High throughput analysis of proteins associating with a proinvasive MT1-MMP in human malignant melanoma A375 cells.
Cancer Sci
; 100(7): 1284-90, 2009 Jul.
Article
em En
| MEDLINE
| ID: mdl-19432894
ABSTRACT
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Metaloproteinase 14 da Matriz
/
Melanoma
/
Proteínas de Neoplasias
Tipo de estudo:
Risk_factors_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2009
Tipo de documento:
Article