MTMR4 Attenuates Transforming Growth Factor β (TGFβ) Signaling by Dephosphorylating R-Smads in Endosomes

Abstract

Homeostasis of Smad phosphorylation at its C-terminal SXS motif is essential for transforming growth factor ␤ (TGF␤) signaling. Whereas it is known that TGF␤ signaling can be terminated by phosphatases, which dephosphorylate R-Smads in the nucleus, it is unclear whether there are any cytoplasmic phosphatase(s) that can attenuate R-Smad phosphorylation and nuclear translocation. Here we demonstrate that myotubularinrelated protein 4 (MTMR4), a FYVE domain-containing dualspecificity protein phosphatase (DSP), attenuates TGF␤ signaling by reducing the phosphorylation level of R-Smads in early endosomes. Co-immunoprecipitation experiments showed that endogenous MTMR4 interacts with phosphorylated R-Smads, and that this interaction is correlated with dephosphorylation of R-Smads. Further analysis showed that overexpression of MTMR4 resulted in the sequestration of activated Smad3 in the early endosomes, thus reducing its nuclear translocation. However, both point mutations at the conserved catalytic site of the phosphatase (MTMR4-C407S) and small interference RNA of endogenous Mtmr4 expression led to sustained Smad3 activation. This work therefore suggests that MTMR4 plays an important role in preventing the overactivation of TGF␤ signaling by dephosphorylating activated R-Smads that have been trafficked to early endosomes. The transforming growth factor ␤ (TGF␤)3 signaling pathway involves two transmembrane serine/threonine kinases, namely type II (T␤RII) and type I TGF␤ receptors (T␤RI) (1, 2). T␤RII, a constitutively active kinase, binds TGF␤ to initiate its heterodimerization with T␤RI. T␤RI then becomes phosphorylated and activates a signaling cascade through a family of intracellular signaling mediators known as Smads.In doing so, T␤RI recruits receptor-regulated Smads (RSmads, including BMP signaling transducers Smad1, -5, and -8 and TGF␤ signaling transducers Smad2, -3) and phosphorylates the two conserved C-terminal serines (SXS) of R-Smads. Activated R-Smads form a trimeric complex with a common mediator Smad (Co-Smad, Smad4), and these complexes translocate into the nucleus to regulate the transcription of an array of target genes (3-7). Recent studies have also indicated that Smad Anchor for Receptor Activation (SARA), an early endosomal protein containing a characteristic FYVE domain (Fab1p, YO1B, Vac1p, and EEA1), serves as an anchor protein by directly recruiting Smad2 to the early endosomes (8, 9) and presenting Smad2 to the internalized T␤RI/II complex (10, 11). Phosphorylated Smad2 then dissociates from SARA and leaves the early endosome. Subsequently, it forms a Smad2-Smad4 complex for nuclear translocation.Despite the fact that TGF␤ signaling activation is surprisingly simple, there are many layers of negative regulation that fine-tune or terminate the signal. The inhibitory Smads (I-Smads, including Smad6 and -7) competitively inhibit the interaction of R-Smads with Smad4 or receptors to provide a negative checkpoint for TGF␤-signaling activation (12). Ubiquitin-dependent degradation of activated...

A phosphoinositide conversion mechanism for exit from endosomes

“…Also, MTMR4 colocalizes with the exocyst subunit Sec15 and affects localization of VAMP3-containing endosomes (Lorenzo, Urbe et al 2006;Naughtin, Sheffield et al 2010). Additionally to PI(3)P, MTMR4 dephosphorylates RSmads at early endosomes and thereby negatively regulates TGF-β (transforming growth factor) (Yu, Pan et al 2010) and BMP (bone morphogenetic protein) signaling pathways (Yu, He et al 2013). …”

Protein phosphatase 5 modulates SMAD3 function in the transforming growth factor‐β pathway

“…Consistent with this notion, PPM1A (also known as PP2C) was reported to be a SMAD2/3-tail phosphatase in the nucleus (11). In contrast, two cytoplasmic phosphatases, namely PP2A and MTMR4, have been proposed to act as SMAD phosphatases (15,16). PP2A has been reported to act as SMAD3-tail phosphatase only under hypoxia (15).…”

“…Our observations raise two possibilities on the role of PPM1A as SMAD2/3-tail phosphatases: i. PPM1A acts as SMAD2/3-phosphatases in the cytoplasm and affects cytoplasmic levels of phospho-SMAD2/3-tail thereby affecting the TGFβ pathway. PPM1A could therefore have an analogous role to PP2A and MTMR4 that have been reported to act as cytoplasmic phosphatases against phospho-SMAD2/3-tail (15,16). ii.…”

“…PP2A has been reported to act as SMAD3-tail phosphatase only under hypoxia (15). MTMR4 has been reported to dephosphorylate SMAD2/3 in the cytoplasm and attenuate TGFβ signalling (16). Despite these reports, evidence for the role of these phosphatases on the TGFβ pathway has not been confirmed in knockout mouse models or cells.…”

Myotubularin-related Protein 4 (MTMR4) Attenuates BMP/Dpp Signaling by Dephosphorylation of Smad Proteins

“…A few nuclear phosphatases for R-Smads have thus far been identified, such as pyruvate dehydrogenase phosphatase (PDP), PPM1A (protein phosphatase magnesiumdependent 1A), and small C-terminal domain phosphatase (SCP2/Os4) (19 -21). However, the existence and roles of cytoplasmic phosphatases have remained elusive; it has been recently revealed by us that a new endosomal phosphatase, myotubularin-related protein 4 (MTMR4), can specifically temper TGF␤ signaling (22). MTMR4 belongs to myotubularin family and contains tyrosine/dual-specificity phospha-tase (DUSP) activity (23) and functions in early endosomes (22,24).…”