Src-dependent Phosphorylation of Membrane Type I Matrix Metalloproteinase on Cytoplasmic Tyrosine 573

Abstract

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a transmembrane MMP that plays important roles in migratory processes underlying tumor invasion and angiogenesis. In addition to its matrix degrading activity, MT1-MMP also contains a short cytoplasmic domain whose involvement in cell locomotion seems important but remains poorly understood. In this study, we show that MT1-MMP is phosphorylated on the unique tyrosine residue located within this cytoplasmic sequence (Tyr The degradation of extracellular matrix (ECM)3 proteins by members of the matrix metalloproteinases (MMPs) plays a crucial role in several biological processes, including cell attachment, cell migration, invasiveness, cell proliferation, apoptosis, and angiogenesis (1-4). Among the various MMPs described to date, there is now considerable evidence that MMPs that are intrinsically associated with the plasma membrane because of the presence of a transmembrane domain within their sequence, the so-called membrane type MMPs, represent key components involved in pericellular proteolysis and subsequent cell locomotion and invasion (5, 6). The prototypical member of this family, MT1-MMP, actively participates in the remodeling of the pericellular ECM by acting as a cellular receptor and activator of proMMP-2 (7) and as a potent matrixdegrading protease that proteolyses a broad spectrum of ECM proteins (8 -10) as well as a number of cell surface-associated adhesion receptors (11,12). These events are likely to be important in vivo because MT1-MMP null mice fail to thrive and have a markedly reduced lifespan (13,14).In addition to its role in normal physiology, MT1-MMP is also overexpressed in many types of tumors (15, 16), and this overexpression appears crucial for tumor cell migration and invasion. For example, MT1-MMP-mediated degradation of some ECM proteins, such as the laminin-5␥2 chain (17), stimulates migration, whereas proteolysis of the dense, cross-linked meshwork of type I collagen fibrils by the enzyme confers neoplastic cells with tissueinvasive activity (18) and sustains tumor cell growth in otherwise growth-restrictive three-dimensional matrices (19).Despite its importance to normal physiology and in the development of malignancy, the mechanisms underlying MT1-MMP-mediated cell invasion remain incompletely understood. During cell migration, MT1-MMP localizes predominantly to the cell adherent edge at the migration front, an appropriate location for the degradation of the ECM barrier (20). In addition, we and others have shown that MT1-MMP is preferentially localized into caveolae, specialized domains of the plasma membrane (21,22), and this localization may contribute to the spatiotemporal regulation of its proteolytic activity by controlling adequate endocytosis and recycling of the enzyme (23,24).In addition to the importance of MT1-MMP-mediated proteolytic breakdown of ECM proteins for the induction of cell migration, recent studies suggested that the cytoplasmic domain of the enzyme may also play a role in this process. For example...

Hic-5 mediates endothelial sprout initiation by regulating a key surface metalloproteinase

“…Endothelial cells were treated with S1P, which is known to promote MT1-MMP translocation to the plasma membrane (Nyalendo et al, 2007). Western blot analyses confirmed Hic-5 silencing and enrichment of surface biotinylated molecules (determined using anti-PECAM1 antibodies), and no contamination from cytoplasmic proteins (by using GAPDH controls) (Fig.…”

“…Endothelial lumen formation is a crucial aspect of endothelial morphogenesis and tubulogenesis (Davis et al, 2011), and MT1-MMP is required for both successful lumen formation and endothelial cell sprouting into collagen matrices (Chun et al, 2004;Stratman et al, 2009;Sacharidou et al, 2010). S1P has been shown to stimulate phosphorylation of MT1-MMP at Y573 (Nyalendo et al, 2007). Because a link between MT1-MMP and Hic-5 has not been established, we first investigated whether Hic-5 colocalized with phosphorylated MT1-MMP ( pMT1-MMP Y573) in endothelial cells in response to activating stimuli.…”

“…Membranes were incubated with HRP-conjugated secondary antibodies (Dako, Carpinteria, CA), washed, and developed with Immobilon Western Chemiluminescent HRP Substrate (Millipore) and HyBlot CL autoradiography film (Denville Scientific, South Plainfield, NJ). The antibodies used were directed against Hic-5 (611164, BD Transduction Laboratories, San Jose, CA; 1:1000 dilution), MT1-MMP (MAB3328, Millipore; 1:2000 dilution), MT1-MMP phospho-tyrosine 573 (custom antisera, 21st Century Biologicals; 1:500 dilution) (Nyalendo et al, 2007), pFAK (ab4803, Abcam, Cambridge, MA; 1:1000 dilution), FAK (05-537, Millipore; 1:1000 dilution), β2M (M8523, Sigma; 1:500 dilution), actin (CP01, Millipore; 1:5000 dilution), α-tubulin (T6199, Sigma; 1:10,000 dilution), GAPDH (ab8245, Abcam; 1:10,000 dilution), Flag (ab18230, Abcam or F7425, Sigma; 1:1000 dilution), integrin β1 (610467, BD Transduction Laboratories; 1:1000 dilution), vinculin (V9131, Sigma; 1:1000 dilution), PECAM1 (sc1505, Santa Cruz Biotechnology, Temecula, CA; 1:5000 dilution), VE-cadherin (sc52751, Santa Cruz Biotechnology; 1:2000 dilution), RACK1 (sc17754, Santa Cruz Biotechnology; 1:1000 dilution), vimentin (sc5565, Santa Cruz Biotechnology; 1:1000 dilution), paxillin (sc365059, Santa Cruz Biotechnology; 1:1000 dilution), zyxin (Cell Signaling, 3553; 1:1000 dilution), and filaminA (Bethyl, A301-134A; 1:1000 dilution).…”

Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Ubiquitination at Lys581 Increases Cellular Invasion through Type I Collagen

“…The MT1-MMP ICD further undergoes palmitoyla-tion at Cys 574 , a lipid post-translational modification that was shown to modulate cell migration and clathrin-mediated internalization (34). Recently, it has been reported that MT1-MMP is phosphorylated within its ICD at Tyr 573 in an Src-dependent mechanism (35). Expression of a non-phosphorylable MT1-MMP Tyr 573 mutant in fibrosarcoma cells resulted in impaired levels of proliferation within three-dimensional type-I collagen gels (36).…”

“…Remaining co-localization was still detected, indicating an overlap in the localization of both proteins although without direct interaction. in a Src-or PKC-dependent manner (35,37), which may be a prerequisite for protein ubiquitination (47). To test whether MT1-MMP phosphorylation affects its ubiquitination, MT1-MYC-Y573A and MT1-MYC-T567A mutants were used.…”