Activation of Smad Transcriptional Activity by Protein Inhibitor of Activated STAT3 (PIAS3)

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

Long, J., Wang, G., Matsuura, I., He, D. and Liu, F. (2004) Proc. Natl. Acad. Sci. USA 101 (1), 99–104.

中興大學生化所蕭育源 TGF-β signalling from cell membrane to nucleus through SMAD proteins

TGF-β Transforming growth factor (TGF- β) regulates a wide variety of biological activities . P P 1.cell growth Smad2/3 2.differentiation Type I Type II 3.apoptosis Receptor complex Smad2/3 P Smad4 cofactor cofactor Smad2/3 P Smad4 1.DNA binding protein Transcriptional complex 2.coactivator P ex.p300/CBP, P/CAF. /3 Activation or repression ad2 Sm Sm ad4 3.corepressor Target gene ex.PIASy 1 SMAD proteins

1.Smad proteins mediating TGF-β transcriptional responses. 2.Smads can activate transcription by recruiting transcriptional coactivators. The Smad family Smad structural domains and functions.

NH2 MH1 Auto-interaction COOH MH2 (no-function)

phosphorylation

NH MH1 MH2 COOH 2 linker 1.MH1 ：DNA binding 2.linker：cofactor interaction 3.MH2 ：protein-protein interaction a.Smad-Smad interaction receptor-regulated Smads(R-Smads) b.Smad-DNA binding cofactor cooperating Smads (Co-Smads) c.Smad-receptor transcription activation inhibitory Smads (Anti-Smads) 2 PIAS protein family

Motives

PIASy can inhibit Smad- interaction mediated transcriptional PIASy Smads responses by interacting with Smads and histone deacetylase inhibit (HDAC).

Target gene PIAS protein family

1. Protein inhibitor of activated STAT (PIAS) 2. Regulating many transcription factors. 3. All PIAS family members possess E3 ligase activity for SUMO.

3 PIAS3 can activate Smad transcriptional activity

Analyze the effects of PIAS proteins on TGF-Smad-dependent transcription. Materials and Methods Luciferase Reporter Gene Assay Plasmid A ??? PIASs expression plasmid PIASs Smads Plasmid B SBE Luc

cotransfected Smad-binding element Target gene ( Luc ) treated TGF-β or not Cell 測Luciferase活性 (ex.PIASy) 4 PIAS3 can activate Smad transcriptional activity

Result: There are two forms of PIAS3 proteins PIAS3 1.short form : 584 aa 2. long form : 619 aa.

activate

inhibit

1.activated：PIAS3 and PIAS1 2.inhibited ：PIASxβ and PIASy no effect ：PIASxβ 3.Two PIAS3 proteins can activate the SBE4-Luc reporter gene. 5 PIAS3 can directly activate Smad- transcriptional activity ??? Luciferase Reporter Gene Assay PIAS3 (GAL4 fusion assay) