Arkadia Enhances Nodal/TGF-β Signaling by Coupling Phospho-Smad2/3 Activity and Turnover

Abstract

Regulation of transforming growth factor-β (TGF-β) signaling is critical in vertebrate development, as several members of the TGF-β family have been shown to act as morphogens, controlling a variety of cell fate decisions depending on concentration. Little is known about the role of intracellular regulation of the TGF-β pathway in development. E3 ubiquitin ligases target specific protein substrates for proteasome-mediated degradation, and several are implicated in signaling. We have shown that Arkadia, a nuclear RING-domain E3 ubiquitin ligase, is essential for a subset of Nodal functions in the embryo, but the molecular mechanism of its action in embryonic cells had not been addressed. Here, we find that Arkadia facilitates Nodal signaling broadly in the embryo, and that it is indispensable for cell fates that depend on maximum signaling. Loss of Arkadia in embryonic cells causes nuclear accumulation of phospho-Smad2/3 (P-Smad2/3), the effectors of Nodal signaling; however, these must be repressed or hypoactive as the expression of their direct target genes is reduced or lost. Molecular and functional analysis shows that Arkadia interacts with and ubiquitinates P-Smad2/3 causing their degradation, and that this is via the same domains required for enhancing their activity. Consistent with this dual function, introduction of Arkadia in homozygous null (−/−) embryonic stem cells activates the accumulated and hypoactive P-Smad2/3 at the expense of their abundance. Arkadia−/− cells, like Smad2−/− cells, cannot form foregut and prechordal plate in chimeras, confirming this functional interaction in vivo. As Arkadia overexpression never represses, and in some cells enhances signaling, the degradation of P-Smad2/3 by Arkadia cannot occur prior to their activation in the nucleus. Therefore, Arkadia provides a mechanism for signaling termination at the end of the cascade by coupling degradation of P-Smad2/3 with the activation of target gene transcription. This mechanism can account for achieving efficient and maximum Nodal signaling during embryogenesis and for rapid resetting of target gene promoters allowing cells to respond to dynamic changes in extracellular signals. In development, cells respond to secreted signals (called morphogens) by turning on or off sets of target genes. How does gene activity adjust quickly in response to rapidly changing extracellular signals? This should require efficient removal of old/used signaling effectors (signal-activated transcription factors) from the promoters of target genes to allow new ones to assume control. We previously discovered Arkadia, an E3 ubiquitin ligase, and showed that it is an essential factor for normal development. (Ubiquitin ligases trigger the addition of ubiquitin residues to proteins, typically marking them for degradation.) Here, we show that Arkadia is required for high activity of the major signaling pathway, TGF-β/Nodal. Arkadia has a dual role to degrade Smads, the TGF-β signaling effectors, and enhance their transcriptional activity. This coupling of degradation with activation provides a mechanism to ensure that only effectors “in use” are degraded, allowing the new ones to proceed. It is possible that very similar mechanisms operate in other pathways to establish dynamic regulation and efficient signaling, while their failure may be associated with developmental abnormalities and disease, including cancer.