March Newsletter: Post-translational Modifications of β-catenin and TFC/LEF-1 Regulate Canonical Wnt Signaling

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Protein post-translational modifications (PTMs) such as phosphorylation, acetylation, ubiquitination, and SUMOylation, to name but a few, have evolved to diversify the functions of a single protein and account for the vast increase in proteome complexity and functional diversity1. A prime example of the complex and dynamic regulatory power PTMs confer is the Wnt/β-catenin signaling pathway2,3. This pathway regulates cellular proliferation, differentiation, and migration during embryonic development and adult cell homeostasis4-6. In addition, dysregulation of Wnt/β-catenin signaling is implicated in multiple pathological conditions, including carcinogenesis and degenerative diseases5,7. In canonical Wnt-mediated signaling, β-catenin is a key effector and interacts, as a co-transcription factor, with the DNA binding proteins TCF (T cell factor) and LEF-1 (lymphoid enhancer factor 1) to activate the transcription of Wnt/β-catenin target genes including cyclin D1, c-jun, and c-myc8-11 (Fig. 1). In this newsletter, the functional regulation of β-catenin and TCF/LEF-1 by PTMs is discussed.

 Post-Translational Regulation of β-Catenin in Wnt Signaling  

In the absence of Wnt binding to the co-receptor complex of Frizzled (Fz) and low-density lipoprotein receptor-related proteins (LRP) 5 and 6, a multi-protein destruction complex consisting of the scaffolding/tumor suppressor proteins Axin and adenomatous polyposis coli (APC), and the serine/threonine kinases casein kinase 1α (CK-1α) and glycogen synthase kinase 3 (GSK3) mediates phosphorylation of β-catenin2,3,9. Under these quiescent conditions, β-cateinin is sequentially phosphorylated on Ser45 by CK-1α, followed by phosphorylation of Ser33, Ser37, and Thr41 by GSK312 (Fig. 1). Upon phosphorylation, the E3 ubiquitin ligase β-transducin repeats containing protein (β-TrCP) ubiquitinates phospho-β-catenin on Lys19 and Lys4913,14, which leads to proteasomal destruction and low β-catenin levels and activity15,16. Conversely, in the presence of Wnt binding, the scaffold protein Dishevelled (Dvl) is recruited to the Fz-LRP5/6 co-receptor complex, which activates a sequence of molecular signals that inhibit the destruction complex and result in stabilization of unphosphorylated, cytosolic β-catenin and β-catenin-dependent transcription2,3,17-19. Stabilization occurs through partial inhibition of CK-1α and GSK318, inhibition of β-TrCP20, phosphorylation of five highly conserved PPPSP motifs in LRP6's intracellular domain21,22 (Fig.1), and subsequent aggregation of these Wnt signaling proteins. The negative Wnt regulator Axin is inhibited through recrutiment and direct binding to this aggregate complex3,17.

Notably, the same PTMs can inhibit or enhance β-catenin activity, depending on the residue(s) modified. For example, ubiquitination of Lys394 stabilizes β-catenin17. Similarly, phosphorylation of Ser675 by PKA, Ser552 by AKT, and Ser191/605 by JNK2 stabilizes β-catenin23-25 (Fig. 1). Acetyltransferases act on multiple Lys residues and each acetylation modulates the functional role and stability of β-catenin in different ways: acetylation of Lys49 by CBP inhibits β-catenin’s transcription activity26, acetylation of Lys345 by p300 enhances β-catenin interaction with TCF27, and acetylation of Lys19 and Lys49 by PCAF increases β-catenin stability28. Interestingly, tyrosine phosphorylation seems to only enhance β-catenin activity17.

Fig. 1 PTMs regulate the stability of ß-catenin and TCF/LEF-1 and their interaction; (ßBD:ß-catenin binding domain, HMG: High mobility group, ↑: favors interaction,     : inhibits interaction).
Fig. 1 PTMs regulate the stability of ß-catenin and TCF/LEF-1 and their interaction; (ßBD:ß-catenin binding domain, HMG: High mobility group, ↑: favors interaction, ⊥: inhibits interaction).

