Control of Smad7 Stability by Competition Between Acetylation and Ubiquitination

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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␤ receptors (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 transfected 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 indicated 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 determined 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 proteins 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 incubated with GST alone (lane 2) or GST-Smad7, either full-length or the indicated deletion mutants (lanes 3–5). The glutathione beads were precipitated and washed, and the precipitated 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- is required for the acetylation of Smad7 in vivo. gested that the N terminus of Smad7 could be a target To map the acetylated lysines in Smad7, we per- for the acetyltransferase activity of p300. formed in vitro acetylation assays using p300 and GST- To determine whether endogenous Smad7 and p300 Smad7, either full-length, Smad7⌬N, or Smad7⌬C. Full- form a complex in vivo, we used anti-Smad7 antibodies length Smad7 and Smad7⌬C were acetylated by p300, to immunoprecipitate Smad7 from HaCaT cell lysates. while p300 failed to acetylate Smad7⌬N, indicating that p300 was detected in the Smad7 immunoprecipitates, the acetylated lysine residues reside in the N terminus indicating that Smad7 and p300 interact in vivo (Figure of Smad7 (Figure 3A). Smad7⌬C interacts poorly with 1D). In contrast, no p300 was detected when an unre- p300 (Figure 1A), and the fact that it is acetylated as lated antibody was used in the immunoprecipitation. efficiently as full-length Smad7 could indicate that the acetylated residues are not fully accessible in full-length The N Terminus of Smad7 Is Acetylated by p300 Smad7 in vitro. In order to identify the acetylated resi- The fact that Smad7 interacted with the HAT domain of dues, we analyzed tryptic fragments of in vitro acet- p300 suggested that Smad7 could be a substrate for ylated GST-Smad7 by mass spectrometry. Compared Acetylation Controls the Stability of Smad7 485

Figure 3. Lysine Residues 64 and 70 in Smad7 Are Targeted by p300-Mediated Acetylation (A) GST-fusion proteins of full-length Smad7, Smad7⌬N, or Smad7⌬C were subjected to in vitro acetylation using p300. After separating the proteins by SDS-PAGE, the gel was dried and exposed to phosphorimage analysis. (B) Amino acid sequence of the acetylated peptides in Smad7 identi- fied by MALDI-TOF-MS. The positions of the acetylated lysines in mouse Smad7 are indicated below each residue. Trypsin cleavage Figure 2. Smad7 Is a Substrate for p300 In Vivo sites are indicated by arrows. (A) Flag-Smad7 was expressed in 293T cells in the absence or presence of the indicated acetyltransferases, either in the absence (lanes 1–4) or presence (lanes 5–8) of constitutively active ALK5 (caALK5). To confirm that K64 and K70 are acetylated, GST- Following immunoprecipitation of Smad7, the samples were re- Smad7 in which lysine residues 64 and 70 were mutated solved by SDS-PAGE, and the acetylation of Smad7 was determined to alanines, either alone (K64A and K70A) or together with anti-acetyl lysine antibodies (␣-AcK). The amount of Smad7 in the immunoprecipitates was determined with anti-Flag antibodies (K64/70A), were generated and used in in vitro acetyla- (␣-Flag). The levels of Gal4-p300, Gal4-CBP, Flag-P/CAF, and HA- tion assays. Mutation of K70 resulted in a minor de- caALK5 in the cell lysates were determined by Western blotting. crease in acetylation (Figure 4A). However, mutation of Flag-Smad7 was expressed in 293T cells in the absence or presence K64 resulted in a significant decrease (50% reduction) of the indicated acetyltransferases in the presence of [3H]acetate. in acetylation, and if both K64 and K70 were mutated, Following immunoprecipitation of Smad7, the samples were re- p300-mediated acetylation of Smad7 was almost com- solved by SDS-PAGE, and the dried gel was exposed to X-ray film (lower panel). pletely prevented. All mutants retained their ability to (B) Myc-Smad7 was expressed in HaCaT cells in the absence or interact with the HAT domain of p300 in GST pull-down presence of siRNA directed against either human p300 (p300 siRNA) experiments (data not shown). or firefly luciferase (GL2 siRNA). Thirty-six hours following transfec- To determine if K64 and K70 are acetylated in vivo, tion, cell lysates were prepared, and Smad7 was immunoprecipi- Smad7, either wild-type or the indicated mutants, was tated with anti-Myc antibodies. The immunoprecipitates were re- expressed in 293T cells alone or together with p300 solved by SDS-PAGE, and the acetylation of Smad7 was determined with anti-acetyl lysine antibodies (␣-AcK). The amount of Smad7 in (Figure 4B). Wild-type Smad7 was acetylated following the immunoprecipitates