Further analysis revealed that GRASP55 associated with the cytoplasmic domain of both proteases and that the LLY573motif in the MT1-MMP intracellular domain was crucial for the interacti
Trang 1membrane-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 English1,2and 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: 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 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 LLY573motif 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 activareduc-tion 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.
Trang 2Extracellular 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
patho-logical 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
pro-teins 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 1 matrix metalloproteinase
(MT1-MMP; MMP14; EC 3.4.24.80) is by far the most
extensively studied member of the MT-MMP
sub-family MT1-MMP is a type 1 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 func-tions [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 intracellu-lar domain (ICD) of MT1-MMP (21 amino acids) plays an important role in multiple MT1-MMP-medi-ated cellular events [15] MT1-MMP ICD has been involved in cell migration [16] and invasion into recon-stituted 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 path-ways [16,25–27] and participates in the homophilic interaction between MT1-MMP monomers [28] Recently, the LL572 di-leucine motif has been reported
l MINT-7897617 , MINT-7897659 , MINT-7897681 , MINT-7897702 , MINT-7897725 ,
MINT-7898032 , MINT-7898011 , MINT-7897907 , MINT-7897884 : GRASP55 (uniprotkb: Q9R064 ) physically interacts ( MI:0915 ) with MT1-MMP (uniprotkb: P50281 ) by two hybrid ( MI:0018 )
l MINT-7898002 : MT1-MMP (uniprotkb: P50281 ) physically interacts ( MI:0914 ) with Furin (uniprotkb: P09958 ) by anti bait coimmunoprecipitation ( MI:0006 )
l MINT-7897500 : MT1-MMP (uniprotkb: P50281 ) and Giantin (uniprotkb: Q14789 ) colocalize ( MI:0403 ) by fluorescence microscopy ( MI:0416 )
l MINT-7897750 , MINT-7897394 : GRASP55 (uniprotkb: Q9R064 ) physically interacts ( MI:0915 ) with MT1-MMP (uniprotkb: P50281 ) by anti tag coimmunoprecipitation ( MI:0007 )
l MINT-7897562 : MT1-MMP (uniprotkb: P50281 ) and GRASP55 (uniprotkb: Q9H8Y8 ) coloca-lize ( MI:0403 ) by fluorescence microscopy ( MI:0416 )
l MINT-7897512 : TGN46 (uniprotkb: O43493 ) and MT1-MMP (uniprotkb: P50281 ) colocalize ( MI:0403 ) by fluorescence microscopy ( MI:0416 )
l MINT-7897921 , MINT-7897975 : GRASP55 (uniprotkb: Q9R064 ) physically interacts ( MI:0915 ) with Furin (uniprotkb: P09958 ) by two hybrid ( MI:0018 )
l MINT-7898052 , MINT-7897410 : MT1-MMP (uniprotkb: P50281 ) physically interacts ( MI:0915 ) with GRASP55 (uniprotkb: Q9R064 ) by anti bait coimmunoprecipitation ( MI:0006 )
l MINT-7897951 : GRASP55 (uniprotkb: Q9R064 ) physically interacts ( MI:0915 ) with PC7 (uniprotkb: Q16549 ) by two hybrid ( MI:0018 )
l MINT-7897866 : GRASP55 (uniprotkb: Q9R064 ) physically interacts ( MI:0915 ) with MT5-MMP (uniprotkb: Q9Y5R2 ) by two hybrid ( MI:0018 )
l MINT-7897633 : GRASP55 (uniprotkb: Q9R064 ) physically interacts ( MI:0915 ) with TGFA (uniprotkb: P01135 ) by two hybrid ( MI:0018 )
l MINT-7897551 : GRASP55 (uniprotkb: Q9H8Y8 ) and Giantin (uniprotkb: Q14789 ) colocalize ( MI:0403 ) by fluorescence microscopy ( MI:0416 )
l MINT-7897938 : GRASP55 (uniprotkb: Q9R064 ) physically interacts ( MI:0915 ) with PC5/6B (uniprotkb: Q04592 ) by two hybrid ( MI:0018 )
Trang 3to influence the O-glycosylation pattern of MT1-MMP
[29] Post-translational modifications of the
MT1-MMP ICD have also been reported with the
palmitoy-lation 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
multifunc-tional protein p32/gC1qR [21], a protein with
homol-ogy to members of the Cupin superfamily (MTCBP-1)
[33], as well as with the l2 subunit of the
clathrin-coated pits adapter protein 2 (AP-2) [18] and
phospho-caveolin-1 in src overexpressing cells [34]
Golgi reassembly stacking protein 55 (GRASP55) is
