Sequence motifs of tissue inhibitor of metalloproteinases 2 (TIMP-2) determining progelatinase A (proMMP-2) binding and activation by membrane-type metalloproteinase 1 (MT1-MMP).
Fundamental cellular processes including angiogenesis and cell migration require a proteolytic cascade driven by interactions of membrane-type matrix metalloproteinase 1 (MT1-MMP) and progelatinase A (proMMP-2) that are dependent on the presence of tissue inhibitor of metalloproteinases 2 (TIMP-2). There are unique interactions between TIMP-2 and MT1-MMP, which we have previously defined, and here we identify TIMP-2 sequence motifs specific for proMMP-2 binding in the context of its activation by MT1-MMP. A TIMP-2 mutant encoding the C-terminal domain of TIMP-4 showed loss of proMMP-2 activation, indicating that the C-terminal domain of TIMP-2 is important in establishing the trimolecular complex between MT1-MMP, TIMP-2 and proMMP-2. This was confirmed by analysis of a TIMP-4 mutant encoding the C-terminal domain of TIMP-2, which formed a trimolecular complex and promoted proMMP-2 processing to the intermediate form. Mutants encoding TIMP-4 from Cys(1) to Leu(185) and partial tail sequence of TIMP-2 showed some gain of activating capability relative to TIMP-4. The identified residues were subsequently mutated in TIMP-2 (E(192)-D(193) to I(192)-Q(193)) and this inhibitor showed a significantly reduced ability to facilitate proMMP-2 processing by MT1-MMP. Furthermore, the tail-deletion mutant Delta(186-194)TIMP-2 was completely incapable of promoting proMMP-2 activation by MT1-MMP. Thus the C-terminal tail residues of TIMP-2 are important determinants for stable trimolecular complex formation between TIMP-2, proMMP-2 and MT1-MMP and play an important role in MT1-MMP-mediated processing to the intermediate and final active forms of MMP-2 at the cell surface.
Worley
Joanna R
JR
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
Thompkins
Philip B
PB
Lee
Meng H
MH
Hutton
Mike
M
Soloway
Paul
P
Edwards
Dylan R
DR
Murphy
Gillian
G
Knäuper
Vera
V
D020816:Amino Acid Motifs / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D004792:Enzyme Precursors / Q000378:metabolism*; D018093:Gelatinases / Q000378:metabolism*; D017353:Gene Deletion; D005796:Genes; D006801:Humans; D053511:Matrix Metalloproteinase 14; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019714:Tissue Inhibitor of Metalloproteinases / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D000097792:Tissue Inhibitor of Metalloproteinase-4
D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D018093:Gelatinases; D053511:Matrix Metalloproteinase 14; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019716:Tissue Inhibitor of Metalloproteinase-2; D019714:Tissue Inhibitor of Metalloproteinases; D000097792:Tissue Inhibitor of Metalloproteinase-4; C507096:Mmp14 protein, mouse; C069860:progelatinase