was determined with anti-Myc antibodies coexpression with p300, and mutation of K64 signifi- (␣-Myc). The amount of p300 in the cell lysates was determined by cantly decreased the acetylation of Smad7, while muta- Western blotting (␣-p300). tion of K70 only had a limited effect (compare lanes 2, 4, and 6 in Figure 4B). However, mutation of both K64 to mock-treated Smad7, the mass spectra of tryptic and K70 prevented acetylation of Smad7 (Figure 4B, fragments of acetylated Smad7 contained two addi- lane 8). When the assay was repeated in the presence of tional peaks (1132.55 and 2469.19 m/z) (data not shown). TSA, a histone deacetylase inhibitor, acetylation of Smad7 The molecular masses of these peaks correspond to was increased, especially Smad7(K64A), demonstrating peptides spanning amino acids 58–67 and 68–93 of that K70 is acetylated and deacetylated in vivo (Figure Smad7, each containing one acetyl group (42 Da) (Figure 4B, lanes 9–16). However, even in the presence of TSA, 3B). Each of these fragments contains one lysine residue no p300-mediated acetylation of Smad7(K64/70A) was that could be modified by acetylation (K64 and K70, seen. This lack of acetylation could not be explained by respectively), suggesting that these residues are targets effects on the interaction between Smad7 and p300, for p300-mediated acetylation in vitro. since Smad7(K64/70A) still interacted with full-length Molecular Cell 486

Figure 4. p300 Acetylates Specific Lysine Residues in Smad7 (A) GST-fusion proteins of wild-type Smad7, Smad7(K64A), Smad7(K70A), or Smad7(K64/70A) were in vitro acetylated in the absence or presence of recombinant p300-HAT. After resolving the reaction products on SDS-PAGE, the gel was dried and exposed to phosphorimage analysis. The minor band seen in lane 9 originates from autoacetylated p300. (B) Flag-Smad7, either wild-type or the indicated lysine mutants, was expressed in 293T cells in the absence or presence of p300-HA. Twenty- four hours post transfection, the cells were left untreated (lanes 1–8) or treated with TSA (200 ng/ml) (lanes 9–16) for 12 hr. Following immunoprecipitation of Smad7, samples were resolved by SDS-PAGE, and the acetylation of Smad7 was detected with anti-acetyl lysine antibodies (␣-AcK). The amount of Smad7 in the immunoprecipitates was determined with anti-Flag antibodies (␣-Flag). The levels of p300- HA in the cell lysates were determined by Western blotting (␣-HA). (C) Myc-Smad7, either wild-type or the K64/70A mutant, 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 coimmunoprecipitated Smad7 was detected by Western blotting (␣-Myc). The levels of p300-HA and the different Smad7 proteins in the cell lysates were determined by Western blotting. (D) Myc-Smad7 was expressed in 293T cells in the absence or presence of p300, either wild-type or HAT-deficient (p300-DY). Following immunoprecipitation, the samples were resolved by SDS-PAGE, and the acetylation of Smad7 was determined with anti-acetyl lysine antibodies (␣-AcK). The amount of Smad7 in the immunoprecipitates was determined with anti-Myc antibodies (␣-Myc). The amount of p300 in the cell lysates was determined by Western blotting (␣-p300). (E) p300, either wild-type (WT) or acetyltransferase-deficient (DY), was expressed in 293T cells. Total cell lysates were used in pull-down experiments with GST alone (lanes 2 and 5) or GST-Smad7 (lanes 3 and 6) coupled to glutathione beads. The complexes were washed, and the proteins separated by SDS-PAGE. Ten percent of the material used in each pull-down was loaded on the gel (lanes 1 and 4). (F) HaCaT cell lysates were immunoprecipitated with anti-Gal4 (lane 1) or anti-Smad7 (lane 2) antibodies. The immunoprecipitates were washed, the proteins were separated by SDS-PAGE, and the acetylation of Smad7 was detected with anti-acetyl lysine antibodies (␣-AcK). The amount of Smad7 in the immunoprecipitates was determined with anti-Smad7 antibodies. p300 in vivo (Figure 4C). In addition, the interaction be- wild-type or the indicated lysine mutants, in the absence tween Smad7 and p300 was not dependent on the ace- or presence of cotransfected p300 (Figure 5A). The tyltransferase activity of p300, since an acetyltransfer- amount of wild-type Smad7 was significantly increased ase-deficient mutant form of p300 (Figure 4D) (Ito et al., in the presence of coexpressed p300 (compare lanes 1 2001) retained its ability to interact with Smad7 (Figure and 2 in Figure 5A). The expression of Smad7(K64A) 4E). was higher than that of wild-type Smad7 (Figure 5A, To confirm that Smad7 is acetylated in vivo, endoge- lanes 1 and 3), and only a weak induction in response nous Smad7 was immunoprecipitated from HaCaT cell to p300 could be observed (lanes 3 and 4). The levels lysates. The anti-Smad7 antibody immunoprecipitated of Smad7(K70A) and the double mutant (K64/70A) were acetylated Smad7, while an unrelated antibody