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.
Sue Ng
Ser
S
Hubrecht Institute, KNAW and University Medical Centre Utrecht, Uppsalalaan 8, NL-3584 CT Utrecht, The Netherlands.
Mahmoudi
Tokameh
T
Hubrecht Institute, KNAW and University Medical Centre Utrecht, Uppsalalaan 8, NL-3584 CT Utrecht, The Netherlands.
Li
Vivian S W
VSW
Hubrecht Institute, KNAW and University Medical Centre Utrecht, Uppsalalaan 8, NL-3584 CT Utrecht, The Netherlands.
Hatzis
Pantelis
P
Hubrecht Institute, KNAW and University Medical Centre Utrecht, Uppsalalaan 8, NL-3584 CT Utrecht, The Netherlands.
Boersema
Paul J
PJ
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.
Mohammed
Shabaz
S
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.
Heck
Albert J
AJ
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.
Clevers
Hans
H
Hubrecht Institute, KNAW and University Medical Centre Utrecht, Uppsalalaan 8, NL-3584 CT Utrecht, The Netherlands.
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