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The epithelial-to-mesenchymal transition (EMT) is a cellular reprogramming process converting epithelial cells into mesenchymal cell morphology. Snail is a critical regulator of EMT by both suppressing epithelial gene expression and promoting mesenchymal gene expression. Expression and activity of Snail are tightly controlled at transcriptional and post-translational levels. It has previously been reported that Snail undergoes phosphorylation and ubiquitin-dependent proteasome degradation. Here, we report nuclear phosphatase SCP4/CTDSPL2 acts as a novel Snail phosphatase. SCP4 physically interacts with and directly dephosphorylates Snail. SCP4-mediated dephosphorylation of Snail suppresses the ubiquitin-dependent proteasome degradation of Snail and consequently enhances TGFβ-induced EMT. The knockdown of SCP4 in MCF10A mammary epithelial cells leads to attenuated cell migration. Collectively, our finding demonstrates that SCP4 plays a critical role in EMT through Snail dephosphorylation and stabilization., (© 2018 The Authors.)

FoxO1 and FoxO3a (collectively FoxO1/3a) proteins regulate a wide array of cellular processes, including hepatic gluconeogenesis. Phosphorylation of FoxO1/3a is a key event that determines its subcellular location and transcriptional activity. During glucose synthesis, the activity of FoxO1/3a is negatively regulated by Akt-mediated phosphorylation, which leads to the cytoplasmic retention of FoxO1/3a. However, the nuclear phosphatase that directly regulates FoxO1/3a remains to be identified. In this study, we discovered a nuclear phosphatase, SCP4/CTDSPL2 (SCP4), that dephosphorylated FoxO1/3a and promoted FoxO1/3a transcription activity. We found that SCP4 enhanced the transcription of FoxO1/3a target genes encoding PEPCK1 and G6PC, key enzymes in hepatic gluconeogenesis. Ectopic expression of SCP4 increased, while knockdown of SCP4 inhibited, glucose production. Moreover, we demonstrated that gene ablation of SCP4 led to hypoglycemia in neonatal mice. Consistent with the positive role of SCP4 in gluconeogenesis, expression of SCP4 was regulated under pathophysiological conditions. SCP4 expression was induced by glucose deprivation in vitro and in vivo and was elevated in obese mice caused by genetic (A vy ) and dietary (high-fat) changes. Thus, our findings provided experimental evidence that SCP4 regulates hepatic gluconeogenesis and could serve as a potential target for the prevention and treatment of diet-induced glucose intolerance and type 2 diabetes., (© 2017 by the American Diabetes Association.)

Ubiquitin-like domain-containing C-terminal domain phosphatase 1 (UBLCP1), an FCP/SCP phosphatase family member, was identified as the first proteasome phosphatase. UBLCP1 binds to proteasome subunit Rpn1 and dephosphorylates the proteasome in vitro However, it is still unclear which proteasome subunit(s) are the bona fide substrate(s) of UBLCP1 and the precise mechanism for proteasome regulation remains elusive. Here, we show that UBLCP1 selectively binds to the 19S regulatory particle (RP) through its interaction with Rpn1, but not the 20S core particle (CP) or the 26S proteasome holoenzyme. In the RP, UBLCP1 dephosphorylates the subunit Rpt1, impairs its ATPase activity, and consequently disrupts the 26S proteasome assembly, yet it has no effects on the RP assembly from precursor complexes. The Rpn1-binding and phosphatase activities of UBLCP1 are essential for its function on Rpt1 dephosphorylation and proteasome activity both in vivo and in vitro Our study establishes the essential role of the UBLCP1/Rpn1/Rpt1 complex in regulating proteasome assembly., (© 2017 The Authors.)

