Small C-terminal Domain Phosphatases Dephosphorylate the Regulatory Linker Regions of Smad2 and Smad3 to Enhance Transforming Growth Factor-β Signaling

Wrighton, Katharine H.; Willis, Danielle; Jiang, Long; Liu, Fang; Lin, Xia; Feng, Xin

doi:10.1074/jbc.m607246200

Cited by 95 publications

(106 citation statements)

References 44 publications

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“…Although our manuscript is in revision, Knockaert et al (27) report dephosphorylation of Smad1 by small C-terminal domain phosphatases (SCPs) (27). Similar to the PDPs, overexpression of SCP1-3 has no effect on Smad1 dephosphorylation in mammalian cells, 4 although SCP1-3 clearly dephosphorylates the linker region of Smad2/3 (28). These discrepancies may be explained by the possibility that PDPs and/or SCPs may require additional cofactors or downstream effectors (which is limiting in mammalian cells) to have their full phosphatase activity toward dephosphorylation of the phospho-SXS motif of Smad1.…”

Section: Discussionmentioning

confidence: 72%

Protein Serine/Threonine Phosphatase PPM1A Dephosphorylates Smad1 in the Bone Morphogenetic Protein Signaling Pathway

Duan

Liang

Feng

et al. 2006

Journal of Biological Chemistry

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Bone morphogenetic proteins (BMPs) are secreted polypeptides belonging to the transforming growth factor-␤ (TGF-␤) 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.Bone morphogenetic proteins (BMPs), 3 originally identified by their ability to cause bone differentiation (1), are signaling molecules that belong to the transforming growth factor-␤ (TGF-␤) superfamily. Presently, the biological functions of BMPs have been greatly expanded. BMPs regulate skeletal development as well as many non-osteogenic developmental processes, such as mesoderm patterning, left-right symmetry, neuronal patterning, and hematopoiesis (2-5). Accumulating evidence indicates that BMPs play an important role in the regulation of stem cell properties (3, 6 -8). Signals from BMP ligands are transduced through binding to type I and II receptors on the cell surface, where type II receptors activate type I receptors, which in turn phosphorylate the downstream Smad1, Smad5, and Smad8. The phospho-R-Smads (P-Smads) form a complex with Smad4 and translocate into the nucleus, where they bind to the Smad binding sites and cooperate with other transcription factors to regulate BMP-induced gene expression (5, 9, 10).Despite substantial effort devoted to understanding the actions of BMP/TGF-␤ and Smads, the precise regulation of Smads remains enigmatic. Regulation of Smads can be accomplished via various post-translational mechanisms, including phosphorylation and ubiquitin-dependent modifications (11,12). Among these, BMP-induced phosphorylation of Smad1/ 5/8, which is carried out by the BMP type I receptor (BMPRIA or BMPRIB) and occurs at the C-terminal SXS motif of Smad1/ 5/8, represents the most critical step in Smad signaling. The SXS phosphorylation triggers a cascade of intracellular events from Smad complex assembly in the cytoplasm to transcriptional control in the nucleus.The reversible phosphorylation and dephosphorylation represents a fundamental strategy used by eukaryotic organisms to regulate a battery of...

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Section: Discussionmentioning

confidence: 72%

Protein Serine/Threonine Phosphatase PPM1A Dephosphorylates Smad1 in the Bone Morphogenetic Protein Signaling Pathway

Duan

Liang

Feng

et al. 2006

Journal of Biological Chemistry

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“…1A, right). Baseline effects have been seen following other manipulations of TGF-␤ signaling components (7,12,20,47,55,57,59) and typically follow the same trend as responses to added TGF-␤. This likely reflects normal receptor signaling caused by low levels of TGF-␤ in the serum-supplemented medium (reference 22 and references within) and/or a TGF-␤ autocrine response (56; reviewed in reference 15).…”

Section: Resultsmentioning

confidence: 81%

Integration of Transforming Growth Factor β and RAS Signaling Silences a RAB5 Guanine Nucleotide Exchange Factor and Enhances Growth Factor-Directed Cell Migration

Hu¹,

Milstein²,

Bliss³

et al. 2008

Molecular and Cellular Biology

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Transforming growth factor ␤ (TGF-␤) receptor (T␤R) signaling contributes to normal development as well as tumorigenesis. Here we report that RIN1, a RAB5 guanine nucleotide exchange factor (GEF) and down regulator of receptor tyrosine kinases (RTKs), promotes T␤R signaling through enhanced endocytosis. T␤R activation induces SNAI1 (Snail), a transcription repressor that reduces RIN1 expression, providing a negative feedback mechanism to control T␤R trafficking and downstream signaling. Persistent RAS signaling disrupts this equilibrium by stabilizing SNAI1 protein, resulting in strong silencing of RIN1 and stabilization of RTKs. TGF-␤-induced RIN1 silencing in breast cancer cells prolonged sensitivity to hepatocyte growth factor, a ligand for the MET-type RTK, and enhanced growth factor-directed cell motility. We conclude that in some tumor cells T␤R and RAS signals are integrated through the silencing of RIN1, leading to a reduction in RAB5-mediated endocytosis. These findings shed new light on the basis for distinct interpretations of TGF-␤ signaling by normal versus transformed cells.Cell surface receptor internalization controls the intensity and duration of signaling and, as a consequence, regulates phenotypes such as differentiation, mitosis, and migration (36, 44). The clathrin-dependent endocytosis of receptor tyrosine kinases (RTKs), for example, typically results in down regulation and signal termination. The GTPase RAB5 is a key regulator of early endosome formation and trafficking and hence a control point for receptor function. This small GTPase is itself activated by the guanine nucleotide exchange factor (GEF) RIN1 (50) and other GEFs (9) and inhibited by GTPaseactivating proteins (24,39).Transforming growth factor ␤ (TGF-␤), signaling through a receptor serine/threonine kinase complex (T␤RI/T␤RII or T␤R), has antiproliferative effects on normal epithelial cells (48). Some late-stage tumor cells escape this cytostatic effect and instead respond to TGF-␤ with increased proliferation and enhanced motility that promote metastatic spread (16). Activated T␤R phosphorylates associated SMAD2 and SMAD3 proteins that then accumulate in the nucleus, inducing expression of SNAI1 (Snail) (42), a promoter of cell motility in development and tumor progression (3), and other genes. This signaling pathway is enhanced by the recruitment of effector proteins during clathrin-mediated endocytosis (18,27,38,43). An alternate, clathrin-independent, T␤R internalization pathway targets T␤R to a degradation shunt (46). We describe a dynamic role for RIN1 in T␤R endocytosis and regulation of downstream signal intensity. In addition, we provide evidence that T␤R and RTK signals integrate through down regulation of RIN1, contributing to the profound difference in TGF-␤ response between normal and transformed cells. MATERIALS AND METHODSCell culture, transfections, and transductions. MCF10A, MDA-MB-231, HeLa, and HepG2 cell lines were cultured under standard conditions. Transfections were performed using Polyfect (Qiagen) (M...