a peripheral Golgi matrix protein that has been
impli-cated, in vitro, in the post-mitotic stacking of Golgi
cis-ternae [35] Cryo-electron microscopy has shown that
GRASP55 is found predominantly in the
medial-cister-nae of the Golgi complex of HeLa cells [35]
GRASP55 interacts with Golgin-45 and the complex is
crucial for maintaining Golgi structure [36] In
addi-tion to its contribuaddi-tion to the Golgi exoskeleton,
GRASP55 has also been reported to be involved in the
intracellular transport of pro-transforming growth
fac-tor (TGF)-a [37], CD8a or the frizzled recepfac-tor Fz4
[38], as well as in the Golgi retention of p24a, a
mem-ber of the p24 family of cargo receptors [39]
In the present study, we report in detail on the
inter-action between the MT1-MMP ICD and GRASP55
using a mammalian two-hybrid (M2H) system Using
this approach, we have identified the GRASP55
bind-ing 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
MT1-MMP 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
interac-tion 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
A
60 50 40
IB: MT1-MMP
MT1-MMP pCDNA3.1 Zeo+
kDa
+ – – +
+ – – +
60 50 40
IB: GRASP55
50 IB: β-actin
1 2
IgG anti MT1-MMP
B
60 50 40
IB: GRASP55
1
–
– +
+
2 kDa
GRASP55F MT1/MYC
IP: FLAG IB: MYC
kDa 60
C
IB: MYC
IB: FLAG
Input lysates 60
60
1 2 3 4
Fig 1 MT1-MMP co-immunoprecipitates with GRASP55F (A) Pro-tein 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 anti-body directed against MT1-MMP (lane 2) and analyzed by IB using
a monoclonal antibody to GRASP55 The arrow identifies immuno-precipitated 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 (immu-noglobulin heavy chain).
Trang 4A B C
Fig 2 Subcellular localization of GRASP55 and MT1-MMP in bbHT1080 Fixed and permeabilized bbHT1080 cells were incubated with anti-bodies 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 = 5 lm.
Trang 5immunoprecipitated with nonspecific rabbit IgGs or
with the rabbit polyclonal antibody directed against
MT1-MMP As shown in Fig 1B,
immunoprecipita-tion of MT1-MMP led to the co-precipitaimmunoprecipita-tion of a
small amount of endogenous GRASP55, as detected
by immunoblotting (IB) No GRASP55 was detected
when the pre-immune IgGs were used
Co-precipita-tion between MT1-MMP and GRASP55 was also
observed in HT1080 cells expressing exogenous
Myc-tagged MT1-MMP (MT1/MYC) and FLAG-Myc-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
extensively with the medial Golgi marker giantin (Fig 2B) and with the trans-Golgi network (TGN) membrane protein marker TGN46 (Fig 2E) Endoge-nous 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 con-struct (MT1/EYFP), where the EYFP tag replaced the entire MT1-MMP catalytic domain In HT1080, the intracellular trafficking of MT1/EYFP was indistin-guishable from that of wild-type MT1-MMP and both constructs were found to accumulate in the Golgi appa-ratus 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
Fig 3 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 fluore-scent 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.
Trang 6VP16-MT1 VP16
PGGGFFAAAHGTPRRLLYCQRSLLDKV
VP16
VP16-MT1 FRR
A
PGGGFFFRRAAAPRRLLYCQRSLLDKV
VP16
VP16-MT1 HGT
PGGGFFFRRHGTAAALLYCQRSLLDKV
VP16
VP16-MT1 PRR
PGGGFFFRRHGTPRRAAACQRSLLDKV
VP16
VP16-MT1 LLY
PGGGFFFRRHGTPRRLLYAAASLLDKV
VP16
VP16-MT1 CQR
PGGGFFFRRHGTPRRLLYCQRAAADKV
VP16
VP16-MT1 SLL
PGGGFFFRRHGTPRRLLYCQRSLLAAA
VP16
VP16-MT1 DKV
B
KHCEWCRALICRHEKPSALLKGRTACCHSETVV
VP16-TGF- α VP16
PGGGFFFRRHGTPRRLLYCQRSLLAAA
VP16
VP16 MT1 DKV VP16-MT1 Y VP16PGGGFFFRRHGTPRRLLACQRSLLDKV
VP16-MT1 LL VP16 PGGGFFFRRHGTPRRAAYCQRSLLDKV
500
0 100 200 300 400
500
0 100 200 300 400
Luminescence (arbitrary units)
1 2 3 4
GAL4 GRASP55 VP16
MT1
VP16
GAL4 GAL4 GRASP55 VP16 MT1
GAL4 + VP16
+ + +
C
VP16
+
GAL4 GRASP55
+ VP16 TGF- α GAL4
+
GAL4 VP16 1
2 3 Luminescence (arbitrary units)
GAL4 GRASP55 + VP16 TGF- α 4
Fig 4 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
(GAL4-GRASP55) and MT1-MMP ICD (VP16-MT1)
or (C) TGF-a ICD (VP16-TGF-a) using the
M2H assay.
