Nitric oxide regulates prolidase activity by serine/threonine phosphorylation

• Published in: Journal of cellular biochemistry 2005-12, Vol.96 (5), p.1086-1094
• Main Authors: Surazynski, Arkadiusz, Liu, Yongmin, Miltyk, Wojciech, Phang, James M.
• Format: Article
• Language: English
• Subjects: Animals; Blotting, Western; cGMP; Cyclic GMP - metabolism; Dipeptidases - biosynthesis; Dipeptidases - chemistry; DNA, Complementary - metabolism; Dose-Response Relationship, Drug; Gene Expression Regulation, Enzymologic; Immunoprecipitation; Inflammation; Matrix Metalloproteinases - chemistry; Mice; Mitogen-Activated Protein Kinase 1 - metabolism; Mitogen-Activated Protein Kinase 3 - metabolism; Neovascularization, Pathologic; NIH 3T3 Cells; Nitric oxide; Nitric Oxide - metabolism; Nitric Oxide Synthase Type II - metabolism; Peptides - chemistry; Phosphorylation; Plasmids - metabolism; prolidase; Serine - chemistry; serine/threonine phosphorylation; Signal Transduction; Thiourea - chemistry; Threonine - chemistry; Time Factors; Transfection; Wound Healing
• ISSN: 0730-2312; 1097-4644
• DOI: 10.1002/jcb.20631

Prolidase [E.C. 3.4.13.9], a member of the matrix metalloproteinase (MMP) family, is a manganese‐dependent cytosolic exopeptidase that cleaves imidodipeptides containing C‐terminal proline or hydroxyproline. It plays an important role in collagen metabolism, matrix remodeling and cell growth. Nitric oxide (NO), a versatile signaling molecule, regulates many processes including collagen synthesis and matrix remodeling and, thereby, may modulate angiogenesis, tumor invasiveness, and metastasis. Thus, we considered that prolidase may be an important target of NO regulation. In our study, SIN I and DETA/NO were used as NO donors. Both donors increased prolidase activity in a time‐dependent and dose‐dependent manner. Prolidase activity increased not only with NO donors but also with endogenous NO in cells transfected with iNOS. The effect of iNOS was abolished by treatment with S‐methylisothiourea (SMT), a selective inhibitor of iNOS. However, with either exogenous or endogenous sources of NO, the increase in prolidase activity was not accompanied by increased prolidase expression. Therefore, we suspected phosphorylation of prolidase as a potential mechanism regulating enzyme activation. We observed increased serine/threonine phosphorylation on prolidase protein in cells treated with NO donors and in cells transfected with iNOS. To determinate the pathways that may mediate prolidase induction by NO, we first used 8‐Br‐cGMP, a cGMP agonist, and found that 8‐Br‐cGMP strongly and rapidly stimulated prolidase activity accompanied by increased phosphorylation. Rp‐8‐Br‐pCPT‐cGMP, an inhibitor of cGMP, reduced NO donor‐stimulated prolidase activity to control levels. To test wheher the MAPK pathway is involved in this NO‐dependent activation, we used an ERK1/2 inhibitor and found that it had no effect on prolidase activity increased by NO donors. These results demonstrate that NO stimulates prolidase activity by increasing serine/threonine phosphorylation through PKG‐cGMP pathway, but independent of MAPK and suggest an interaction between inflammatory signaling pathways and regulation of the terminal step of matrix degradation. J. Cell. Biochem. © 2005 Wiley‐Liss, Inc.