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Regulation of matrix metalloproteinase-2 (MMP-2) activity by phosphorylation

Meltem Sariahmetoglu^*,, Bryan D. Crawford^*, Hernando Leon, Jolanta Sawicka^*, Laiji Li, Barbara J. Ballermann, Charles Holmes, Luc G. Berthiaume^||, Andrew Holt^*, Grzegorz Sawicki^*,1 and Richard Schulz^*,,2

Departments of Pharmacology,
^* Pediatrics,

Biochemistry,

Medicine,

and Cell Biology,

^|| Cardiovascular Research Group, University of Alberta, Edmonton, Alberta, Canada

²Correspondence: Departments of Pediatrics and Pharmacology, 462 Heritage Medical Research Center, University of Alberta, Edmonton, AB T6G 2S2, Canada, E-mail: richard.schulz{at}ualberta.ca

The regulation of matrix metalloproteinases (MMP) has been studied extensively due to the fundamental roles these zinc-endopeptidases play in diverse physiological and pathological processes. However, phosphorylation has not previously been considered as a potential modulator of MMP activity. The ubiquitously expressed MMP-2 contains 29 potential phosphorylation sites. Mass spectrometry reveals that at least five of these sites are phosphorylated in hrMMP-2 expressed in mammalian cells. Treatment of HT1080 cells with an activator of protein kinase C results in a change in MMP-2 immunoreactivity on 2D immunoblots consistent with phosphorylation, and purified MMP-2 is phosphorylated by protein kinase C in vitro. Furthermore, MMP-2 from HT1080 cell-conditioned medium is immunoreactive with antibodies directed against phosphothreonine and phosphoserine, which suggests that it is phosphorylated. Analysis of MMP-2 activity by zymography, gelatin dequenching assays, and measurement of kinetic parameters shows that the phosphorylation status of MMP-2 significantly affects its enzymatic properties. Consistent with this, dephosphorylation of MMP-2 immunoprecipitated from HT1080 conditioned medium with alkaline phosphatase significantly increases its activity. We conclude that MMP-2 is modulated by phosphorylation on multiple sites and that protein kinase C may be a regulator of this protease in vivo.

Key Words: dephosphorylation • gelatinase • alkaline phosphate • protein kinase C • mass spectrometry