+ VP16 MT1
GAL4
500
0 100 200 300 400 1
Luminescence (arbitrary units)
A
B
GAL4 GRASP55 + VP16 MT1 FRR
GAL4 GRASP55 + VP16 MT1 HGT
GAL4 GRASP55 + VP16 MT1 PRR
GAL4 GRASP55 + VP16 MT1
GAL4 GRASP55 + VP16 MT1 LLY
2 3 4 5 6
+
GAL4 GRASP55 + VP16 MT1 CQR
GAL4 GRASP55 + VP16 MT1 SLL
+
GAL4 GRASP55 VP16 MT1 DKV
7 8 9
MT1/MYC +
+
+ + + +
MT1 LLY/MYC
IP: FLAG IB: MYC 50
– –
WB: MYC
WB: FLAG
Input lysates
60
60
Fig 5 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
immunoprecipi-tated with the FLAG M2 antibody.
MT1-MMP present in the
immunoprecipi-tate was detected by IB using the MYC tag
antibody Levels of transfected proteins
were monitored in input lysates using
specific antibodies.
Trang 7Materials 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-ter-minal 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
tran-siently co-transfected in HT1080 cells together with the
reporter plasmid pG5luc, which contains the firefly
lucif-erase 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
meth-ods, and the values obtained were normalized according
to transfection efficiency using the Renilla reniformis
luciferase expressed by the pBIND vector
Co-expres-sion of VP16-MT1 and GAL4-GRASP55 fusion
proteins (Fig 4B, lane 4) in HT1080 resulted in the
production of significantly higher firefly luciferase lumi-nescence 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 gener-ated (Fig 4A) and used in the M2H system System-atic 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 reduc-tion 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
MT3 MT3 ILY MT5
VP16-MT1
VP16-MT2
PGGGFFFRRHGTPRRLLYCQRSLLDKV
VP16
PGGGVQMQRKGAPRVLLYCKRSLQEWV
VP16
% homology with MT1-MMP ICD -57.1%
VP16-MT3
VP16-MT5
PGGGFQFKRKGTPRHILYCKRSMQEWV
VP16
PGGGFQFKNKTGPQPVTYYKRPVQEWV
VP16
57.1%
23.8%
500
A
B Luminescence (arbitrary units)
0 100 200 300 400
+
GAL4 VP16
+
GAL4 GRASP55 VP16
+
GAL4 GRASP55 VP16
4 5 6
+
GAL4 VP16
GAL4 GRASP55 + VP16
+
GAL4 GRASP55 VP16
1 2 3
GAL4 GRASP55 VP16
+
GAL4 VP16
+
GAL4 GRASP55 MT5 VP16
+
GAL4 GRASP55 VP16 MT5 VTY
7 8 9
MT2
MT3
MT2 MT2 LLY
Fig 6 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.
