TGF-β-activated Kinase 1 (Tak1) Mediates Agonist-induced Smad Activation and Linker Region Phosphorylation in Embryonic Craniofacial Neural Crest-derived Cells

Kenji Yumoto,Penny S Thomas,Jamie Lane,Kouichi Matsuzaki,Maiko Inagaki,Jun Ninomiya-Tsuji,Gregory J Scott,Manas K Ray,Mamoru Ishii,Robert Maxson,Yuji Mishina,Vesa Kaartinen

doi:10.1074/jbc.m112.431775

Abstract

The role of Smad-independent TGF-β signaling in craniofacial development is poorly elucidated. In craniofacial mesenchymal cells, Tak1 regulates both R-Smad C-terminal and linker region phosphorylation in TGF-β signaling. Tak1 plays an irreplaceable role in craniofacial ecto-mesenchyme during embryogenesis. Understanding the mechanisms of TGF-β signaling contributes to knowledge of pathogenetic mechanisms underlying common craniofacial birth defects. Although the importance of TGF-β superfamily signaling in craniofacial growth and patterning is well established, the precise details of its signaling mechanisms are still poorly understood. This is in part because of the concentration of studies on the role of the Smad-dependent (so-called "canonical") signaling pathways relative to the Smad-independent ones in many biological processes. Here, we have addressed the role of TGF-β-activated kinase 1 (Tak1, Map3k7), one of the key mediators of Smad-independent (noncanonical) TGF-β superfamily signaling in craniofacial development, by deleting Tak1 specifically in the neural crest lineage. Tak1-deficient mutants display a round skull, hypoplastic maxilla and mandible, and cleft palate resulting from a failure of palatal shelves to appropriately elevate and fuse. Our studies show that in neural crest-derived craniofacial ecto-mesenchymal cells, Tak1 is not only required for TGF-β- and bone morphogenetic protein-induced p38 Mapk activation but also plays a role in agonist-induced C-terminal and linker region phosphorylation of the receptor-mediated R-Smads. Specifically, we demonstrate that the agonist-induced linker region phosphorylation of Smad2 at Thr-220, which has been shown to be critical for full transcriptional activity of Smad2, is dependent on Tak1 activity and that in palatal mesenchymal cells TGFβRI and Tak1 kinases mediate both overlapping and distinct TGF-β2-induced transcriptional responses. To summarize, our results suggest that in neural crest-derived ecto-mesenchymal cells, Tak1 provides a critical point of intersection in a complex dialogue between the canonical and noncanonical arms of TGF-β superfamily signaling required for normal craniofacial development.

Highlights

The role of Smad-independent TGF-␤ signaling in craniofacial development is poorly elucidated
In light of the important role played by the Smad-independent pathway implied by these studies, as well as the well established importance on Tgf-␤ and Bmp signaling in normal craniofacial development in mouse models [17], we examined the role of Tak1 in craniofacial neural crest development by deleting Tak1 function in premigratory neural crest cells using the Wnt1-Cre driver line
Neural Crest-specific Tak1 Mutants Display Mandibular Hypoplasia and Cleft Palate—A recent study demonstrated that haploinsufficiency of Tak1 in neural crest cells rescued the cleft palate phenotype of Tgfbr2 mutant mice [17]

Summary

Background

The role of Smad-independent TGF-␤ signaling in craniofacial development is poorly elucidated. Our studies show that in neural crest-derived craniofacial ecto-mesenchymal cells, Tak is required for TGF-␤- and bone morphogenetic protein-induced p38 Mapk activation and plays a role in agonist-induced C-terminal and linker region phosphorylation of the receptor-mediated R-Smads. Tak Signaling in the Craniofacial Neural Crest become phosphorylated as the result of distinct pathways: antagonists, such as EGF, which enable the activated R-Smads to remain in the cytoplasm and be degraded [8]; and agonistinduced linker phosphorylation that is required for maximal transcriptional activity of R-Smad-Smad complexes, as well as for rapid turnover of corresponding R-Smads [7, 9] In this “action turnover switch” model, the Thr/Ser residues adjacent to proline-rich sequences in R-Smads (Thr-220 in Smad2) are phosphorylated by nuclear Cdks creating a docking site for transcriptional co-regulators (Pin in the case of TGF-␤ R-Smads and Yap in the case of Bmp R-Smads). These results imply that in neural crestderived mesenchymal cells, Tak mediates both canonical and noncanonical arms of the TGF-␤ superfamily signaling

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION