GRASP55 has been found to increase the amount of complex containing MT1MMP and furin, and the expression of a catalytically inactive... with a full-length furin cDNA, were permeabilized and stained with polyclonal antibodies against furin and GRASP55 Arrows show examples of membrane compartment containing GRASP55 and furin Scale... general mechanism for furin- mediated activation of transmembrane substrates The activation of ADAM17 by furin [68] and our observation of an interaction between GRASP55 and the ICD of ADAM17