High throughput analysis of proteins associating with a proinvasive MT1-MMP in human malignant melanoma A375 cells.
Membrane-type 1 matrix metalloproteinase (MT1-MMP), a powerful modulator of the pericellular environment, promotes migration, invasion, and proliferation of cells. To perform its potent proteolytic activity in a controlled manner, MT1-MMP has to be regulated precisely. However, our knowledge about substrates and regulatory proteins is still very limited. In this study we identify a catalog of proteins that directly or indirectly interact with MT1-MMP. We expressed a FLAG-tagged MT1-MMP stably in human malignant melanoma A375 cells. We prepared cell lysate using Brij98 and MT1-MMP was affinity purified together with associating proteins using an anti-FLAG antibody. A distinct set of membrane proteins was found to copurify with MT1-MMP when biotin-labeled proteins were monitored. The proteins were analyzed with an integrated system composed of nano-flow liquid chromatography and tandem mass spectrometry. We identified 158 proteins including several previously reported to bind MT1-MMP, although most had not previously been identified. Six of these membrane proteins, including one previously shown to interact with MT1-MMP, were co-expressed with MT1-MMP in HT1080 cells. Five of the latter were found to associate with MT1-MMP in an immunoprecipitation assay. Immunostaining of cells expressing each of these test proteins revealed that one colocalized with MT1-MMP at the ruffling membrane and the other at the perinuclear vesicles. In contrast, another protein which did not coprecipitate with MT1-MMP showed no colocalization. Recombinant MT1-MMP cleaved two of the tested proteins at least in vitro. Thus, we provide a valuable resource to identify substrates and regulators of MT1-MMP in tumor cells.
Department of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.
Taizo
T
Tomari
Naohiko
N
Koshikawa
Takayuki
T
Uematsu
Takashi
T
Shinkawa
Daisuke
D
Hoshino
Nagayasu
N
Egawa
Toshiaki
T
Isobe
Motoharu
M
Seiki
D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D006801:Humans; D053511:Matrix Metalloproteinase 14 / Q000378:metabolism*; D008545:Melanoma / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology*; D009361:Neoplasm Invasiveness / Q000473:pathology; D009363:Neoplasm Proteins / Q000032:analysis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014162:Transfection
D009363:Neoplasm Proteins; D011994:Recombinant Proteins; C507086:MMP14 protein, human; D053511:Matrix Metalloproteinase 14