was un- very high in the absence of p300, and no significant able to do so (Figure 4F). increase was observed in response to p300 expression (Figure 5A, lanes 5–8). These results indicate that acet- Acetylation of Smad7 Inhibits Its Degradation ylation by p300 or mutation of K64 and K70 increases Theoretically, acetylation of specific lysines could in- the stability of Smad7. In support of this notion, the crease the stability of a protein, since acetylation would p300-mediated stabilization of Smad7 was dependent prevent ubiquitination of the same lysine residues. In on the acetyltransferase activity of p300, since an acetyl- order to determine if p300 could affect the expression of transferase-deficient mutant form of p300 (p300-DY) Smad7, 293T cells were transfected with Smad7, either was unable to stabilize Smad7 (Figure 5B). As shown Acetylation Controls the Stability of Smad7 487

Figure 5. Acetylation of Smad7 Protects It from caALK5-Induced Degradation (A) Flag-Smad7, either wild-type or the indicated mutants, was expressed in 293T cells in the absence or presence of p300-HA. Thirty-six hours following transfection, cell lysates were prepared and resolved by SDS-PAGE. The levels of Smad7 were detected by Western blotting (␣-Flag). (B) Flag-Smad7 was expressed in 293T cells in the absence or presence of p300, either wild-type or acetyltransferase-deficient (p300-DY). Thirty-six hours following transfection, cell lysates were prepared and resolved by SDS-PAGE. The levels of Smad7 and p300 were determined by Western blotting using anti-Flag (␣-Flag) and anti-p300 (␣-p300) antibodies, respectively. (C) Flag-Smad7, either wild-type or the indicated mutants, was expressed in 293T cells in the absence or presence of HA-caALK5. Thirty-six hours following transfection, cell lysates were prepared and resolved by SDS-PAGE. The levels of Smad7 and caALK5 in the cell lysates were determined by Western blotting using anti-Flag (␣-Flag) and anti-HA (␣-HA) antibodies, respectively. To ensure that equal amounts of protein were loaded in each well, the levels of tubulin in the samples were estimated by Western blotting using anti-tubulin antibodies (␣-Tub). (D) Flag-Smad7, either wild-type or Smad7(K64A), was expressed in 293T cells in the absence or presence of HA-caALK5 and p300-HA. Thirty- six hours following transfection, cell lysates were prepared and resolved by SDS-PAGE. The levels of Smad7 were detected by Western blotting using anti-Flag antibodies (␣-Flag). The levels of p300-HA and HA-caALK5 in the cell lysates were determined by Western blotting (␣-HA). To ensure that equal amounts of protein were loaded in each well, the levels of tubulin in the samples were estimated by Western blotting using anti-tubulin antibodies (␣-Tub). (E) Flag-Smad7 was expressed in 293T cells in the absence or presence of p300, either wild-type or acetyltransferase-deficient (p300-DY). Thirty-six hours following transfection, cells were treated with TGF␤ for 4 hr. Total cell lysates were prepared and resolved by SDS-PAGE. The levels of Smad7 and p300 were determined by Western blotting using anti-Flag (␣-Flag) and anti-p300 (␣-p300) antibodies, respectively. To ensure that equal amounts of protein were loaded in each well, the levels of tubulin in the samples were estimated by Western blotting using anti-tubulin antibodies (␣-Tub). (F) GST-Smad7, either wild-type or the K64/70A mutant, was used as substrate in in vitro ubiquitination reactions in the absence or presence of rabbit reticulocyte lysate (RRL). Where indicated, GST-Smad7 was acetylated prior to the ubiquitination reaction. The samples were resolved by SDS-PAGE, and the ubiquitination of Smad7 was determined with anti-ubiquitin antibodies (␣-Ub). The amount of Smad7 and its acetylation were determined with anti-Smad7 (␣-Smad7) and anti-acetyl lysine (␣-AcK) antibodies, respectively. above, p300-DY is able to interact with Smad7 (Figure 5C), while all three lysine mutants were resistant to the 4E), demonstrating that the interaction between Smad7 effects of caALK5 (Figure 5C, lanes 3–8). To determine and p300 is not sufficient to stabilize Smad7. if p300-mediated acetylation of these lysine residues is To determine if K64 and K70 are involved in regulating able to protect Smad7 from caALK5-mediated degrada- the stability of Smad7 in response to TGF␤ signaling, tion, the experiment described in Figure 5C was re- wild-type Smad7 and the different lysine mutants were peated in the absence or presence of cotransfected expressed in 293T cells in the absence or presence p300 (Figure 5D). Again, cotransfection of caALK5 de- of constitutively active TGF␤ type I receptor (caALK5). creased the levels of Smad7 in the absence of p300 Expression of caALK5 drastically reduced the amounts (compare lanes 1 and 2 in Figure 5D). However, the of wild-type Smad7 (compare lanes 1 and 2 in Figure enhanced level of Smad7 in the presence of p300 was Molecular Cell 488

