Regulation of matrix metalloproteinase-2 (MMP-2) activity by phosphorylation.
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.
Sariahmetoglu
Meltem
M
Department of Pediatrics, 462 Heritage Medical Research Center, University of Alberta, Edmonton, AB T6G 2S2, Canada.
Crawford
Bryan D
BD
Leon
Hernando
H
Sawicka
Jolanta
J
Li
Laiji
L
Ballermann
Barbara J
BJ
Holmes
Charles
C
Berthiaume
Luc G
LG
Holt
Andrew
A
Sawicki
Grzegorz
G
Schulz
Richard
R
D000469:Alkaline Phosphatase / Q000378:metabolism; D001665:Binding Sites; D045744:Cell Line, Tumor; D015180:Electrophoresis, Gel, Two-Dimensional; D005354:Fibrosarcoma; D006706:Homeostasis; D006801:Humans; D013058:Mass Spectrometry; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D009363:Neoplasm Proteins; D010446:Peptide Fragments; D011493:Protein Kinase C; D000469:Alkaline Phosphatase; D014357:Trypsin; D020778:Matrix Metalloproteinase 2