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“…Western Blotting and Immunoprecipitation-For direct Western blotting of whole cell lysates, cells were harvested in 2ϫ Laemmli sample buffer. Immunoprecipitation for the detection of exogenous Smad phosphorylation, and caALK5-Smad interactions, was performed essentially as described previously (17). Western blotting was performed using anti-Smad and anti-phospho-Smad antibodies.…”

Section: Methodsmentioning

confidence: 99%

Transforming Growth Factor β Can Stimulate Smad1 Phosphorylation Independently of Bone Morphogenic Protein Receptors

Wrighton

Lin

et al. 2009

Journal of Biological Chemistry

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114

126

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Transforming growth factor-␤ (TGF␤) superfamily ligands control a diverse set of cellular processes by activating type I and type II serine-threonine receptor kinases. Canonical TGF␤ signaling is mediated via the T␤RI/ALK5 type I receptor that phosphorylates Smad2 and Smad3 in their SXS motif to facilitate their activation and subsequent role in transcriptional regulation. Canonical bone morphogenic protein (BMP) signaling is mediated via the ALK1/2/3/6 type I receptors that phosphorylate Smad1, Smad5, and Smad8 in their SXS motif. However, studies in endothelial cells have shown that TGF␤ can also lead to the phosphorylation of Smad1, dependent on ALK1 receptor activity. Here we present data showing that TGF␤ can significantly induce Smad1 phosphorylation in several non-endothelial cell lineages. Additionally, by using chemical inhibitors specific for the TGF␤/activin/nodal (ALK4/5/7) and BMP (ALK1/ 2/3/6) type I receptors, we show that in some cell types TGF␤ induces Smad1 phosphorylation independently of the BMP type I receptors. Thus, TGF␤-mediated Smad1 phosphorylation appears to occur via different receptor complexes in a cell typespecific manner. Transforming growth factor (TGF)2 ␤ superfamily members signal through heteromeric complexes of transmembrane serine/threonine kinase receptors to control diverse developmental processes and the pathogenesis of many diseases. The heteromeric receptor complex usually comprises two type II receptors and two type I receptors, and the receptors are classified based on their structural and functional properties. Type II receptors are constitutively active, and they phosphorylate type I receptors on serine/threonine residues in the GS domain in response to ligand binding. Activated type I receptors then phosphorylate specific downstream receptor-activated Smad (R-Smads) proteins in their distal C-terminal SXS motif, activating them to transduce the signal to the nucleus (1, 2).There are five mammalian type II receptors: T␤RII, ActR-II, ActR-IIB, BMPR-II, AMHR-II, and seven type I receptors, which are referred to as activin receptor-like kinases (ALK) 1-7 (1, 3). Of the ALKs, ALK5 is thought to be specific for TGF␤ ligands, and ALK4 and ALK7 are thought to mediate signaling via nodal and activins (2). In canonical signaling, the activated ALK4/ALK5/ALK7 phosphorylate downstream R-Smads 2 and 3. The remaining ALKs, ALK1/2/3/6, phosphorylate R-Smads 1, 5, and 8 following specific activation by bone morphogenic proteins (BMP) (2). Once activated, R-Smads undergo heterooligomeric complex formation with Smad4, named the common Smad due to its role in all branches of TGF␤ superfamily signaling, and this complex accumulates in the nucleus to regulate gene transcription in conjunction with a variety of transcriptional cofactors (1, 2).The specificity of different ALKs for different Smad proteins is based on the L45 loop and phosphorylated GS motif in the specific type I receptor, and the L3 loop in the MH2 domain of the relevant R-Smad (4, 5). The L45 loops of ALK4/5/7 that pho...

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Small C-terminal Domain Phosphatases Dephosphorylate the Regulatory Linker Regions of Smad2 and Smad3 to Enhance Transforming Growth Factor-β Signaling

Cited by 95 publications

References 44 publications

Protein Serine/Threonine Phosphatase PPM1A Dephosphorylates Smad1 in the Bone Morphogenetic Protein Signaling Pathway

Protein Serine/Threonine Phosphatase PPM1A Dephosphorylates Smad1 in the Bone Morphogenetic Protein Signaling Pathway

Integration of Transforming Growth Factor β and RAS Signaling Silences a RAB5 Guanine Nucleotide Exchange Factor and Enhances Growth Factor-Directed Cell Migration

Transforming Growth Factor β Can Stimulate Smad1 Phosphorylation Independently of Bone Morphogenic Protein Receptors

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