Trang 8the 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
produc-tion or stability Our data therefore suggest that most
of the MT1-MMP ICD is implicated in the interaction
with GRASP55, with the LLY573motif playing a
criti-cal 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),
dem-onstrating that the mutation of the whole LLY573
motif is needed to abolish the interaction of
MT1-MMP ICD with GRASP55
The importance of the LLY573 motif in the
MT1-MMP ICD observed in the M2H assay was next
con-firmed 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
MT1-MMP ICD led to a marked reduction in the amount
of MT1-MMP present in the immunoprecipitated
material (Fig 5B, lane 4), thus confirming the
impor-tant 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-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 dif-ferent 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], suggest-ing that the Golgi localization of GRASP55 is crucial for its interaction with MT1-MMP
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, 5 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, MT2 LLY, VP16-MT3 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
Luminescence (arbitrary units)
500
0 100 200 300 400
+ VP16 MT1 GAL4
VP16
+
GAL4 GRASP55 GAL4 GRASP55 + VP16 MT1 P1
GAL4 + VP16 MT1
+ VP16 MT1 GAL4 P2
+ VP16 MT1 GAL4
1 2 3 4 5 6
+ VP16 MT1
+ VP16 TGF-α GAL4
+
GAL4 GRASP55 VP16
Luminescence (arbitrary units)
500
0 100 200 300 400 1
2
+
GAL4 GRASP55 VP16
+
P1 GAL4 VP16 TGF-α
2 3 TGF-α
A
B
R3
Fig 7 GRASP55 PDZ2 and region 3 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 3 (GAL4-R3) and
(B) between VP16-TFG a and
GAL4-GRASP55 or GAL4-P1 were tested using
the M2H system.
Trang 9contrast, mutation of MT3-MMP ILY598 (Fig 6B,
lane 6) and MT5-MMP VTY636to AAA (Fig 6B, lane
9) had no effect on GRASP55 binding
MT1-MMP ICD binds to PDZ2 domain and
region 3 of GRASP55
GRASP55 contains two non-overlapping and
structur-ally 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
inter-acts with MT1-MMP ICD GRASP55 PDZ1 (amino
acids 1–107), GRASP55 PDZ2 (amino acids 84–172)
and GRASP55 region 3 (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 3
(R3; Fig 7A, lane 6) However, no interaction was
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 co-immunoprecipitate 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 impli-cated 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
500
0 100 200 300 400
+ VP16 Furin
GAL4
Luminescence (arbitrary units)
2 3 4 5 6 GAL4 + VP16 PC7
+
GAL4 VP16 PC5/6B
GAL4 GRASP55 + VP16 Furin
GAL4 GRASP55 + VP16 PC5/6B
PC7
VP16
GAL4 GRASP55 +
Luminescence (arbitrary units)
+
GAL4 VP16 Furin
+
GAL4 GRASP55 VP16
GAL4 GRASP55 + VP16 Furin
P1
GAL4 + VP16 Furin
500
0 100 200 300 400 1
2 3 4
+
GAL4 P2 VP16 Furin
+
R3
GAL4 VP16 Furin
5 6
A
B
C
Furin GRASP55
Fig 8 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 (GAL4-P1), GRASP55 PDZ2 (GAL4-P2) or GRASP55 Region 3 (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.
Trang 10where 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
trans-membrane pro-convertases were also fused to VP16
Comparable to furin, PC5/6B and PC7 have
previ-ously 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 con-structs described previously As shown in Fig 8B, we detected an interaction between furin ICD and PDZ2 (Fig 8B, lane 5) and region 3 (Fig 8B, lane 6) of GRASP55 No interaction was detected between GRASP55 PDZ1 (Fig 8B, lane 4) and furin ICD,
A
GRASP55F Furin
IP: MT1-MMP IB: Furin
kDa 98 64
*
C
IB: MT1-MMP
EGFP-furin ICD 64
50
kDa
pro-MT1-MMP active-MT1-MMP
IB: FLAG IB: Furin
Input lysate
IB: MT1-MMP 50
64
64
98
50
36
GRASP55F MT1/MYC
EGFP-furin ICD (μg)
B
50
D
IP: FLAG IB: MYC EGFP furin ICD (μg)
60
60 50
kDa
5 10 15 20
n.s
***
IB: FLAG IB: GFP
Input lysate
60 50
60
60
0
5 )
IB: MYC
50
–
– –
Fig 9 GRASP55 is important for MT1-MMP–furin complex formation and activation of pro-MT1-MMP (A) Lysates of bbHT1080 cells trans-fected 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 immunopre-cipitated 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 immunoprecipi-tated 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, fu-rin + GRASP55, MT1-MMP, MT1-MMP + fufu-rin, MT1-MMP + GRASP55 and MT1-MMP + GRASP55 + fufu-rin 4b-Phorbol 12-myristate 13-ace-tate was used at 50 ngÆlL)1 After 24 h, supernatants were collected and analyzed by zymography Data represent the mean ± SEM of two independent experiments.