SCP1 as a nuclear transcriptional regulator acts globally to silence neuronal genes and to affect the dephosphorylation of RNA Pol ll. However, we report the first finding and description of SCP1 as a plasma membrane-localized protein in various cancer cells using EGFP- or other epitope-fused SCP1. Membrane-located SCP1 dephosphorylates AKT at serine 473, leading to the abolishment of serine 473 phosphorylation that results in suppressed angiogenesis and a decreased risk of tumorigenesis. Consistently, we observed increased AKT phosphorylation and angiogenesis followed by enhanced tumorigenesis in Ctdsp1 (which encodes SCP1) gene - knockout mice. Importantly, we discovered that the membrane localization of SCP1 is crucial for impeding angiogenesis and tumor growth, and this localization depends on palmitoylation of a conserved cysteine motif within its NH2 terminus. Thus, our study discovers a novel mechanism underlying SCP1 shuttling between the plasma membrane and nucleus, which constitutes a unique pathway in transducing AKT signaling that is closely linked to angiogenesis and tumorigenesis.

Twist1 is a basic helix-loop-helix transcription factor that strongly promotes epithelial-to-mesenchymal transition, migration, invasion, and metastasis of cancer cells. The MAPK-phosphorylated Twist1 on its serine 68 (Ser(P)(68)-Twist1) has a significantly enhanced stability and function to drive cancer cell invasion and metastasis. However, the phosphatase that dephosphorylates Ser(P)(68)-Twist1 and destabilizes Twist1 has not been identified and characterized. In this study, we screened a serine/threonine phosphatase cDNA expression library in HEK293T cells with ectopically coexpressed Twist1. We found that the small C-terminal domain phosphatase 1 (SCP1) specifically dephosphorylates Ser(P)(68)-Twist1 in both cell-free reactions and living cells. SCP1 uses its amino acid residues 43-63 to interact with the N terminus of Twist1. Increased SCP1 expression in cells decreased Ser(P)(68)-Twist1 and total Twist1 proteins, whereas knockdown of SCP1 increased Ser(P)(68)-Twist1 and total Twist1 proteins. Furthermore, the levels of SCP1 are negatively correlated with Twist1 protein levels in several cancer cell lines. SCP1-dephosphorylated Twist1 undergoes fast degradation via the ubiquitin-proteasome pathway. Importantly, an increase in SCP1 expression in breast cancer cells with either endogenous or ectopically expressed Twist1 largely inhibits the Twist1-induced epithelial-to-mesenchymal transition phenotype and the migration and invasion capabilities of these cells. These results indicate that SCP1 is the phosphatase that counterregulates the MAPK-mediated phosphorylation of Ser(68)-Twist1. Thus, an increase in SCP1 expression and activity may be a useful strategy for eliminating the detrimental roles of Twist1 in cancer cells., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The auto-phosphorylation of murine receptor-interacting protein 3 (Rip3) on Thr 231 and Ser 232 in the necrosome is required to trigger necroptosis. However, how Rip3 phosphorylation is regulated is still largely unknown. Here we identified protein phosphatase 1B (Ppm1b) as a Rip3 phosphatase and found that Ppm1b restricts necroptosis in two settings: spontaneous necroptosis caused by Rip3 auto-phosphorylation in resting cells, and tumour necrosis factor-α (TNF)-induced necroptosis in cultured cells. We revealed that Ppm1b selectively suppresses necroptosis through the dephosphorylation of Rip3, which then prevents the recruitment of mixed lineage kinase domain-like protein (Mlkl) to the necrosome. We further showed that Ppm1b deficiency (Ppm1b(d/d)) in mice enhanced TNF-induced death in a Rip3-dependent manner, and the role of Ppm1b in inhibiting necroptosis was evidenced by elevated Rip3 phosphorylation and tissue damage in the caecum of TNF-treated Ppm1b(d/d) mice. These data indicate that Ppm1b negatively regulates necroptosis through dephosphorylating Rip3 in vitro and in vivo.

