Furin Directly Cleaves proMMP-2 in the trans-Golgi Network Resulting in a Nonfunctioning Proteinase

Cao, Jian; Rehemtulla, Alnawaz; Pavlaki, Maria; Kozarekar, Pallavi; Chiarelli, Christian

doi:10.1074/jbc.m412370200

Cited by 50 publications

(53 citation statements)

References 49 publications

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“…Consistent with previous data (14,15), we showed that MMP-2 processed by thrombin and plasmin was also enzymatically active (data not shown). Cleavage of pro-MMP-2 at Arg 101 -Cys by furin and at Arg 98 -Lys by trypsin-2 has also been reported (40,45). In contrast to our data, furin-cleaved MMP-2 did not exhibit enzymatic activity in COS-1 cells, and trypsin-2-cleavage of the propeptide resulted in only partial activation of pro-MMP-2 (40, 45).…”

Section: Discussioncontrasting

confidence: 61%

Regulatory Mechanism of Matrix Metalloprotease-2 Enzymatic Activity by Factor Xa and Thrombin

Koo

Park

Jeon³

et al. 2009

Journal of Biological Chemistry

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Matrix metalloprotease (MMP)-2 plays a key role in many biological and pathological processes related to cell migration, invasion, and mitogenesis. MMP-2 is synthesized as a zymogen that is activated through either a conformational change or proteolysis of the propeptide. Several activating enzymes for pro-MMP-2 have been proposed, including metalloproteases and serine proteases. The mechanism of pro-MMP-2 activation by metalloproteases is well established, and the most studied activation mechanism involves cleavage of the propeptide by membrane type 1-MMP (MT1-MMP). In contrast, serine protease activation has not been thoroughly studied, although studies suggest that MT1-MMP may be involved in activation by thrombin and plasmin. Here, we demonstrate that factor Xa mediates MT1-MMP-independent processing of pro-MMP-2 in vascular smooth muscle cells and endothelial cells. Factor Xa and thrombin directly cleaved the propeptide on the carboxyl terminal sides of the Arg 98 and Arg 101 residues, whereas plasmin only cleaved the propeptide downstream of Arg 101 . Moreover, processed MMP-2 showed enzymatic activity that was enhanced by intermolecular autoproteolytic processing at the Asn 109 -Tyr peptide bond. In addition to its role in activation, factor Xa rapidly degraded MMP-2, thereby restricting excessive MMP-2 activity. Thrombin also degraded MMP-2, but the degradation was reduced greatly under cell-associated conditions, resulting in an increase in processed MMP-2. Overall, factor Xa and thrombin regulate MMP-2 enzymatic activity through its activation and degradation. Thus, the net enzymatic activity results from a balance between MMP-2 activation and degradation. Matrix metalloprotease (MMP)3 -2 is a member of the zincdependent endopeptidase family, which comprises 24 enzymes (1). MMP-2 plays a key role in many biological and pathological processes, including organ growth, endometrial cycling, wound healing, bone remodeling, tumor invasion, and metastasis (2). This enzyme functions through proteolysis of non-structural extracellular molecules and components of the basement membrane, including type IV collagen, fibronectin, elastin, laminin, aggrecan, and fibrillin (3).Like most MMPs, MMP-2 is synthesized as a zymogen that is activated by conformational change (4) or proteolysis within the propeptide, which may involve membrane type MMPs (MT-MMPs) (5-9). The most studied activation mechanism for pro-MMP-2 is cleavage of the propeptide by MT1-MMP, which requires cooperative activity between MT1-MMP and tissue inhibitor of metalloprotease (TIMP)-2 (5, 10 -12). Serine proteases, such as thrombin, factor Xa, activated protein C, and plasmin as well as the cysteine protease legumain are all known activators of pro-MMP-2 (13-17). (25,26). Both proteases can also elicit endothelial cell and SMC migration through pro-MMP-2 activation and subsequent extracellular matrix degradation (13,27,28). However, despite studies suggesting that MT1-MMP is involved in thrombin-mediated activation of pro-MMP-2, a detailed mechanism...

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Section: Discussioncontrasting

confidence: 61%

Regulatory Mechanism of Matrix Metalloprotease-2 Enzymatic Activity by Factor Xa and Thrombin

Koo

Park

Jeon³

et al. 2009

Journal of Biological Chemistry

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“…Inactivation of the furin sites by the ARAA and the R89A mutations were convincingly established by earlier works (Yana and Weiss, 2000;Cao et al, 2005). Glioma cells expressing these constructs were lysed and the lysates were analysed by Western blotting with an MT1-MMP antibody.…”

