Activin and TGF-β signaling: Differential role of a Serine/Threonine phosphate in the regulation of SMAD2 activity

Abstract

Activins are the most prominent members of the Activin/Inhibin branch of the TGF-β superfamily. Similar to TGF-β, they play an essential role in regulating multiple physiological processes such as development, reproductive physiology, and immune responses. Besides, Activins demonstrate a vital role in wound healing and diseases like fibrosis and cancer. It contributes to various cancer processes, such as cell migration, metastasis, immune evasion, angiogenesis, drug resistance, and cancer cachexia. In recent studies, several differences have been reported between Activin and TGF-β mediated signaling and response. For example, Activins have been shown to function as a morphogen gradient in early embryonic development, unlike TGF-and are crucial for maintaining pluripotency in embryonic stem cells Despite differences, the primary notion in the field is that Activin signaling mirrors TGF-β signaling, where both the ligands activate the same SMAD2 and SMAD3 proteins and induce similar cellular responses. However, if we look closely at the available literature, it is intriguing that all known TGF-β superfamily ligands (over 40) signal through merely seven type I and five type II receptors and a set of 8 SMAD proteins. This undoubtedly results in a high degree of context-dependent promiscuity, where ligands of one subfamily could signal through receptors of other subfamilies. This could potentially result from additional complexity at the level of signaling, which is poorly understood and represent some of the main standing questions in the field. One of the major non-canonical pathways induced by Activin and TGF-β is ERK/MAPK pathway. The ERK/MAPK pathway forms an essential component of tumorigenesis and is active in most cancers. In addition to being Serine/Threonine kinases, the two TGF-β receptors, type I and type II, also function as tyrosine kinases and undergo auto-phosphorylation at tyrosine residues. These dual-specificity kinases employ various mechanisms to initiate the non-canonical ERK/MAPK pathway; many of them have been extensively studied. However, the regulation of the ERK/MAPK pathway by Activin and its role in Activin-mediated signaling is not well studied. We demonstrate that the non-canonical ERK/MAPK pathway is indispensable for Activin-induced EMT markers and cell migration. Next, we investigated the effect of ERK inhibition on Activin-mediated SMAD signaling. We found that the non-canonical ERK/MAPK pathway is crucial for Activin-mediated phosphorylation of SMAD2 and had no effect on phosphorylation of SMAD3. Furthermore, we demonstrate that the active ERK/MAPK pathway is essential for the nuclear translocation of SMAD2 and transcription of specific gene targets. In addition to this, the activity of GSK3β was identified to be critical for the regulation of receptor-mediated phosphorylation of SMAD2. A Ser/Thr phosphatase, PHLPP1, was determined as the common target of both ERK/MAPK and GSK3β kinases, which differentially targets the phosphorylation of SMAD2 upon Activin stimulation. ERK/MAPK and GSK3β kinases inhibit the phosphatase activity of PHLPP1, thereby facilitating SMAD2 phosphorylation. This signaling interaction was recorded only upon stimulation with Activin and was absent in TGF-β signaling. Therefore, through this study, we present novel differences between Activin and TGF-β-mediated signal transduction. Next, we attempted to understand how the non-canonical ERK/MAPK pathway is induced in response to the two ligands. We found that Activin and TGF-β differ in the mechanisms that they employ for ERK activation. We demonstrate that Activin signaling requires ectodomain shedding and EGFR transactivation to induce ERK/MAPK pathway; however, TGF-β promotes ligand-independent EGFR transactivation. Besides ERK/MAPK and GSK3β, we identified a unique regulatory role for PI3K, and observed that the phosphatase activity of PHLPP1, is regulated by all three kinases independently. We identified a positive regulation of PHLPP1 by PI3K, whereas both ERK/MAPK and GSK3β were found to regulate it negatively. Therefore, we propose that an “incoherent feedforward loop” is functioning between these kinases that regulate the PHLPP1 activity, and its outcome governs the eventual phosphorylation of Activin-mediated SMAD2. To understand the effect of this regulation on Activin response, we performed a stable knockdown of PHLPP1 in cells and examined their EMT phenotype and migratory ability. Weobserved that upon knockdown of PHLPP1 expression, the migratory ability of cells was retained even in the presence of ERK inhibition. Therefore, we demonstrate a unique role for PHLPP1 in Activin-mediated signaling and response. In conclusion, this study provides evidence of some fundamental differences in the signal transduction pathways initiated by Activin and TGF-β. We demonstrate that the two ligands employ different mechanisms to induce the non-canonical ERK/MAPK pathway. ERK/MAPK pathway plays a unique regulatory role in Activin-mediated SMAD2 and not SMAD3. We also identified a novel regulation of PHLPP1 by ERK/MAPK, along with GSK3β and PI3K kinases, and found this crosstalk indispensable for Activin-mediated response.