MAP3K1 functionally interacts with Axin1 in the canonical Wnt signalling pathway.
A central point of regulation in the Wnt/beta-catenin signalling pathway is the formation of the beta-catenin destruction complex. Axin1, an essential negative regulator of Wnt signalling, serves as a scaffold within this complex and is critical for rapid turnover of beta-catenin. To examine the mechanism by which Wnt signalling disables the destruction complex, we used an immunoprecipitation-coupled proteomics approach to identify novel endogenous binding partners of Axin1. We found mitogen-activated protein kinase kinase kinase 1 (MAP3K1) as an Axin1 interactor in Ls174T colorectal cancer (CRC) cells. Importantly, confirmation of this interaction in HEK293T cells indicated that the Axin1-MAP3K1 interaction is induced and modulated by Wnt stimulation. siRNA depletion of MAP3K1 specifically abrogated TCF/LEF-driven transcription and Wnt3A-driven endogenous gene expression in both HEK293T as well as DLD-1 CRC. Expression of ubiquitin ligase mutants of MAP3K1 abrogated TCF/LEF transcription, whereas kinase mutants had no effect in TCF-driven activity, highlighting the essential role of the MAP3K1 E3 ubiquitin ligase activity in regulation of the Wnt/beta-catenin pathway. These results suggest that MAP3K1, previously reported as an Axin1 inter-actor in c-Jun NH(2)-terminal kinase pathway, is also involved in the canonical Wnt signalling pathway and positively regulates expression of Wnt target genes.
Hubrecht Institute, KNAW and University Medical Centre Utrecht, Uppsalalaan 8, NL-3584 CT Utrecht, The Netherlands.
Ser
S
Sue Ng
Hubrecht Institute, KNAW and University Medical Centre Utrecht, Uppsalalaan 8, NL-3584 CT Utrecht, The Netherlands.
Tokameh
T
Mahmoudi
Hubrecht Institute, KNAW and University Medical Centre Utrecht, Uppsalalaan 8, NL-3584 CT Utrecht, The Netherlands.
Vivian S W
VSW
Li
Hubrecht Institute, KNAW and University Medical Centre Utrecht, Uppsalalaan 8, NL-3584 CT Utrecht, The Netherlands.
Pantelis
P
Hatzis
Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands.
Paul J
PJ
Boersema
Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands.
Shabaz
S
Mohammed
Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands.
Albert J
AJ
Heck
Hubrecht Institute, KNAW and University Medical Centre Utrecht, Uppsalalaan 8, NL-3584 CT Utrecht, The Netherlands.
Hans
H
Clevers
D000595:Amino Acid Sequence; D060466:Axin Protein; D002460:Cell Line; D045744:Cell Line, Tumor; D006801:Humans; D047468:Immunoprecipitation; D048728:MAP Kinase Kinase Kinase 1 / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000494:pharmacology; D012097:Repressor Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism*; D051176:beta Catenin / Q000378:metabolism
C555451:AXIN1 protein, human; D060466:Axin Protein; C495270:CTNNB1 protein, human; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D051153:Wnt Proteins; D051176:beta Catenin; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human