The bone morphogenetic protein (BMP) signaling pathway regulates a wide range of cellular responses in metazoans. A key step in the canonical BMP signaling is the phosphorylation and activation of transcription factors Smad1, Smad5, and Smad8 (collectively Smad1/5/8) by the type I BMP receptors. We previously identified PPM1A as a phosphatase toward dephosphorylation of all receptor-regulated Smads (R-Smads), including Smad1/5/8. Here we report another nuclear phosphatase named SCP4/CTDSPL2, belonging to the FCP/SCP family, as a novel Smad phosphatase in the nucleus. SCP4 physically interacts with and specifically dephosphorylates Smad1/5/8, and as a result attenuates BMP-induced transcriptional responses. Knockdown of SCP4 in multipotent mesenchymal C2C12 cells leads to increased expression of BMP target genes and consequently promotes BMP-induced osteogenic differentiation. Collectively, our results demonstrate that SCP4, as a Smad phosphatase, plays a critical role in BMP-induced signaling and cellular functions., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Bone morphogenetic proteins (BMPs) belong to the TGF-β superfamily of structurally related signaling proteins that regulate a wide array of cellular functions. The key step in BMP signal transduction is the BMP receptor-mediated phosphorylation of transcription factors Smad1, 5, and 8 (collectively Smad1/5/8), which leads to the subsequent activation of BMP-induced gene transcription in the nucleus. In this study, we describe the identification and characterization of PPM1H as a novel cytoplasm-localized Smad1/5/8-specific phosphatase. PPM1H directly interacts with Smad1/5/8 through its Smad-binding domain, and dephosphorylates phospho-Smad1/5/8 (P-Smad1/5/8) in the cytoplasm. Ectopic expression of PPM1H attenuates BMP signaling, whereas loss of PPM1H activity or expression greatly enhances BMP-dependent gene regulation and mesenchymal differentiation. In conclusion, this study suggests that PPM1H acts as a gatekeeper to prevent excessive BMP signaling through dephosphorylation and subsequent nuclear exclusion of P-Smad1/5/8 proteins.

BMP signals play pivotal roles in dorsoventral patterning of vertebrate embryos. The role of Ppp4c, the catalytic subunit of ubiquitous protein phosphatase 4, in vertebrate embryonic development and underlying mechanisms is poorly understood. Here, we demonstrate that knockdown of zebrafish ppp4cb and/or ppp4ca inhibits ventral development in embryos and also blocks ventralizing activity of ectopic Smad5. Biochemical analyses reveal that Ppp4c is a direct binding partner and transcriptional coactivator of Smad1/Smad5. In response to BMP, Ppp4c is recruited to the Smad1-occupied promoter, and its phosphatase activity is essential in inhibiting HDAC3 activity and, consequently, potentiating transcriptional activation. Consistently, genetic or chemical interference of Hdac3 expression or activity compromises the dorsalizing phenotype induced by ppp4cb knockdown. We conclude that Ppp4c is a critical positive regulator of BMP/Smad signaling during embryonic dorsoventral pattern formation in zebrafish., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Smad2 and Smad3 (Smad2/3) are essential signal transducers and transcription factors in the canonical transforming growth factor-β (TGF-β) signalling pathway. Active Smad2/3 signalling in the nucleus is terminated by dephosphorylation and subsequent nuclear export of Smad2/3. Here we report that protein phosphatase PPM1A regulates the nuclear export of Smad2/3 through targeting nuclear exporter RanBP3. PPM1A directly interacted with and dephosphorylated RanBP3 at Ser 58 in vitro and in vivo. Consistently, RanBP3 phosphorylation was elevated in PPM1A-null mouse embryonic fibroblasts. Dephosphorylation of RanBP3 at Ser 58 promoted its ability to export Smad2/3 and terminate TGF-β responses. Our findings indicate the critical role of PPM1A in maximizing exporter activity of RanBP3 for efficient termination of canonical TGF-β signalling.

Tight regulation of TGF-beta (transforming growth factor-beta) superfamily signalling is important for normal cellular functions and tissue homoeostasis. Since TGF-beta superfamily signalling pathways are activated by a short phosphorylation cascade, from receptor phosphorylation to subsequent phosphorylation and activation of downstream signal transducer R-Smads (receptor-activated Smads), reversible phosphorylation serves as a critical step to assure proper TGF-beta signalling. The present article will review the current progress on the understanding of dynamic phosphorylation in TGF-beta signalling and the essential role of protein phosphatases in this process.

