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Control of Smad7 Stability by Competition Between Acetylation and Ubiquitination

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Control of Smad7 Stability by Competition Between Acetylation and Ubiquitination Molecular Cell, Vol. 10, 483–493, September, 2002, Copyright 2002 by Cell Press Control of Smad7 Stability by Competition between Acetylation and Ubiquitination Eva Gro¨ nroos, Ulf Hellman, Carl-Henrik Heldin, 1998), where it binds the receptors and inhibits further and Johan Ericsson1 signaling by at least two different mechanisms. First, Ludwig Institute for Cancer Research Smad7 is able to interfere with TGF␤ signaling by Box 595 blocking the interactions between the R-Smads and the Husargatan 3 activated receptors (Hayashi et al., 1997; Nakao et al., S-751 24 Uppsala 1997). Second, Smad7 interacts with the E3-ubiquitin Sweden ligases Smurf1 or Smurf2 in the nucleus; after TGF␤ stimulation, the Smad7-Smurf complex translocates from the nucleus to the plasma membrane, where Smurf Summary induces ubiquitination and degradation of the TGF␤ re- ceptors (Ebisawa et al., 2001; Kavsak et al., 2000). Smad proteins regulate gene expression in response Acetylation is a dynamic posttranslational modifica- to TGF␤ signaling. Here we present evidence that tion of lysine residues. Proteins with intrinsic histone Smad7 interacts with the transcriptional coactivator acetyltransferase (HAT) activity act as transcriptional p300, resulting in acetylation of Smad7 on two lysine coactivators by acetylating histones and thereby induce residues in its N terminus. Acetylation or mutation of an open chromatin conformation, which allows the tran- these lysine residues stabilizes Smad7 and protects scriptional machinery access to promoters (Roth et al., it from TGF␤-induced degradation. Furthermore, we 2001). The best characterized HATs are p300, CBP, and demonstrate that the acetylated residues in Smad7 P/CAF (Roth et al., 2001). These proteins interact with also are targeted by ubiquitination and that acetylation a large number of transcription factors, such as the of these lysine residues prevents subsequent ubiquiti- Smads (Feng et al., 1998; Itoh et al., 1998, 2000a; Jank- nation. Specifically, acetylation of Smad7 protects it necht et al., 1998; Nishihara et al., 1998; Pouponnot et al., against ubiquitination and degradation mediated by 1998), as well as components of the basic transcriptional the ubiquitin ligase Smurf1. Thus, our data suggest machinery. Thus, it is believed that HATs are central that competition between ubiquitination and acetyla- integrators of various signaling pathways in the nucleus. tion of overlapping lysine residues constitutes a novel Since the observation that p53 is a direct target for mechanism to regulate protein stability. acetylation by the coactivators p300 and P/CAF (Gu and Roeder, 1997), a number of transcription factors and Introduction nuclear proteins have been found to be modified in this manner (Bannister and Miska, 2000; Kouzarides, 2000; TGF␤ belongs to a superfamily of cytokines that regulate Sterner and Berger, 2000). The functional consequences diverse biological functions, ranging from differentia- of acetylation are diverse and include increased DNA tion, motility, and apoptosis to the inhibition of cell binding (p53 [Gu and Roeder, 1997]; GATA-1 [Boyes growth (Massague and Wotton, 2000; Moustakas et al., et al., 1998]; and MyoD [Polesskaya et al., 2000; Sarto- 2001). Inappropriate regulation of TGF␤ signaling has relli et al., 1999]), decreased DNA binding (HMGI(Y) been implicated in multiple human diseases, such as [Munshi et al., 1998]), increased stability (E2F-1 [Marti- fibrosis, rheumatoid arthritis, and carcinogenesis (Blobe nez-Balbas et al., 2000]), inhibition of nuclear export et al., 2000; Piek and Roberts, 2001). TGF␤ exerts its (importin-␣ and HNF4 [Bannister et al., 2000; Soutoglou cellular effects via binding to type I and type II serine/ et al., 2000]), and changes in protein-protein interactions threonine kinase receptors; within the heterotetrameric (TCF [Waltzer and Bienz, 1998]; ACTR [Chen et al., 1999]; receptor complex, the type II receptor phosphorylates and EKLF [Zhang et al., 2001]). and activates the type I receptor, leading to activation In this paper, we demonstrate that Smad7 is a direct of the Smad signaling pathway (Itoh et al., 2000b; Mas- target of the transcriptional coactivator p300 in vivo and sague and Wotton, 2000; Miyazono et al., 2000; Mousta- in vitro, and we identify the acetylated lysine residues. kas et al., 2001). The TGF␤ type I receptor phosphory- Acetylation of these residues enhances the stability of lates the receptor-regulated Smads (R-Smads; Smad2 Smad7 by interfering with Smurf-mediated degradation. and Smad3), and subsequently, a heteromeric complex Our results identify a mechanism that regulates the ac- with the