Figure 6. The Acetylated Lysine Residues in Smad7 Control Its Stability Cos7 cells, transfected with wild-type or mutant Flag-Smad7, were pulse-labeled with [35S]methionine/cysteine and chased for the indicated time periods. 35S-labeled Smad7 was immunoprecipitated, resolved by SDS- PAGE, and quantified by phosphorimage analysis. The experiment was performed both in the absence (left panel) and presence (right panel) of caALK5. The amount of 35S-labeled Flag-Smad7 at each time point is plotted as percent of the amount at the start of the chase. The data represent the average Ϯ SEM of three independent experiments. not reduced by caALK5 (Figure 5D, lanes 3 and 4). The indicating that this mutant interferes with p300-medi- expression of Smad7(K64A) was higher in the absence ated acetylation of Smad7 in a dominant-negative man- of p300 compared to wild-type Smad7, and coexpres- ner. The dominant-negative p300-DY decreased the sion of caALK5 or p300 had no major effects on the expression of Smad7 when the two proteins were coex- expression of this mutant (Figure 5D, lanes 5–8). Trans- pressed in HeLa cells (Figure 7B). Interestingly, this ef- fected Smad7 was degraded in response to TGF␤ treat- fect was dependent on the acetylated lysine residues ment (Figure 5E, lanes 1 and 2). Interestingly, expression of p300 blocked TGF␤-dependent degradation of Smad7, and this effect was dependent on the acetyltransferase activity of p300, since the acetyltransferase- deficient p300-DY was unable to stabilize Smad7 (Figure 5E, lanes 3–6). Our results suggest that acetylation of Smad7 enhances its expression, potentially by interfering with the ubiquitin-proteasome pathway. To test this hypothesis, we used GST-Smad7, either wild-type or K64/70A, in a reconstituted in vitro ubiquitination assay (Figure 5F). Wild-type Smad7 was ubiquitinated in the presence of rabbit reticulocyte lysate, while a similar amount of Smad7(K64/70A) was significantly less ubiquitinated (compare lanes 2 and 4 in Figure 5F). To determine if p300-mediated acetylation is able to affect the ubiquitination of Smad7, the experiment was repeated with acetylated and nonacetylated GST-Smad7 (Figure 5F, lanes 5–10). Interestingly, acetylation of wild-type Smad7 prevented its ubiquitination (compare lanes 6 and 7 in Figure 5F), while no such effect was seen with Smad7(K64/70A) (Figure 5F, lanes 9 and 10). These results suggest that K64 and K70 are targeted by ubiquitination and that p300-mediated acetylation of these residues prevents this process. The results presented in Figure 5 demonstrate that p300 is able to enhance the expression of Smad7, espe- Figure 7. Inhibition of p300 Negatively Affects the Acetylation and cially during caALK5-induced signaling. Based on this Stability of Smad7 observation, we hypothesized that acetylation of lysine (A) Myc-Smad7 was expressed in HeLa cells in the absence or residues 64 and 70 in Smad7 is important for its stability. presence of HAT-deficient p300-DY. Following immunoprecipitation To address this possibility, we determined the half-life of Smad7, the samples were resolved by SDS-PAGE, and the acet- of wild-type and acetylation-deficient (K64A and K70A) ylation of Smad7 was determined with anti-acetyl lysine antibodies ␣ Smad7, both in the absence and presence of caALK5. ( -AcK). The amount of Smad7 in the immunoprecipitates was determined with anti-Myc antibodies (␣-Myc). The amount of p300 in the Both wild-type and mutant Smad7 were relatively stable cell lysates was determined by Western blotting (␣-p300). in the absence of caALK5 expression (Figure 6). How- (B) Flag-Smad7, either wild-type or the K64/70A mutant, was ex- ever, the degradation of wild-type Smad7 was enhanced pressed in HeLa cells in the absence or presence of p300-DY. Thirty- following caALK5 expression, while K64A and K70A six hours following transfection, cell lysates were prepared and were insensitive to caALK5-induced degradation. resolved by SDS-PAGE. The levels of Smad7 and p300 were deter- ␣ Smad7 was acetylated by endogenous acetyltransfer- mined by Western blotting using anti-Flag ( -Flag) and anti-p300 (␣-p300) antibodies, respectively. To ensure that equal amounts of ases when expressed in HeLa cells (Figure 7A). Interest- protein were loaded in each well, the levels of tubulin in the samples ingly, the acetylation of Smad7 was prevented in cells were estimated by Western blotting using anti-tubulin antibodies expressing a HAT-deficient version of p300 (p300-DY), (␣-Tub). Acetylation Controls the Stability of Smad7 489

in Smad7, since the K64/70A mutant was insensitive to the destabilizing activity of p300-DY. These results confirm that p300 is required for the acetylation of Smad7 in vivo and indicate that Smad7 is destabilized and degraded when the activity of this acetyltransferase is compromised in cells.