Aim: To investigate the expressions of PTEN, PPM1A and P-Smad2 in hepatocellular carcinoma (HCC) and their significance., Methods: The expressions of PTEN, PPM1A and P-Smad2 in 31 HCC tissues, 25 adjacent liver tissues and 13 non-tumor liver tissues were detected by using Envision immunohistochemical technique., Results: The positive expression (64.52%) and staining intensity (4.19 +/- 3.31) of PTEN in the cytoplasm of HCC were significantly lower and weaker than those in the adjacent or non-tumor liver tissues (97.37%, 7.88 +/- 0.93; 100%, 7.77 +/- 0.93, respectively) (P < 0.05), and its staining intensity in the cytoplasm of HCC, which belongs to Edmondson pathologic grades II-III and above, was also lower than that of grade I and I-II. Furthermore, its location in the nucleus or cytoplasm of liver cells was negatively correlated with the progression of liver disease (r = -0.339, P = 0.002); most of PPM1A might be only expressed in the nucleus of adjacent liver tissues, non-HCC tissues or Edmondson grade I and I-II HCC, but it was mainly expressed in the cytoplasm of HCC with Edmondson grade > or = II, weakly or negatively expressed in the nucleus (P < 0.05), and its location was negatively correlated with the progression of liver disease (r = -0.45, P = 0.0000). P-Smad2, which was mostly located in the nucleus and cytoplasm of grade I and I-II HCC, surrounding or non-tumor liver tissues, was only in the nucleus of HCC with Edmondson grade II and above (P < 0.001), and its location was positively correlated with the disease progression (r = 0.224, P = 0.016). Spearman correlation analysis revealed that P-Smad2 was significantly negatively correlated with PTEN and PPM1A (r = -0.748, P = 0.000; r = -0.366, P = 0.001, respectively); and PTEN and PPM1A were positively correlated with HCC carcinogenesis (r = 0.428, P = 0.000)., Conclusion: The aberrant location of expression and staining intensity of PTEN, PPM1A and P-Smad2 in HCC and their relationship might have an impact on the pathogenesis of HCC.

Bone morphogenetic proteins (BMPs) are secreted polypeptides belonging to the transforming growth factor-beta (TGF-beta) superfamily that activates a broad range of biological responses in the metazoan organism. The BMP-initiated signaling pathway is under tight control by processes including regulation of the ligands, the receptors, and the key downstream intracellular effector Smads. A critical point of control in BMP signaling is the phosphorylation of Smad1, Smad5, and Smad8 in their C-terminal SXS motif. Although such phosphorylation, which is mediated by the type I BMP receptor kinases in response to BMP stimulation, is well characterized, biochemical mechanisms underlying Smad dephosphorylation remain to be elucidated. In this study, we have found that PPM1A, a metal ion-dependent protein serine/threonine phosphatase, physically interacts with and dephosphorylates Smad1 both in vitro and in vivo. Functionally, overexpression of PPM1A abolishes BMP-induced transcriptional responses, whereas RNA interference-mediated knockdown of PPM1A enhances BMP signaling. Collectively, our study suggests that PPM1A plays an important role in controlling BMP signaling through catalyzing Smad dephosphorylation.

TGFbeta signaling controls diverse normal developmental processes and pathogenesis of diseases including cancer and autoimmune and fibrotic diseases. TGFbeta responses are generally mediated through transcriptional functions of Smads. A key step in TGFbeta signaling is ligand-induced phosphorylation of receptor-activated Smads (R-Smads) catalyzed by the TGFbeta type I receptor kinase. However, the potential of Smad dephosphorylation as a regulatory mechanism of TGFbeta signaling and the identity of Smad-specific phosphatases remain elusive. Using a functional genomic approach, we have identified PPM1A/PP2Calpha as a bona fide Smad phosphatase. PPM1A dephosphorylates and promotes nuclear export of TGFbeta-activated Smad2/3. Ectopic expression of PPM1A abolishes TGFbeta-induced antiproliferative and transcriptional responses, whereas depletion of PPM1A enhances TGFbeta signaling in mammalian cells. Smad-antagonizing activity of PPM1A is also observed during Nodal-dependent early embryogenesis in zebrafish. This work demonstrates that PPM1A/PP2Calpha, through dephosphorylation of Smad2/3, plays a critical role in terminating TGFbeta signaling.