Yao-Yun Liang

Direct Interaction of c-Myc with Smad2 and Smad3 to Inhibit TGF-β-Mediated Induction of the CDK Inhibitor p15Ink4B

Summary rynck and Feng, 1997; Massague´ et al., 2000; Roberts and Sporn, 1990). Mutations or deregulation of compo- The c-Myc oncogene has been implicated in the gene- nents of the TGF- growth arrest pathway have beensis of diverse human tumors. Ectopic expression of shown to be responsible for the inability of tumor cells the c-Myc gene in cultured epithelial cells causes re- torespondtoTGF- Ink4B.Inactivationofthegeneforp15 ,sistancetotheantiproliferativeeffectsofTGF- .How- Smad4, Smad2, or TGF- receptors can allow cells to ever, little is known about the precise mechanisms of escape from TGF- control and drive progression in ac-Myc-mediated TGF- resistance. In this study, we specific tumor (Barrett et al., 1996; Eppert et al., 1996; reveal that c-Myc physically interacts with Smad2 and Hahn et al., 1996; Riggins et al., 1997; Schutte et al.,Smad3, two specific signal transducers involved in 1996; for review see Massague´ et al., 2000). TGF- signaling. Through its direct interaction with In human cancers, the c-Myc gene is often amplifiedSmads, c-Myc binds to the Sp1-Smad complex on the

Opposed Regulation of Corepressor CtBP by SUMOylation and PDZ Binding

The transcription corepressor CtBP is often recruited to the target promoter via interaction with a conserved PxDLS motif in the interacting repressor. In this study, we demonstrate that CtBP1 was SUMOylated and that its SUMOylation profoundly affected its subcellular localization. SUMOylation occurred at a single Lys residue, Lys428, of CtBP1. CtBP2, a close homolog of CtBP1, lacked the SUMOylation site and was not modified by SUMO-1. Mutation of Lys428 into Arg (K428R) shifted CtBP1 from the nucleus to the cytoplasm, while it had little effect on its interaction with the PxDLS motif. Consistent with a change in localization, the K428R mutation abolished the ability of CtBP1 to repress the E-cadherin promoter activity. Notably, SUMOylation of CtBP1 was inhibited by the PDZ domain of nNOS, correlating with the known inhibitory effect of nNOS on the nuclear accumulation of CtBP1. This study identifies SUMOylation as a regulatory mechanism underlying CtBP1-dependent transcriptional repression.

Smad6 Recruits Transcription Corepressor CtBP To Repress Bone Morphogenetic Protein-Induced Transcription

ABSTRACT Smad6 and Smad7 are inhibitory Smads induced by transforming growth factor β-Smad signal transduction pathways in a negative-feedback mechanism. Previously it has been thought that inhibitory Smads bind to the type I receptor and block the phosphorylation of receptor-activated Smads, thereby inhibiting the initiation of Smad signaling. Conversely, few studies have suggested the possible nuclear functions of inhibitory Smads. Here, we present compelling evidence demonstrating that Smad6 repressed bone morphogenetic protein-induced Id1 transcription through recruiting transcriptional corepressor C-terminal binding protein (CtBP). A consensus CtBP-binding motif, PLDLS, was identified in the linker region of Smad6. Our findings show that mutation in the motif abolished the Smad6 binding to CtBP and subsequently its repressor activity of transcription. We conclude that the nuclear functions and physical interaction of Smad6 and CtBP provide a novel mechanism for the transcriptional regulation by inhibitory Smads.

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

Bone morphogenetic proteins (BMPs) are secreted polypeptides belonging to the transforming growth factor-β (TGF-β) 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.

Ubiquitination and Proteolysis of Cancer-Derived Smad4 Mutants by SCFSkp2

ABSTRACT Smad4/DPC4, a common signal transducer in transforming growth factor beta (TGF-β) signaling, is frequently inactivated in human cancer. Although the ubiquitin-proteasome pathway has been established as one mechanism of inactivating Smad4 in cancer, the specific ubiquitin E3 ligase for ubiquitination-mediated proteolysis of Smad4 cancer mutants remains unclear. In this report, we identified the SCFSkp2 complex as candidate Smad4-interacting proteins in an antibody array-based screen and further elucidated the functions of SCFSkp2 in mediating the metabolic instability of cancer-derived Smad4 mutants. We found that Skp2, the F-box component of SCFSkp2, physically interacted with Smad4 at the physiological levels. Several cancer-derived unstable mutants exhibited significantly increased binding to Skp2, which led to their increased ubiquitination and accelerated proteolysis. These results suggest an important role for the SCFSkp2 complex in switching cancer mutants of Smad4 to undergo polyubiquitination-dependent degradation.

