TGF-beta-induced phosphorylation of Smad3 regulates its interaction with coactivator p300/CREB-binding protein

Abstract

Smads are intermediate effector proteins that transduce the TGF-beta signal from the plasma membrane to the nucleus, where they participate in transactivation of downstream target genes. We have shown previously that coactivators p300/CREB-binding protein are involved in TGF-beta-mediated transactivation of two Cdk inhibitor genes, p21 and p15. Here we examined the possibility that Smads function to regulate transcription by directly interacting with p300/CREB-binding protein. We show that Smad3 can interact with a C-terminal fragment of p300 in a temporal and phosphorylation-dependent manner. TGF-beta-mediated phosphorylation of Smad3 potentiates the association between Smad3 and p300, likely because of an induced conformational change that removes the autoinhibitory interaction between the N- and C-terminal domains of Smad3. Consistent with a role for p300 in the transcription regulation of multiple genes, overexpression of a Smad3 C-terminal fragment causes a general squelching effect on multiple TGF-beta-responsive reporter constructs. The adenoviral oncoprotein E1A can partially block Smad-dependent transcriptional activation by directly competing for binding to p300. Taken together, these findings define a new role for phosphorylation of Smad3: in addition to facilitating complex formation with Smad4 and promoting nuclear translocation, the phosphorylation-induced conformational change of Smad3 modulates its interaction with coactivators, leading to transcriptional regulation.

Figure 1

p300 C-terminal region interacts with Smad3C and Smad4C. (A) p300C interacts with Smad3C. HA-tagged full-length Smad3 and Smad3 C-terminal fragment (aa 199–424) constructs were transfected into COS cells as indicated. Cells were harvested 48 h after transfection, and GST pull-downs were performed using GST-p300M (aa 744-1571) and GST-p300C (aa 1572–2414). The bound proteins were analyzed by immunoblotting with antibodies against HA. The lysates in lanes 1 and 2 represent ∼12% of the amount used for the pull-down. (B) A region between aa 199 and 381 of Smad3 interacts with p300. Smad3 and truncated forms were in vitro-translated using rabbit reticulocyte lysates. Ten microliters of the ³⁵S-labeled proteins were incubated with GST-p300C beads for 2 h at 4°C, and the bound proteins were analyzed by SDS-PAGE followed by fluorography. (C) Smad4C can interact with p300C. HA-Smad4 and Smad4C (aa 266–552) were transfected into COS cells, and the lysates were pulled down with GST-p300C as in A.

Figure 2

TGF-β regulates the interaction of Smad3 and p300 in a temporal and phosphorylation-dependent manner. (A) The time course of interaction between Smad3 and p300 after TGF-β treatment. HaCaT cells were treated with TGF-β for 0–4 h. Total cell lysates were used for the GST-p300C pull-down assay as described in MATERIALS AND METHODS. Bound proteins were separated by SDS-PAGE and immunoblotted with antibodies against Smad3. (B) The association of Smad3 and p300 is phosphorylation dependent. HaCaT cells were treated with TGF-β for 0 and 30 min, and then lysates were treated with phosphatases CIAP and PAP, as described in MATERIALS AND METHODS, and used for the GST-p300C pull-down assay as in A. Bound proteins were separated by SDS-PAGE and immunoblotted with antibodies against Smad3. (C) Endogenous Smad3, but not Smad4, interacts with GST-p300C after TGF-β treatment. HaCaT lysates treated with TGF-β for 0 and 30 min were precipitated by GST-p300C and then immunoblotted with antibodies against Smad3 and Smad4. (D) Smad3 interacts with p300 in vivo. HaCaT cells were incubated with TGF-β for 30 min. Lysate (300 μg) was used for immunoprecipitation using agarose-conjugated antibodies against p300 and then immunoblotted with antibodies against Smad3. Thirty micrograms of lysates were loaded on the gel to shown the correct size of Smad3.

Figure 3

Overexpression of Smad3C has a squelching effect on multiple TGF-β–responsive reporter genes. Three micrograms of HA-tagged Smad3C were cotransfected with 3 μg of different reporter constructs into HaCaT cells as indicated. The total DNA amount was kept constant by adding pCDNA3.

Figure 4

E1A inhibits Smad-dependent transcriptional activation. (A) E1A, but not the p300 binding mutant E1AΔ2–36, blocks transcriptional activation of 3TP-Lux and PAI-1-Luc. HaCaT cells were cotransfected with 3 μg of 3TP-Lux or PAI-1-Luc reporter constructs and 4 μg of the indicated E1A expression constructs. The total amount of DNA was kept constant with the addition of vector control pCDNA3. TGF-β was added 12 h after transfection, and luciferase activity was measured 24 h later. Error bars represent the SD for duplicate transfections in a single experiment. “E1A” stands for wild-type E1A, and “Δ2–36” stands for E1AΔ2–36 mutant. (B) The transcriptional activation induced by Smad3/Smad4 overexpression is inhibited by E1A in a p300-dependent manner. HaCaT cells were cotransfected with 3 μg of PAI-1, 3 μg of Smad3/Smad4, and 4 μg of E1A expression constructs as indicated. The total DNA amount was kept constant with the addition of pCDNA3. After transfection and TGF-β treatment, luciferase activity was measured as above. (C) E1A can compete with Smad3 for interaction with p300. COS-overexpressed HA-tagged Smad3C (aa 199–424) was used to access the ability of Smad3 to interact with p300 in the presence of E1A. Eluted bacterial-produced 6XHis E1A was added in increasing amounts from lanes 2 to 4 to the GST-p300C pull-down reaction. After incubation at 4°C for 2 h, the bound proteins were washed three times with lysis buffer and immunoblotted with antibodies against HA.

Figure 5

Proposed model for Smad-dependent TGF-β signal transduction pathway. TGF-β treatment initiates a receptor kinase cascade that results in the phosphorylation of Smad3. The phosphorylation of Smad3 weakens the interaction between the N and C terminal regions of Smad3, enabling its interaction with Smad4 and subsequent nuclear translocation of the complex. Once in the nucleus, Smad3 is able to associate with p300 because of the phosphorylation-induced unmasking of the p300 interaction region of Smad3. The Smad3-p300/CBP interaction synergizes with AP1-p300/CBP interaction to activate transcription for PAI-1 promoter. The mechanism underlying the TGF-β–induced transactivation of the p21 and p15 promoters may require both the Smad3-p300/CBP interaction and additional signals that may modulate the interaction between Sp1 and the rest of the transcriptional complex.