Acetylation Protects Smad7 against Smurf-Mediated Degradation Smad7 interacts with the ubiquitin ligases Smurf1 and Smurf2, and this interaction furthers the degradation of Smad7. To address the question whether K64 and K70 are involved in Smurf-mediated degradation of Smad7, wild-type and mutant Smad7 were expressed in the absence or presence of Smurf1 (Figure 8A). The levels of Smad7 were decreased in response to Smurf1 expression (compare lanes 1 and 2 in Figure 8A). In contrast, Smad7(K64A), Smad7(K70A), and the double mutant were protected against Smurf-dependent decreases in expression (Figure 8A, lanes 3–8), although all mutants tested still interacted with Smurf1 (data not shown). To investigate if p300 is able to protect Smad7 from Smurf- mediated degradation, the experiment described in Fig- ure 8A was repeated in the absence or presence of cotransfected p300 (Figure 8B). Again, cotransfection of Smurf1 decreased the levels of Smad7 in the absence of p300. In the presence of cotransfected p300, Smurf1 was unable to reduce the levels of Smad7 (compare lanes 3 and 4 in Figure 8B). Transfected Smad7 was ubiquitinated in response to Smurf1 expression in 293T cells (Figure 8C). Interestingly, the Smurf-mediated poly- ubiquitination of Smad7 was blocked when K64 and K70 were mutated. Taken together with the results presented in Figures 5–7, our results suggest that K64 and K70 in Smad7 are targeted by Smurf-mediated ubiquitination and that acetylation prevents this process.

Discussion

Here we present evidence that Smad7 interacts with the transcriptional coactivator p300, resulting in acetylation Figure 8. Acetylation of Smad7 Protects It from Smurf-1-Induced of Smad7 on two lysine residues in its N terminus. Acet- Degradation ylation of these residues stabilizes Smad7 and protects (A) Flag-Smad7, either wild-type or the indicated mutants, was ex- it against TGF␤- and Smurf-dependent degradation, in- pressed in 293T cells in the absence or presence of Flag-Smurf1. dicating that competition between acetylation and ubi- Thirty-six hours following transfection, cell lysates were prepared quitination controls the stability of Smad7. and resolved by SDS-PAGE. The levels of Smad7 and Smurf1 were We demonstrate that Smad7 interacts with several detected by Western blotting (␣-Flag). domains of the coactivator p300. The C-terminal part of (B) Flag-Smad7 was expressed in 293T cells in the absence or presence of Flag-Smurf1 and Gal4-p300. Thirty-six hours following Smad7, containing the MH2 domain, interacted strongly transfection, cell lysates were prepared and resolved by SDS-PAGE. with full-length p300 in vivo (Figure 1). This is similar to The levels of Smad7 and Smurf1 were detected by Western blotting Smad2 and Smad3, which also interact with p300 and/ (␣-Flag). The levels of Gal4-p300 were determined by Western blot- or CBP through their MH2 domains (Feng et al., 1998; ting (␣-Gal4). To ensure that equal amounts of protein were loaded Janknecht et al., 1998; Nishihara et al., 1998). In contrast, in each well, the levels of tubulin in the samples were estimated by ␣ Smad4 has been shown to interact with p300 through Western blotting using anti-tubulin antibodies ( -Tub). (C) Myc-Smad7, either wild-type or K64/70A, and His-tagged ubiqui- a sequence in its linker region (de Caestecker et al., tin were expressed in 293T cells in the absence or presence of Flag- 2000). The R- and Co-Smads interact with the C terminus Smurf1. Cells were treated with 10 ␮M lactacystin for 4 hr prior to of p300 and/or CBP, and we found that this was also lysis. Following immunoprecipitation of Smad7, the samples were true for Smad7. However, Smad7 demonstrated a much resolved by SDS-PAGE, and the ubiquitination of Smad7 detected broader binding specificity and also interacted with with anti-His antibodies (␣-His). The amount of Smad7 in the immu- ␣ N-terminal domains of p300. Acetylation of Smad7 oc- noprecipitates was determined with anti-Myc antibodies ( -Myc). curs at the N terminus of the protein, both in vitro and in vivo (Figures 2 and 3). In vitro, acetylation of Smad7 required only the N terminus of Smad7 and the HAT Molecular Cell 490