Phosphatase PPM1A Regulates Phosphorylation of Thr-186 in the Cdk9 T-loop

Cdk9 is the catalytic subunit of a general RNA polymerase II elongation factor known as positive transcription elongation factor b (P-TEFb). The kinase function of P-TEFb requires phosphorylation of Thr-186 in the T-loop of Cdk9 to allow substrates to access the catalytic core of the enzyme. To identify human phosphatases that dephosphorylate the T-loop of Cdk9, we used a Thr-186-phosphospecific antiserum to screen a phosphatase expression library. Overexpression of PPM1A and the related PPM1B greatly reduced Cdk9 T-loop phosphorylation in vivo. PPM1A and Cdk9 appear to associate in vivo as the proteins could be co-immunoprecipitated. The short hairpin RNA depletion of PPM1A resulted in an increase in Cdk9 T-loop phosphorylation. In phosphatase reactions in vitro, purified PPM1A could dephosphorylate Thr-186 both with and without the association of 7SK RNA, a small nuclear RNA that is bound to ∼50% of total cellular P-TEFb. PPM1B only efficiently dephosphorylated Cdk9 Thr-186 in vitro when 7SK RNA was depleted from P-TEFb. Taken together, our data indicate that PPM1A and to some extent PPM1B are important negative regulators of P-TEFb function.

Essential Phosphatases and a Phospho-Degron Are Critical for Regulation of SRC-3/AIB1 Coactivator Function and Turnover

SRC-3/AIB1 is a master growth coactivator and oncogene, and phosphorylation activates it into a powerful coregulator. Dephosphorylation is a potential regulatory mechanism for SRC-3 function, but the identity of such phosphatases remains unexplored. Herein, we report that, using functional genomic screening of human Ser/Thr phosphatases targeting SRC-3s known phosphorylation sites, the phosphatases PDXP, PP1, and PP2A were identified to be key negative regulators of SRC-3 transcriptional coregulatory activity in steroid receptor signalings. PDXP and PP2A dephosphorylate SRC-3 and inhibit its ligand-dependent association with estrogen receptor. PP1 stabilizes SRC-3 protein by blocking its proteasome-dependent turnover through dephosphorylation of two previously unidentified phosphorylation sites (Ser101 and S102) required for activity. These two sites are located within a degron of SRC-3 and are primary determinants of SRC-3 turnover. Moreover, PP1 regulates the oncogenic cell proliferation and invasion functions of SRC-3 in breast cancer cells.

Smad3 mediates immediate early induction of Id1 by TGF-beta.

Id1 is a member of the inhibitor of differentiation (Id) protein family that regulates a wide range of cell functions. Previous studies have shown that expression of the Id1 gene is down-regulated by TGF-β in epithelial cells, whereas it is up-regulated by BMP in a variety of cell types. During our study of the biological function of TGF-β1, we found that Id1 can be strongly up-regulated by TGF-β1 in the human mammary gland epithelial cell line MCF10A. Quantitative real-time RT-PCR has revealed as high as 7.5-fold induction of Id1 mRNA by TGF-β1 in MCF10A cells after 1 h of TGF-β1 stimulation, and this induction does not require de novo protein synthesis. Using Smad knockdown and knockout approaches, we have identified Smad3 as the responsible R-Smad for mediating transcriptional activation of the Id1 gene. Chromatin immunoprecipitation assay confirms that Smad3 and Smad4 bind to the upstream region of the Id1 gene. Our results demonstrate that Smad3, but not Smad2, mediates TGF-β1-dependent early transcriptional induction of Id1.

Editorial NotePPM1A Functions as a Smad Phosphatase to Terminate TGFβ Signaling

(Cell 125, 915–928; June 2, 2006) We, the editors of Cell, published an Editorial Expression of Concern (http://dx.doi.org/10.1016/j.cell.2016.03.038) earlier this year regarding issues raised about Figures 2F, 2H, and 3G of the above article. Dr. Yao-Yun Liang, who performed the experiments in question, claimed to have manipulated his experiments to achieve pre-determined results. The corresponding author, Dr. Xin-Hua Feng, refuted the claims of falsification. In the intervening time, two independent labs have carried out experiments attempting to reproduce the data in question. This Editorial Note is to inform the community about the outcome of this process. Those two labs have now completed their experiments, and their data largely confirm the central conclusions drawn from the original figures. Although this does not resolve the conflicting claims, based on the information available to us at this time, we will take no further action. We would like to thank the independent labs who invested significant time and effort in ensuring the accuracy of the scientific record.

Editorial Expression of ConcernDirect Interaction of c-Myc with Smad2 and Smad3 to Inhibit TGF-β-Mediated Induction of the CDK Inhibitor p15Ink4B

Abstract The c-Myc oncogene has been implicated in the genesis of diverse human tumors. Ectopic expression of the c-Myc gene in cultured epithelial cells causes resistance to the antiproliferative effects of TGF-β. However, little is known about the precise mechanisms of c-Myc-mediated TGF-β resistance. In this study, we reveal that c-Myc physically interacts with Smad2 and Smad3, two specific signal transducers involved in TGF-β signaling. Through its direct interaction with Smads, c-Myc binds to the Sp1-Smad complex on the promoter of the p15 Ink4B gene, thereby inhibiting the TGF-β-induced transcriptional activity of Sp1 and Smad/Sp1-dependent transcription of the p15 Ink4B gene. These results suggest that oncogenic c-Myc promotes cell growth and cancer development partly by inhibiting the growth inhibitory functions of Smads.