common-mediator Smad (Co-Smad; Smad4) is tivity of Smad7 and indicate that competition between formed. The Smad complexes then translocate into the ubiquitination and acetylation of common lysine resi- nucleus where they, in cooperation with coactivators dues controls the stability of Smad7. and corepressors, act as transcription factors regulating gene expression. Results The inhibitory Smads (I-Smads; Smad6 and Smad7) are induced by receptor signaling and act as negative Smad7 Interacts with p300 regulators of the pathway. Smad7 resides in the nucleus To analyze if Smad7 is directly associated with compo- in unstimulated cells and translocates to the plasma nents of the transcriptional machinery, we performed membrane following receptor activation (Itoh et al., coimmunoprecipitation experiments in transiently trans- fected 293T cells, using full-length p300 and three differ- 1Correspondence: [email protected] ent Smad7 constructs. Full-length Smad7 and Smad7⌬N Molecular Cell 484 Figure 1. Smad7 Interacts with the Coactiva- tor p300 (A) Myc-Smad7, either full-length or the indi- cated deletion mutants, was expressed in 293T cells in the absence or presence of p300-HA. Following immunoprecipitation with anti-HA antibodies, the samples were resolved by SDS-PAGE, and the presence of coimmunoprecipitated Smad7 was detected by Western blotting with anti-Myc antibodies. The levels of p300-HA and the different Smad7 proteins in the cell lysates were deter- mined by Western blotting. (B) The indicated fragments of p300 were in vitro translated and incubated with GST alone (lanes 2, 5, 8, and 11) or GST-Smad7 (lanes 3, 6, 9, and 12) coupled to glutathione beads. The complexes were washed, and the pro- teins separated by SDS-PAGE. Ten percent of the material used in each pull-down was loaded on the gel (lanes 1, 4, 7, and 10). The gel was dried and the 35S-labeled proteins detected by phosphorimage analysis. (C) Poly-histidine p300-HAT (amino acids 1135–1810) was in vitro translated and incu- bated with GST alone (lane 2) or GST-Smad7, either full-length or the indicated deletion mu- tants (lanes 3–5). The glutathione beads were precipitated and washed, and the precipi- tated proteins were resolved by SDS-PAGE. Ten percent of the material used in each pull-down was loaded on the gel (lane 1). The gel was dried, and the 35S-labeled proteins detected by phosphorimage analysis. (D) HaCaT cell lysates were immunoprecipitated with anti-Gal4 (lane 1) or anti-Smad7 (lane 2) antibodies. The immunoprecipitates were washed, and the proteins separated by SDS-PAGE. Coimmunoprecipitated p300 was detected by Western blotting using anti-p300 antibodies. The amount of Smad7 in the immunoprecipitates was determined with anti-Smad7 antibodies. (amino acids 204–402) interacted strongly with p300, the acetyltransferase activity of p300. In order to test while Smad7⌬C (amino acids 2–261) failed to do so this possibility, 293T cells were transfected with Smad7 (Figure 1A), indicating that the C-terminal domain of alone or together with p300, CBP, or P/CAF, and the Smad7 is required for binding to full-length p300. acetylation of Smad7 was monitored by anti-acetyl ly- To further map the interaction between Smad7 and sine antibodies (Figure 2A). Although CBP and p300 p300, we performed GST pull-down experiments using are homologous and have overlapping functions, p300 GST-Smad7 together with four in vitro translated p300 induced a robust acetylation of Smad7, whereas CBP fragments (amino acids 2–411, 412–1150, 1151–1673, and P/CAF failed to acetylate Smad7 (Figure 2A, lanes and 1674–2414). Full-length Smad7 interacted with all 1–4). CBP and P/CAF were able to acetylate p53, both p300 fragments except the region between amino acids in vivo and in vitro (data not shown), confirming their 412–1150 (Figure 1B). activity toward other substrates. To investigate whether The intrinsic HAT activity of p300 is contained within acetylation of Smad7 is regulated in response to TGF␤ one of the fragments used in Figure 1B (amino acids signaling, the experiment was also performed in the pres- 1151–1673), indicating that Smad7 interacts with the ence of constitutively active TGF␤ receptor (caALK5); HAT domain of p300. To further map this interaction, coexpression of caALK5 decreased the acetylation of the HAT domain of p300 was in vitro translated and Smad7 by p300 (compare lanes 2 and 6 in Figure 2A). used in pull-down experiments with various fragments Smad7 was acetylated by endogenous acetyltransfer- of Smad7. Both full-length and the N-terminal fragment ases when expressed in HaCaT cells (Figure 2B). Inter- of Smad7 interacted with the HAT domain of p300, estingly, RNAi-mediated inactivation of endogenous whereas the C-terminal part of Smad7 was unable to p300 blocked this acetylation, demonstrating that p300 interact with this domain of p300 (Figure 1C). This sug-
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