domain of p300, suggesting that the interaction of degraded. Further studies are, therefore, necessary to Smad7 with other domains of p300 may serve other determine if competition between ubiquitination and functions. We and others have been unable to demon- acetylation of common lysine residues is responsible strate acetylation of R- or Co-Smads (Fukuchi et al., for the stabilization of E2F1 in response to P/CAF ex- 2001; Itoh et al., 2000a; E.G. and J.E., unpublished data), pression. There have also been reports of a potential making Smad7 the first member of the Smad family correlation between the acetylation and stability of the found to be modified by acetylation. Interestingly, CBP p53 tumor suppressor protein (Ito et al., 2001; Nakamura and P/CAF were unable to acetylate Smad7 in vivo. We et al., 2000; Rodriguez et al., 2000). However, the stabili- were able to reproduce the specificity in acetylation zation was not linked to individual lysine residues in using metabolic labeling of cells with [3H]acetate; incor- p53, and it is unclear if acetylation of p53 is the only poration of [3H]acetate into Smad7 was only detected factor contributing to the observed effects. The issue is in the presence of coexpressed p300 (Figure 2A, lower further complicated by the fact that p53 is destabilized panel). In addition, RNAi-mediated inactivation of en- through its interaction with p300/CBP under certain con- dogenous p300 in HaCaT cells prevented acetylation of ditions (Grossman et al., 1998). Smad7, confirming the role of p300 in the acetylation Further evidence for a link between acetylation and of Smad7 (Figure 2B). A similar pattern was recently ubiquitination of Smad7 comes from our observation reported for the acetylation of p73 following DNA dam- that mutation or acetylation of K64 and K70 protected age (Costanzo et al., 2002). Smad7 against Smurf1-mediated degradation (Figure 8). The acetylation of Smad7 was significantly decreased Members of the Smurf family of E3 ubiquitin ligases following ectopical expression of constitutively active interact with Smads, including Smad7. In the case of ALK5, indicating that the acetylation of Smad7 is nega- Smad7, translocation of the Smad7-Smurf complex from tively regulated by TGF␤ signaling (Figure 2A). The inter- the nucleus to the plasma membrane induces ubiquiti- action between R- and Co-Smads and p300/CBP is en- nation and degradation of the TGF␤ receptors. At the hanced following TGF␤ treatment, and it is possible that plasma membrane, Smad7 is ubiquitinated and de- the interaction between Smad7 and p300 is disrupted graded, while the nuclear Smad7-Smurf complex is sta- following activation of the TGF␤ pathway, since Smad7 ble (Kavsak et al., 2000). Interestingly, we found that is translocated out of the nucleus following activation Smurf-mediated poly-ubiquitination of Smad7 was of the TGF␤ pathway. blocked when K64 and K70 were mutated (Figure 8C). Using a combination of in vitro acetylation assays One possibility is that mutation or acetylation of K64 followed by mass spectrometry analyses and mutations and K70 affects the localization of Smad7 and, thereby, of specific Smad7 lysines in vivo, we could map the its stability. However, we were unable to detect any acetylated residues in Smad7 to K64 and K70 in the N effects on the localization of Smad7 in response to muta- terminus of the protein (Figures 3 and 4). One of these tion of the acetylated lysine residues or as a result of lysine residues, K64, conforms to the consensus site for coexpression of p300 (see Supplemental Figure S1 at acetylation, i.e., a lysine preceded by a glycine or a http://www.molecule.org/cgi/content/full/10/3/483/ serine residue. Both sites were also acetylated in vivo, DC1). Rather, our results suggest that p300-mediated with K64 being the major acceptor site. Interestingly, acetylation of Smad7 protects it against premature the acetylation of Smad7, and in particular K70, was Smurf-mediated degradation while in the nucleus. In greatly enhanced following treatment with the deacety- support of this model, we found that both the acetylation lase inhibitor TSA. This indicates that the acetylation of and stability of Smad7 was decreased in HeLa cells Smad7 is a dynamic process that is regulated by both expressing an acetyltransferase-deficient p300 mutant acetyltransferases and deacetylases. (Figure 7). Mutation or acetylation of K64 and/or K70 blocked The potential link between acetylation/deacetylation the ALK5- and TGF␤-dependent decrease in Smad7 ex- and ubiquitination received further support from the re- pression, indicating that these residues are involved in cent identification of the components of the murine TGF␤-induced degradation of Smad7 (Figure 5). Indeed, HDAC6 complex (Seigneurin-Berny et al., 2001). It was when we determined the half-life of Smad7, we found demonstrated that a number of the proteins in this com- that the degradation of the wild-type protein increased plex are involved in the ubiquitin signaling pathway. significantly following ALK5 expression, while the mu- Active deacetylation of p53 by HDAC1 and Sir2 plays tants were insensitive to ALK5 expression