No 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...

Retinoic acid-inducible protein I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are cytosolic viral RNA sensors that induce type I interferon production (IFN). In this study, we found that MDA5 undergoes inducible SUMOylation by small ubiquitin-like modifier-1 (SUMO-1) in response to polyI:C stimulation. Enhanced SUMOylation of MDA5 by exogenously expressed SUMO-conjugating enzyme Ubc9 correlated with upregulation of IFN expression and repressed virus replication. Conversely, overexpression of a SUMOylation-deficient mutant of Ubc9 or knockdown of endogenous Ubc9 reduced IFN production. Furthermore, we showed that PIAS2β, a SUMOylation E3 ligase, could specifically interact with and enhance the SUMOylation of MDA5. Consequently, PIAS2β knockdown reduced the SUMOylation of MDA5 and the IFN production. Collectively, these findings suggest that SUMO-1 modification of MDA5 possibly via PIAS2β may play a role in the MDA5-mediated IFN response to viral infections.