Section: Resultsmentioning

confidence: 77%

Furin regulates the intracellular activation and the uptake rate of cell surface-associated MT1-MMP

et al. 2006

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Invasion-promoting membrane type-1 matrix metalloproteinase (MT1-MMP) functions in cancer cells as an oncogene and as a mediator of proteolytic events on the cell surface. To exert its functional activity, MT1-MMP requires proteolytic removal of the prodomain sequence. There are two potential furin cleavage motifs, R 89 -R-P-R-C 93 and R 108 -R-K-R-Y 112 , in the prodomain sequence of MT1-MMP. Our data suggest an important role of furin and related proprotein convertases (PCs) in mediating both the activation of MT1-MMP and the levels of functionally active MT1-MMP at the surface of cancer cells. We have determined that the peptide sequence that spans the first cleavage site is susceptible to furin and PC5/6, whereas the second sequence is susceptible to furin and also to PC5/6, PC7 and PACE4. In the structure of the MT1-MMP proenzyme, the R 89 -R-P-R-C 93 site, however, is inaccessible to PCs. Our studies also demonstrated a direct functional link between the activation and the uptake rate of the proenzyme and the enzyme of MT1-MMP. Thus, the uptake rate of the latent MT1-MMP proenzyme noticeably exceeded that of the active enzyme. We conclude that furin and related PCs are the essential components of the specialized cellular machinery that controls the levels of the functionally active, mature, MT1-MMP enzyme on the cell surface to continually support the potency of pericellular proteolysis.

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“…We are aware of two other examples in the literature in which furin processing was associated with loss of enzymatic activity (40,41), but in both these instances cleavage occurred at sites unrelated to the propeptide. The mechanism by which furin processing reduces ADAMTS9 catalytic activity is presently unclear.…”

Section: Discussionmentioning

confidence: 98%

Regulation of ADAMTS9 Secretion and Enzymatic Activity by Its Propeptide

Koo

Longpré

Somerville

et al. 2007

Journal of Biological Chemistry

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ADAMTS9 is a secreted, cell-surface-binding metalloprotease that cleaves the proteoglycans versican and aggrecan. Unlike most precursor proteins, the ADAMTS9 zymogen (pro-ADAMTS9) is resistant to intracellular processing. Instead, pro-ADAMTS9 is processed by furin at the cell surface. Here, we investigated the role of the ADAMTS9 propeptide in regulating its secretion and proteolytic activity. Removal of the propeptide abrogated secretion of the ADAMTS9 catalytic domain, and secretion was inefficiently restored by expression of the propeptide in trans. Substitution of Ala for Asn residues within each of three consensus N-linked glycosylation sites in the propeptide abrogated ADAMTS9 secretion. Thus, the propeptide is an intramolecular chaperone whose glycosylation is critical for secretion of the mature enzyme. In addition to two previously identified furin-processing sites (Arg 74 2 and Arg 287 2) the ADAMTS9 propeptide was also furin-processed at Arg 209 . Substitution of Ala for Arg 74 , Arg 209 , and Arg 287 resulted in secretion of an unprocessed zymogen. Unexpectedly, versican incubated with cells expressing this pro-ADAMTS9 was processed to a greater extent than when incubated with cells expressing wild-type, furin-processable ADAMTS9. Moreover, cells and medium treated with the proprotein convertase inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone had greater versican-cleaving activity than untreated cells. Following furin processing of pro-ADAMTS9, propeptide fragments maintained a non-covalent association with the catalytic domain. Collectively, these observations suggest that, unlike other metalloproteases, furin processing of the ADAMTS9 propeptide reduces its catalytic activity. Thus, the propeptide is a key functional domain of ADAMTS9, mediating an unusual regulatory mechanism that may have evolved to ensure maximal activity of this protease at the cell surface. ADAMTS4 proteases have critical roles in many biological processes and in inherited and acquired human disorders (1-5). The 19 enzymes of this family share a conserved organization comprising an N-terminal metalloprotease domain and a C-terminal ancillary domain, which contains the thrombospondin type-1 repeats that are the hallmark of the family (6). ADAMTS9 is the largest enzyme of the family, containing 15 thrombospondin type-1 repeats, and its mRNA is widely expressed during embryonic development and in adult tissues (7-9). In previously published work, we showed that when expressed in COS-1 or HEK293F cells, ADAMTS9 is located at the cell surface or within the pericellular matrix (8), suggesting that, despite the lack of a membrane anchor, it could be considered as an operational cell-surface protease. ADAMTS9 can cleave the large aggregating proteoglycans aggrecan and versican (8), suggesting a role in turnover of extracellular matrix. Thus, like its Caenorhabditis elegans ortholog, Gon-1, which is required for cell migration during gonadal morphogenesis (10), it is possible that ADAMTS9 participates in extracellular proteolysi...

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Furin Directly Cleaves proMMP-2 in the trans-Golgi Network Resulting in a Nonfunctioning Proteinase

Cited by 50 publications

References 49 publications

Regulatory Mechanism of Matrix Metalloprotease-2 Enzymatic Activity by Factor Xa and Thrombin

Regulatory Mechanism of Matrix Metalloprotease-2 Enzymatic Activity by Factor Xa and Thrombin

Furin regulates the intracellular activation and the uptake rate of cell surface-associated MT1-MMP

Regulation of ADAMTS9 Secretion and Enzymatic Activity by Its Propeptide

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