and remained important roles in the regulation of p53-dependent pro- stable (Figure 6). One interpretation of our results is that cesses, including transcription, cell cycle regulation, K64 and K70 confer instability to Smad7, presumably and apoptosis (Luo et al., 2000, 2001; Vaziri et al., 2001). because they are also targeted by ubiquitination. The Future studies will address whether acetylated Smad7 latter hypothesis gained support from our observation is a substrate of HDACs. that mutation or acetylation of K64 and K70 prevented It was recently demonstrated that TGF␤ stimulation ubiquitination of Smad7 in vitro. Martinez-Balbo´ s et al. induces the assembly of Smad2-Smurf2 and Smad3- (2000) demonstrated that the expression levels of E2F1 APC ubiquitin ligase complexes that target SnoN for are enhanced following coexpression with P/CAF. This degradation (Bonni et al., 2001; Stroschein et al., 2001; enhanced expression was dependent on the HAT do- Wan et al., 2001). These observations indicate that cer- main of P/CAF, indicating that acetylation of E2F1 is tain proteins are targeted for degradation through their important for this effect. However, when the acetylated interactions with Smad proteins. It is striking that Smad7 lysines in E2F1 were mutated, the mutant protein did undergoes two different modifications which both target not accumulate. Rather, and in contrast to the case for the same lysine residues. We propose that acetylation Smad7, the lysine-mutated version of E2F1 was rapidly of specific lysines in Smad7 prevents subsequent ubiq- Acetylation Controls the Stability of Smad7 491

uitination of the same residues, thereby blocking protea- ies, washed, and incubated with horseradish peroxidase-coupled some-mediated degradation of Smad7. The ubiquitin- secondary antibodies. After washing, the blots were visualized by proteasome pathway regulates a large number of nu- using Western Blotting Chemiluminescence Luminol Reagent (Santa Cruz). clear proteins, and many of these are also acetylated. Therefore, it will be of utmost importance to determine In Vitro Acetylation Assay if competition between acetylation and ubiquitination is GST-Smad7 (1 ␮g) was incubated for 2 hr at 30ЊC in acetylation a general mechanism to regulate protein stability. buffer (50 mM Tris-HCl [pH 8.0], 10% glycerol, 1 mM DTT, 1 mM 14 MgCl2, 1 mM PMSF, 20 mM sodium butyrate, and 10 nCi [ C]acetyl- Experimental Procedures CoA) in the absence or presence of the HAT domain of p300 (50 ng). To avoid unspecific interactions, the samples were incubated Cell Culture with unlabeled acetyl-CoA (final concentration, 1 mM) for an addi- All tissue culture media and antibiotics were from Invitrogen and tional 15 min. SDS sample buffer was added, and the samples were Sigma. Cells were from ATCC and maintained at 37ЊC in DMEM boiled and separated by SDS-PAGE. The gels were dried and ana- supplemented with 10% fetal calf serum, sodium pyruvate, nones- lyzed by phosphorimage analysis (Fuji). sential amino acids, 50 U/ml penicillin, and 50 ␮g/ml streptomycin, in 5% CO . 2 In Vitro Ubiquitination Assay GST-Smad7, either wild-type or K64/70A, was used as substrate in TGF␤, Reagents, and Antibodies in vitro ubiquitination reactions in the absence or presence of 5 ␮l TGF␤ was a generous gift from N. Ferrara (Genentech). [14C]acetyl- crude rabbit reticulocyte lysate (Promega) as described (Shenoy et CoA (51.6 mCi/mmol) was from ICN. Promix (14 ␮Ci/␮l), peroxidase- al., 2001). Where indicated, GST-Smad7 was acetylated prior to the conjugated sheep anti-mouse and donkey anti-rabbit IgG were from ubiquitination reaction, using recombinant p300-HAT and unlabeled Amersham-Pharmacia Biotech. Trichostatin A (TSA), acetyl-CoA, acetyl-CoA. Reaction mixtures were incubated for 1 hr at 30ЊC. GST- ubiquitin, and anti-Flag antibody (M5) were from Sigma. Monoclonal Smad7 was captured on glutathione beads and washed extensively. anti-Myc (9E10), anti-ubiquitin (P4D1), goat and rabbit anti-Smad7 The samples were resolved by SDS-PAGE and transferred to nitro- (N-19 and H-79), anti-HA (Y-11), anti-p300 (N-15), and anti-Gal4 cellulose membranes, and the ubiquitination of Smad7 was deter- DBD (RK5C1) antibodies were from Santa Cruz. Monoclonal anti- mined by Western blotting. HA (12CA5) antibodies were from Roche. Anti-acetyl lysine antibodies were from Cell Signaling Technology and Upstate Biotechnology. Protein A was obtained from Zymed. Determination of Protein Half-Life Cos7 cells were transfected using the reagents and methods de- Plasmids and DNA Transfections scribed above. 