TGFβ-stimulated Smad1/5 phosphorylation requires the ALK5 L45 loop and mediates the pro-migratory TGFβ switch

Liu, Irwin M.; Schilling, Stephen H.; Knouse, Kristin A.; Choy, Lisa; Derynck, Rik; Wang, Xiaofan

doi:10.1038/emboj.2008.266

Cited by 157 publications

(135 citation statements)

References 35 publications

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“…Significantly, we found that in these cell lines TGF␤ leads to Smad1 phosphorylation independently of ALK1/2/3/6, exclusively relying on the ALK4/5/7 subset as a type I receptor. This is in line with a study published while our manuscript was in revision, which revealed ALK5-mediated TGF␤-induced Smad1 phosphorylation in mammary epithelial cells (12). Our data provide firm evidence that TGF␤-induced Smad1 phosphorylation can occur independently of involvement of one of its canonical BMP type I receptors.…”

supporting

confidence: 80%

“…While our manuscript was under revision, Liu et al (12) reported that TGF␤ could induce Smad1 phosphorylation in the mouse mammary epithelial cell line 4T1. This phosphorylation event was strictly dependent on ALK5 kinase activity, and the authors rule out involvement of BMP receptors using short hairpin RNA.…”

Section: Discussionmentioning

confidence: 99%

“…In Vitro Kinase Assays-Assays were performed largely as previously described (12). In brief, His-caALK5 was immunoprecipitated from transfected HEK293T cells after lysis in FLAG lysis buffer (25 mM Tris-HCl (pH 7.5), 300 mM NaCl, 1% Triton X-100, with protease inhibitors (aprotinin, leupeptin, and phenylmethylsulfonyl fluoride) and phosphatase inhibitors (sodium fluoride and Na 3 VO 4 )).…”

Section: Methodsmentioning

confidence: 99%

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

Wrighton

Lin

et al. 2009

Journal of Biological Chemistry

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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supporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

Wrighton

Lin

et al. 2009

Journal of Biological Chemistry

114

126

View full text Add to dashboard Cite

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“…Conversely, signals derived from the BMP family of cytokines use BMPR2 and a host of type I receptors (ALK1, -2, -3, and -6) to activate SMAD1/5/8. However, noncanonical signals linking TGF-β1 to SMAD1/5 have been recently described in endothelial and epithelial tissues (12)(13)(14)(15). Because miR-155 specifically targets SMAD5, we investigated whether this alternative route was active in malignant B lymphocytes.…”

Section: Smad5mentioning

confidence: 99%

“…The noncanonical TGF-β1 signaling in endothelial and epithelial cells requires both BMP and TGF-β type I receptors (12)(13)(14)(15). To study this issue in DLBCL, we first measured the expression of ALK1-7, TGFRB2, and BMPR2.…”

Section: Smad5mentioning

confidence: 99%

Targeting of SMAD5 links microRNA-155 to the TGF-β pathway and lymphomagenesis

Rai¹,

Kim²,

McKeller³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

187

164

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The mechanisms by which microRNA dysfunction contributes to the pathogenesis of diffuse large B cell lymphoma (DLBCL) are not well established. The identification of the genes and pathways directly targeted by these small regulatory RNAs is a critical step to advance this field. Using unbiased genome-wide approaches in DLBCL, we discovered that the oncogenic microRNA-155 (miR-155) directly targets the bone morphogenetic protein (BMP)-responsive transcriptional factor SMAD5. Surprisingly, we found that in DLBCL a noncanonical signaling module linking TGF-β1 signals to SMAD5 is also active. In agreement with these data, miR-155 overexpression rendered DLBCLs resistant to the growth-inhibitory effects of both TGF-β1 and BMPs, via defective induction of p21 and impaired cell cycle arrest. In confirmatory experiments, RNAi-based SMAD5 knockdown recapitulated in vitro and in vivo the effects miR-155 overexpression. Furthermore, in primary DLBCLs, miR-155 overexpression inhibited SMAD5 expression and disrupted its activity, as defined by individual and global analyses of its transcriptional targets. Together, our data helped explain miR-155 function, highlighted a hitherto unappreciated role of SMAD5 in lymphoma biology, and defined a unique mechanism used by cancer cells to escape TGF-β's growth-inhibitory effects.lymphoma | miR-155 | transforming growth factor β | B lymphocytes | bone morphogenetic protein S ignificant progress has been made recently in elucidating the molecular basis of diffuse large B cell lymphoma (DLBCL) (1). These studies were primarily centered on classic mRNA genes, whereas the role of microRNAs (miRNA) in lymphomagenesis remains to be fully appreciated. MiRNAs are non-protein-coding RNAs that function by regulating the expression of target transcripts (2). Thus, to capture the biologic impact of these regulatory molecules it is necessary to identify the genes that they inhibit. Micro-RNA-155 (miR-155) is overexpressed in aggressive DLBCLs (3, 4), and its aberrant expression in a transgenic, Eμ-miR-155, mouse model was associated with the development of lymphoblastic leukemia/high-grade lymphoma (5). However, although several bona fide miR-155 targets have been identified (6-10), it is unclear how their excessive down-regulation in DLBCLs that overexpress miR-155 contribute to lymphomagenesis. A direct way to address this problem is to propose that miR-155 physiologically targets B cell growth-suppressing pathways and that its abnormal expression in DLBCL curtails these inhibitory signals, contributing to an advantageous prosurvival behavior.Herein, we report that miR-155 binds to the 3′ UTR of the SMAD5 gene. Detailed characterization of this interaction showed that genetic modulation of miR-155 expression in DLBCL cell lines concomitantly changed SMAD5 levels. Although SMAD5 activity is classically associated with signals transduced by the BMP (bone morphogenetic protein) family of cytokines (11), we found that in DLBCL TGF-β1 also activated SMAD5. Thus, DLBCL cell lines engineered to expres...

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Dynamic analysis of BMP‐responsive smad activity in live zebrafish embryos

Laux

Febbo

Roman

2011

Developmental Dynamics

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Bone morphogenetic proteins (BMPs) are critical players in development and disease, regulating such diverse processes as dorsoventral patterning, palate formation, and ossification. These ligands are classically considered to signal via BMP receptor-specific Smad proteins 1, 5, and 8. To determine the spatiotemporal pattern of Smad1/5/8 activity and thus canonical BMP signaling in the developing zebrafish embryo, we generated a transgenic line expressing EGFP under the control of a BMP responsive element. EGFP is expressed in many established BMP signaling domains and is responsive to alterations in BMP type I receptor activity and smad1 and smad5 expression. This transgenic Smad1/5/8 reporter line will be useful for determining ligand and receptor requirements for specific domains of BMP activity, as well as for genetic and pharmacological screens aimed at identifying enhancers or suppressors of canonical BMP signaling.

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TGFβ-stimulated Smad1/5 phosphorylation requires the ALK5 L45 loop and mediates the pro-migratory TGFβ switch

Cited by 157 publications

References 35 publications

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

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

Targeting of SMAD5 links microRNA-155 to the TGF-β pathway and lymphomagenesis

Dynamic analysis of BMP‐responsive smad activity in live zebrafish embryos

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