 Post-Translational Regulation of TCF/LEF-1 in Wnt Signaling 

TCF and LEF-1 form multimeric transcription complexes with cofactors such as CtBP, HBP1, and β-catenin, which function together to either repress or activate gene expression29,30. The binding affinity between TCF/LEF-1 and co-regulators is significantly affected by each protein’s respective PTM status (Fig. 1). TCF is phosphorylated on multiple Thr/Ser residues with opposing functional effects. For instance, phosphorylation of Ser154 on TCF by the TNIK kinase is required for its transcriptional activity3, while phosphorylation of Thr178 and Thr189 by Nemo-like kinase (NLK) inhibits TCF/DNA binding and suppresses its transcriptional activity31. The phosphorylation of TCF by NLK is linked to TCF ubiquitination. LEF-1 is phosphorylated on Thr155 and Ser166 by NLK, which inhibits DNA binding of the TCF/LEF-1/β-catenin complex3. Conversely, phosphorylation of Ser42 and Ser61 by CK-1 enhances β-catenin binding and transactivation32. The E3 ligase NLK-associated Ring Finger Protein (NARF) ubiquitinates TCF/LEF-1 in vitro and in vivo, which leads to TCF/LEF-1 degradation33. Both TCF and LEF-1 undergo SUMOylation by PIASy, but the functional consequences are distinct. SUMOylation of Lys297 on TCF confers enhanced activity34, while  SUMOylation of Lys25 and Lys267 residues on LEF-1 results in loss of activity35.

 Summary   

Like most, if not all, proteins, the Wnt signaling effector β-catenin and its co-transcriptional regulators TCF and LEF-1 are regulated by multiple PTMs, with the same PTM able to both enhance and inhibit activity, depending on which residue(s) are modified. In addition, PTMs can be cooperative or mutually exclusive. To study such complex and diverse functional regulation of cells' proteomes requires sensitive and quantitative reagents. At Cytoskeleton, Inc., the Signal-Seeker kits offer an unparalleled view into how PTMs regulate protein localization and function with the ability to measure tyrosine phosphorylation, ubiquitination, SUMOylation, and acetylation of endogenous proteins.

Related Products & Resources

Signal Seeker™ Kits    

New Signal-Seeker™ Acetyl-Lysine Detection Kit (30 assay) (Cat. # BK163)

New Signal-Seeker™ Acetyl-Lysine Detection Kit (10 assay) (Cat. # BK163-S)

Signal-Seeker™ Phosphotyrosine Detection Kit (30 assay) (Cat. # BK160)

Signal-Seeker™ Phosphotyrosine Detection Kit (10 assay) (Cat. # BK160-S)

Signal-Seeker™ Ubiquitination Detection Kit (30 assay) (Cat. # BK161)

Signal-Seeker™ Ubiquitination Detection Kit (10 assay) (Cat. # BK161-S)

Signal-Seeker™ SUMOylation 2/3 Detection Kit (30 assay) (Cat. # BK162)

Signal-Seeker™ SUMOylation 2/3 Detection Kit (10 assay) (Cat. # BK162-S)

PTM Antibodies, Beads, Etc   

New Acetyl-Lysine Antibody Mouse Monoclonal (7B5A1) (Cat. # AAC02)

New Acetyl-Lysine-HRP Antibody Mouse Monoclonal (19C4B2.1) (Cat. # AAC03-HRP)

Phosphotyrosine Affinity Beads (Cat. # APY03-beads)

Phosphotyrosine-HRP Mouse Monoclonal Antibody 27B10 (Cat. # APY03-HRP)

SUMOylation 2/3 Affinity Beads (Cat. # ASM24-beads)

Ubiquitin Antibody Mouse Monoclonal (Cat. # AUB01)

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