24 hr after transfection, cells were incubated for 1 The expression vectors for Flag- and Myc-tagged Smad7, Smad7⌬N hr with 35S-labeled cysteine and methionine (Promix, 20 ␮Ci/ml) in (amino acids 204–402), and Smad7⌬C (amino acids 2–261) in the methionine- and cysteine-free MCDB medium. The cells were mammalian expression vector pcDNA3 (Invitrogen) were kind gifts washed and incubated in MCDB medium supplemented with 50 from P. ten Dijke (The Netherlands Cancer Institute, Amsterdam). ␮g/ml methionine and cysteine for the indicated time periods. The The corresponding GST-tagged Smad7 constructs were generated immunoprecipitated samples were separated by SDS-PAGE. The by subcloning into pGEX4T-1 (Amersham-Pharmacia Biotech). gels were dried and analyzed by phosphorimage analysis. Smad7 point mutants were generated by site-directed mutagenesis (QuickChange, Stratagene). The various fragments of human p300 Generation of Recombinant Proteins (amino acids 2–411, 412–1150, 1151–1673, and 1674–2414) were and In Vitro Interaction Assays generated by PCR and cloned into HA-pCDNA3. The expression GST-Smad7 fusion proteins and poly-histidine p300-HAT were ex- vector for HA-tagged constitutively active ALK5 has been described pressed in BL21 E. coli and purified according to standard protocols. previously (Nakao et al., 1997). The expression vectors for p300-HA Proteins were in vitro translated using the T7 TNT kit (Promega) and and Flag-P/CAF were kind gifts from T. Kouzarides (Wellcome/CRC 35S-labeled methionine and cysteine (Promix), and incubated with Institute, Cambridge, UK). Gal4-tagged p300 and CBP in the GST-fusion proteins prebound to glutathione beads at 4ЊC for 2 hr. pVR1012 vector (Vical) were from N. Perkins (University of Dundee, The samples were washed extensively and resolved by SDS-PAGE. Dundee, UK). The bacterial expression vector for the poly-histidine- The gel was Commassie stained, incubated in enhancer (Amplify, tagged p300 HAT domain (amino acids 1135–1810) was a kind gift Amersham-Pharmacia Biotech), dried, and analyzed by phosphoi- from Y. Nakatani (Dana-Farber Cancer Institute, Boston, MA). The mage analysis. expression vector for Flag-Smurf1 was a kind gift from K. Miyazono (University of Tokyo, Tokyo, Japan). The expression vector for the acetyltransferase-deficient p300 mutant (p300-DY) was a kind gift Mass Spectrometry and Analysis of Acetylated Lysine Residues from P. Yao (Duke University, Durham, NC). Transient transfections The Coomassie stained band was excised and treated for in-gel were performed using the MBS transfection kit (Stratagene). The digestion as described (Hellman, 2000). The peptide mixture was transfection efficiency was monitored by transfection of pEGFP- analyzed by matrix-assisted laser desorption ionization time of flight C1 (Clontech) and was routinely found to be 90%–95%. Transient mass spectrometry (MALDI-TOF-MS) on a Bruker Biflex III instru- transfections of siRNA were performed using lipofectamine PLUS ment (Bruker) using standard settings for peptide mass fingerprint- (Invitrogen). The p300 siRNA (directed against nucleotides 4751– ing (PMF). The identity of Smad7 was confirmed by PMF using 4773 in human p300; accession number U01877) and the control ProFound. To identify acetylated lysine residues, the peptide mass GL2 siRNA (directed against firefly luciferase) were used according list was imported into GPMAW (version 4.22, Lighthouse Data) where to the manufacturer’s recommendations (Dharmacon Research). the sequence of Smad7 was scanned for the addition of 42 Da on a lysine residue. A similar search for acetylated lysines was also done in ProFound, long form. Immunoprecipitations and Immunoblotting Cells were lysed in ice-cold lysis buffer (50 mM HEPES [pH 7.2], 150 mM NaCl, 1 mM EDTA, 20 mM NaF, 2 mM sodium orthovanadate, 1% Acknowledgments (v/v) Triton X-100, 10% (v/v) glycerol, 1 mM PMSF, 10 mM sodium butyrate, and 1% aprotinin) and cleared by centrifugation. Immuno- We are grateful to P. ten Dijke, N. Perkins, T. Kouzarides, Y. Nakatani, precipitations were carried out by adding the appropriate antibodies N. Ferrara, P. Yao, and K. Miyazono for kindly providing expression plus protein A or protein G Sepharose beads, followed by incubation vectors and reagents and S. Souchelnytskyi, R. Heuchel, and A. at 4ЊC. The immunoprecipitates were washed extensively, resolved Moustakas for stimulating discussions. This work was in part sup- by SDS-PAGE, and transferred to nitrocellulose membranes (Milli- ported by a grant from the Swedish Research Council to J.E. J.E. pore). The membranes were incubated with the appropriate antibod- is a Research Fellow of the Royal Swedish Academy of Sciences. Molecular Cell 492

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