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. 2004 Jan 6;101(1):99-104.
doi: 10.1073/pnas.0307598100. Epub 2003 Dec 22.

Activation of Smad transcriptional activity by protein inhibitor of activated STAT3 (PIAS3)

Jianyin Long  1 , Guannan Wang, Isao Matsuura, Dongming He, Fang Liu
Affiliations

Activation of Smad transcriptional activity by protein inhibitor of activated STAT3 (PIAS3)

Jianyin Long et al. Proc Natl Acad Sci U S A. .

Abstract

Smad proteins play pivotal roles in mediating the transforming growth factor beta (TGF-beta) transcriptional responses. We show in this report that PIAS3, a member of the protein inhibitor of activated STAT (PIAS) family, activates TGF-beta/Smad transcriptional responses. PIAS3 interacts with Smad proteins, most strongly with Smad3. PIAS3 and Smad3 interact with each other at the endogenous protein level in mammalian cells and also in vitro, and the association occurs through the C-terminal domain of Smad3. We further show that PIAS3 can interact with the general coactivators p300/CBP, the first evidence that a PIAS protein can associate with p300/CBP. In contrast, PIASy, which inhibits Smad transcriptional activity and other transcriptional responses, is unable to interact with p300/CBP. The RING domain of PIAS3 is essential for interaction with p300/CBP, and a RING domain mutant PIAS3, which cannot bind p300/CBP, no longer activates TGF-beta/Smad-dependent transcription. Furthermore, we show that PIAS3, Smad3, and p300 can form a ternary complex, which is markedly increased by TGF-beta treatment. Taken together, our studies indicate that on TGF-beta treatment, PIAS3 can form a complex with Smads and p300/CBP and activate Smad transcriptional activity.

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Figures

Fig. 1.
Fig. 1.
PIAS3 can activate Smad transcriptional activity. (A) Different PIAS proteins have distinct effects on the SBE4-Luc reporter gene activity. HaCaT cells were cotransfected with the SBE4-Luc reporter gene and a Flag-PIAS plasmid as indicated. Cells were treated with or without TGF-β and analyzed for luciferase activity. (B) The short and long forms of PIAS3 have comparable activity to stimulate the SBE4-Luc reporter gene. (C) PIAS3 can activate the transcriptional activity of GAL4-Smad fusion proteins. HaCaT cells were cotransfected with the GAL4-Luc reporter gene along with the GAL4 DNA-binding domain (DBD), GAL4-Smad2, GAL4-Smad3, or GAL4-Smad4 in the absence or presence of Flag-PIAS3 plasmid, treated with or without TGF-β, and analyzed for luciferase activity. (D) The stimulatory effect of PIAS3 occurs through the transcriptional activation domains of Smad proteins.
Fig. 2.
Fig. 2.
Interaction of PIAS3 with Smad3 and other Smad proteins. (A) Analysis of PIAS3 interaction with Smad2, Smad3, and Smad4 by immunoprecipitation (IP)-immunoblot assay in COS cells. TβRI (T204D) was cotransfected for TGF-β stimulation. Lysate controls are shown for expression levels of Smads. (B) PIAS3 interacts with the C-terminal domain of Smad3. FL, full length; N, N-terminal domain; LC, linker and C-terminal domain; C, C-terminal domain. (C) The RING domain of PIAS3 is involved in the interaction with Smad3. (Upper) Different domains of PIAS3 were cotransfected with Smad3 (199–424) into COS cells and analyzed as indicated. (Lower) The structures of the various PIAS3 deletion mutants. (D) 35S-labeled PIAS3 synthesized by in vitro translation (IVT) binds to GST-Smad proteins. (E) Endogenous Smad3 and Smad2 interact with PIAS3. HaCaT cells were treated with or without 200 pM TGF-β for 1 h. Cell lysates were immunoprecipitated with a goat antibody that specifically recognizes Smad3 and Smad2, followed by immunoblot with a rabbit PIAS3 antibody. The immunoblot was exposed for half an hour in the enhanced chemiluminescence (ECL, Amersham Biosciences) detection assay to obtain a strong signal for the interaction.
Fig. 3.
Fig. 3.
The RING domain of PIAS3 is essential for interaction with p300/CBP. (A) PIAS3 can interact with p300/CBP. Myc-PIAS3 along with p300-HA or CBP-HA and TβRI (T204D) for TGF-β stimulation was cotransfected into 293T cells. (B) The RING domain of PIAS3 can interact with p300. (C) The RING domain mutant PIAS3 cannot bind to p300. COS cells were cotransfected with p300-HA along with Flag-tagged wild-type PIAS3 or RING mutant PIAS3 (C299S, H301A) in the absence or presence of TβRI (T204D) for TGF-β stimulation. (D) The RING domain mutant PIAS3 has a reduced ability to bind Smad3.
Fig. 4.
Fig. 4.
The RING domain mutant PIAS3 cannot activate Smad-dependent transcription. (A) RING domain mutant PIAS3 cannot activate SBE4-luc reporter gene. (Upper) HaCaT cells were cotransfected with the SBE4-Luc reporter gene and increasing amount of wild type or RING mutant (C299S, H301A) of Myc-PIAS3 as indicated. (Lower) A representative immunoblot with the Myc antibody using the transfected extracts from Upper. Higher amount of RING domain mutant PIAS3 plasmids were used to achieve comparable expression as the wild-type PIAS3. (B) RING domain mutant PIAS3 cannot activate GAL4-Smad3 transcriptional activity. HaCaT cells were cotransfected with the GAL4-Luc reporter gene, GAL4-Smad3, and increasing dose of wild type or RING mutant (C299S, H301A) of PIAS3 as indicated. Higher doses of RING domain mutant PIAS3 plasmids were used to obtain comparable expression levels as the wild-type PIAS3. (C) The PIAS3 RING-like domain (amino acids 274–392) functions as a dominant-negative mutant to inhibit full-length PIAS3. (Upper) HaCaT cells were cotransfected with the SBE4-Luc reporter gene and the full-length Flag-PIAS3 along with increasing amount of Myc-PIAS3 (274–392) as indicated. (Lower) Representative immunoblots with the PIAS3 and the Myc antibodies.
Fig. 5.
Fig. 5.
PIAS3, Smad3, and p300 can form a ternary complex. Myc-PIAS3, Flag-Smad3, p300-HA, and TβRI (T204D) for TGF-β stimulation were cotransfected into 293T cells. Ternary complex was detected by sequential immunoprecipitation with Flag and HA beads, followed by immunoblot with the Myc antibody.
Fig. 6.
Fig. 6.
Analysis of PIAS3 and PIASy for interactions with p300 and HDAC1. (A) PIAS3 but not PIASy can interact with p300. Myc-PIAS3 or Myc-PIASy along with p300-HA was cotransfected into 293T cells. Cell lysates were immunoprecipitated with the HA antibody and immunoblotted with the Myc antibody. The part of the membrane containing p300-HA was immunoblotted with the HA antibody to detect the immunoprecipitated p300-HA. (B) Both PIAS3 and PIASy can interact with HDAC1. Myc-PIAS3 or Myc-PIASy along with Flag-HDAC1 was cotransfected into 293T cells. Cell lysates were immunoprecipitated with the Flag antibody, followed by immunoblot with the Flag and the Myc antibodies.
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