Biological importance of TIMP-2 phosphorylation on MMP-2 activity

Abstract

Matrix metalloproteinases (MMPs) are proteolytic enzymes that are secreted from the cell and play an important role in embryonic development and tissue remodeling. In cancer, MMPs are hyperactive, promoting degradation of the ex-tracellular matrix. Enhancement of MMP proteolytic activity allows tumor cells to migrate and invade surrounding tissues, increasing the chance of metastasis. Tissue inhibitor of metalloproteinases (TIMPs) are also known to act extracellu-larly, and are the endogenous inhibitors of MMPs. To inhibit the protease activi-ty of MMPs, the N-terminus of the TIMP protein binds to the catalytic domain of MMP at a ratio of 1:1. Studies from our lab have found that TIMP-2 is phosphor-ylated on three tyrosine residues, and this phosphorylation increases the inter-action with MMP-2. This is the first time that phosphorylation of TIMP-2 has been reported. Fascinatingly, the proto-oncogene tyrosine kinase c-Src was found to phosphorylate TIMP-2. This is significant in that c-Src has not yet been shown to act extracellularly, and there are no details within the current lit-erature describing how this protein may function outside of the cell. In this the-sis, we usedmammalian cells as a model to decipher whether TIMP-2 phosphor-ylation wasable to occur extracellularly,as well as the effect that phosphoryla-tion of TIMP-2 hadon its functionto both inhibit/activate MMP-2. We found that(1) c-Src is able to phosphorylate TIMP-2 extracellularly in conditioned me-vidia; and (2) phosphorylation of TIMP-2 enhances its function of inhibiting MMP-2 proteolytic activity, as well as assisting in the activation of pro-MMP-2. Our results suggest the presence of anovel mechanismin whichphosphoryla-tion of TIMP-2is able to regulate the extracellular environment through en-hanced interaction with MMP-2. The information gained from this research couldlead to development of novel therapies that use phosphorylated TIMP-2 as a means of decreasing cellular migration and invasion with the overall goal of preventing metastasis.