Regulation of Wnt signaling by the nuclear pore complex.
OBJECTIVE
The function of beta-catenin as a transcriptional coactivator of T-cell factor-4 (TCF-4) is crucial for colorectal carcinogenesis. However, beta-catenin has no nuclear localization signal, and the mechanisms by which beta-catenin is imported into the nucleus and forms a complex with the TCF-4 nuclear protein are poorly understood.


METHODS
Proteins of 2 colorectal cancer cell lines, HCT-116 and DLD1, were immunoprecipitated with anti-TCF-4 antibody and analyzed directly by nanoflow liquid chromatography and mass spectrometry. The functional significance of nuclear pore complex (NPC) proteins in Wnt signaling was evaluated by in vitro and in vivo sumoylation, luciferase reporter, and colony formation assays.


RESULTS
TCF-4 interacted with a large variety of NPC proteins including ras-related nuclear protein (Ran), Ran binding protein-2 (RanBP2), and Ran GTPase-activating protein-1 (RanGAP1). The NPC protein RanBP2 functioned as the small ubiquitin-related modifier (SUMO) E3 ligase of TCF-4, and sumoylation of TCF-4 enhanced the interaction between TCF-4 and beta-catenin. The overexpression of NPC proteins increased the nuclear import of the TCF-4 and beta-catenin proteins and enhanced the transcriptional activity. NPC proteins increased the growth of colorectal cancer cells, whereas sentrin-specific protease-2 inhibited it.


CONCLUSIONS
Through a comprehensive proteomics approach, we revealed that NPC functions as a novel regulator of Wnt signaling and is possibly involved in colorectal carcinogenesis. A new drug targeting the interactions of TCF-4 with NPC proteins as well as their sumoylation activity might be effective for suppressing aberrant Wnt signaling and the proliferation of colorectal cancer cells.
Shitashige
Miki
M
Chemotherapy Division and Cancer Proteomics Project, National Cancer Center Research Institute, Tokyo, Japan.
Satow
Reiko
R
Honda
Kazufumi
K
Ono
Masaya
M
Hirohashi
Setsuo
S
Yamada
Tesshi
T
D021581:Active Transport, Cell Nucleus; D049109:Cell Proliferation; D015179:Colorectal Neoplasms / Q000201:enzymology / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D045325:HCT116 Cells; D006801:Humans; D018832:Molecular Chaperones / Q000378:metabolism; D022022:Nuclear Pore / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism; D011485:Protein Binding; D040901:Proteomics / Q000379:methods; D025842:SUMO-1 Protein / Q000378:metabolism; D015398:Signal Transduction; D051820:TCF Transcription Factors / Q000235:genetics / Q000378:metabolism*; D058118:Transcription Factor 7-Like 2 Protein; D014158:Transcription, Genetic; D014162:Transfection; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D051153:Wnt Proteins / Q000378:metabolism*; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism*; D000098843:Ubiquitin-Conjugating Enzyme UBC9
C495270:CTNNB1 protein, human; D018832:Molecular Chaperones; D028861:Nuclear Pore Complex Proteins; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; D051820:TCF Transcription Factors; C544894:TCF7L2 protein, human; D058118:Transcription Factor 7-Like 2 Protein; D051153:Wnt Proteins; D051176:beta Catenin; C094493:ran-binding protein 2; D044763:Ubiquitin-Conjugating Enzymes; D003546:Cysteine Endopeptidases; C501833:SENP2 protein, human; D000098843:Ubiquitin-Conjugating Enzyme UBC9