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

Meltem Sariahmetoglu1, Bryan D Crawford, Hernando Leon

  • 1Department of Pediatrics, 462 Heritage Medical Research Center, University of Alberta, Edmonton, AB T6G 2S2, Canada.

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology
|April 17, 2007
PubMed

Insights

Matrix metalloproteinase-2 (MMP-2) is regulated by phosphorylation, a previously unrecognized mechanism. Protein kinase C phosphorylates MMP-2, altering its enzymatic activity and suggesting a new regulatory pathway for this protease.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Matrix metalloproteinases (MMPs) are crucial zinc-endopeptidases involved in numerous physiological and pathological processes.
  • The regulatory mechanisms of MMPs are extensively studied, yet phosphorylation has not been recognized as a modulator of their activity.

Purpose of the Study:

  • To investigate the role of phosphorylation in the regulation of matrix metalloproteinase-2 (MMP-2) activity.
  • To identify potential phosphorylation sites and kinases involved in MMP-2 regulation.

Main Methods:

  • Mass spectrometry was used to identify phosphorylated sites on human recombinant MMP-2 (hrMMP-2).
  • HT1080 cells were treated with protein kinase C activators, and MMP-2 phosphorylation was assessed via 2D immunoblotting.
  • Purified MMP-2 was incubated with protein kinase C in vitro.
  • MMP-2 activity was analyzed using zymography, gelatin dequenching assays, and kinetic parameter measurements.
  • Dephosphorylation of MMP-2 was performed using alkaline phosphatase.

Main Results:

  • Mass spectrometry identified at least five phosphorylated sites on hrMMP-2 expressed in mammalian cells.
  • Activation of protein kinase C in HT1080 cells induced changes in MMP-2 immunoreactivity indicative of phosphorylation.
  • Protein kinase C phosphorylated purified MMP-2 in vitro.
  • MMP-2 from cell-conditioned medium showed immunoreactivity with anti-phosphothreonine and anti-phosphoserine antibodies.
  • The phosphorylation status of MMP-2 significantly influenced its enzymatic properties, with dephosphorylation increasing its activity.

Conclusions:

  • MMP-2 is modulated by phosphorylation at multiple sites.
  • Protein kinase C is identified as a potential in vivo regulator of MMP-2 activity through phosphorylation.
  • Phosphorylation represents a novel regulatory mechanism for MMP-2 function.

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