Binding of Active (57 kDa) Membrane Type 1-Matrix Metalloproteinase (MT1-MMP) to Tissue Inhibitor of Metalloproteinase (TIMP)-2 Regulates MT1-MMP Processing and Pro-MMP-2 Activation

doi:10.1074/jbc.275.16.12080

Binding of Active (57 kDa) Membrane Type 1-Matrix Metalloproteinase (MT1-MMP) to Tissue Inhibitor of Metalloproteinase (TIMP)-2 Regulates MT1-MMP Processing and Pro-MMP-2 Activation*

From the Department of Pathology and Karmanos Cancer Institute, Wayne State University, Detroit, Michigan 48201 and the^§Department of Chemistry, Biochemistry Division, Florida State University, Tallahassee, Florida 32306-4390

Abstract

Previous studies have shown that membrane type 1-matrix metalloproteinase (MT1-MMP) (MMP-14) initiates pro-MMP-2 activation in a process that is tightly regulated by the level of tissue inhibitor of metalloproteinase (TIMP)-2. However, given the difficulty in modulating TIMP-2 levels, the direct effect of TIMP-2 on MT1-MMP processing and on pro-MMP-2 activation in a cellular system could not be established. Here, recombinant vaccinia viruses encoding full-length MT1-MMP or TIMP-2 were used to express MT1-MMP alone or in combination with various levels of TIMP-2 in mammalian cells. We show that TIMP-2 regulates the amount of active MT1-MMP (57 kDa) on the cell surface whereas in the absence of TIMP-2 MT1-MMP undergoes autocatalysis to a 44-kDa form, which displays a N terminus starting at Gly²⁸⁵ and hence lacks the entire catalytic domain. Neither pro-MT1-MMP (N terminus Ser²⁴) nor the 44-kDa form bound TIMP-2. In contrast, active MT1-MMP (N terminus Tyr¹¹²) formed a complex with TIMP-2 suggesting that regulation of MT1-MMP processing is mediated by a complex of TIMP-2 with the active enzyme. Consistently, TIMP-2 enhanced the activation of pro-MMP-2 by MT1-MMP. Thus, under controlled conditions, TIMP-2 may act as a positive regulator of MT1-MMP activity by promoting the availability of active MT1-MMP on the cell surface and consequently, may support pericellular proteolysis.

Footnotes

↵* This work was supported by National Institutes of Health Grant CA-61986 and Department of Defense Grant DAMD17-99-1-9440 (to R. F.), National Insititues of Health Grant CA-78646 (to Q-X. S.), and Department of Defense Predoctoral Fellowship DAMD17-99-1-9441 (to S. H-B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ Contributed equally to the results of this work.
↵¶ To whom all correspondence should be addressed: Dept. of Pathology, Wayne State University, 540 E. Canfield Ave., Detroit, MI 48201. Tel.: 313-577-1218; Fax: 313-577-8180; E-mail: rfridman@med.wayne.edu.
↵2 M. Toth, D. Gervasi, Y. A. De Clerck, and R. Fridman, unpublished results.
Abbreviations:

ECM

extracellular matrix

MMP

matrix metalloproteinase

TIMP

tissue inhibitor of metalloproteinase

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

mAb

monoclonal antibody

pAb

polyclonal antibody

pfu

plaque-forming units

FBS

fetal bovine serum

ECL

enhanced chemiluminescence

PM

plasma membrane

MT

membrane-type
- Received July 13, 1999.
- Revision received January 21, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.