Modulation of the Membrane Type 1 Matrix Metalloproteinase Cytoplasmic Tail Enhances Tumor Cell Invasion and Proliferation in Three-dimensional Collagen Matrices

Abstract

Increasing evidence suggests that the cytoplasmic tail of membrane type 1 matrix metalloproteinase (MT1-MMP) is subject to phosphorylation and that this modification may influence its enzymatic activity at the cell surface. In this study, phosphorylated MT1-MMP is detected using a phospho-specific antibody recognizing a protein kinase C consensus sequence (phospho-TXR), and a MT1-MMP tail peptide is phosphorylated by exogenous protein kinase C. To characterize the potential role of cytoplasmic residue Thr 567 in these processes, mutants that mimic a state of either constitutive (T567E) or defective phosphorylation (T567A) were expressed and analyzed for their functional effects on MT1-MMP activity and cellular behavior. Phospho-mimetic mutants of Thr 567 exhibit enhanced matrix invasion as well as more extensive growth within a three-dimensional type I collagen matrix. Together, these findings suggest that MT1-MMP surface action is regulated by phosphorylation at cytoplasmic tail residue Thr 567 and that this modification plays a critical role in processes that are linked to tumor progression.Largely composed of a mixture of collagens, laminins, and vitronectin, the extracellular matrix (ECM) 2 serves as both a physical scaffold and a barrier against cell invasion. It has become increasingly evident that the structural condition of the ECM plays a unique role in regulating cell behavior. Proteolysis of integral components of the basement membrane disturbs the barrier provided by the ECM. Without physical restriction, cells invade the surrounding environment in an unregulated manner. The ability of matrix metalloproteinases (MMPs) to collectively degrade nearly all ECM constituents allows this class of enzymes to function in a diverse range of physiological processes (1, 2). Of the anchored MMPs, membrane type 1 matrix metalloproteinase (MT1-MMP) was the first to be discovered and has been most thoroughly characterized. Unlike soluble MMPs, MT1-MMP has a stretch of hydrophobic amino acids that traverse the cell membrane, followed by a short cytoplasmic tail composed of 20 amino acids (3). The advantage of cell surface localization is 2-fold. Surface restriction allows MT1-MMP to modify the immediate pericellular environment, overcoming physical constraints imposed by the ECM (2). Localization at the cell surface also places tethered MMPs in an optimal position to function at invadapodia, highly specialized areas of the cell membrane that form during focalized cell invasion (4). Although information regarding the role of the cytoplasmic tail is relatively limited (5, 6), this domain may function as a bridge to the intracellular machinery.MT1-MMP has an essential role in matrix remodeling during physiological processes (7,8). Conversely, its enzymatic activity is key to acquiring a metastatic phenotype in a variety of tumor cells, including lung, colon, breast, and cervical carcinomas (2, 9 -11). The ability to alter the physical structure of the pericellular environment, while triggering the activation and modific...

Lack of an Endogenous Anti-inflammatory Protein in Mice Enhances Colonization of B16F10 Melanoma Cells in the Lungs

“…Because it has been reported that the S100A8 and S100A9 heterodimerize to yield maximum the stimulation of gene expression, we treated B16F10 cells with a combination of recombinant S100A8 (1 g/ml) and S100A9 (1 g/ml) and determined the mRNA and protein levels of MMP-2, MMP-9, and MMP-14. We chose to study these three MMPs because it has been previously reported that these proteinases confer invasiveness to tumor cells promoting metastasis (42,43). The results of real time RT-PCR revealed that the mRNA levels of MMP-2, MMP-9, and MMP-14 in B16F10 cells treated with a combination of S100A8 and S100A9 were mark-FIGURE 6.…”

Phosphorylation of Membrane Type 1-Matrix Metalloproteinase (MT1-MMP) and Its Vesicle-associated Membrane Protein 7 (VAMP7)-dependent Trafficking Facilitate Cell Invasion and Migration

“…Trafficking of MT1-MMP occurs rapidly because cell surface levels of MT1-MMP transiently increase within 20 min of PMA treatment (17,26). Recently, it was shown using an in vitro kinase assay that MT1-MMP is phosphorylated in a PKCdependent manner (27). Here, we tested whether MT1-MMP is phosphorylated in vivo upon PMA treatment and examined the effects of this phosphorylation on MT1-MMP trafficking, cell migration, and cell invasion.…”

Membrane Type 1-Matrix Metalloproteinase (Mt1-Mmp) Identified as a Multifunctional Regulator of Vascular Responses

“…cating that MT1 -MMP functions as a signaling molecule 71,72) . TIMP -2 and MT1 -MMP siRNA enhanced the increased levels of TF antigen and activity, and further reduced the decreased levels of TM antigen in TNF -α -stimulated ECs, indicating that MT1 -MMP may be critical for the modulation of procoagulant states in TNF -α stimulation.…”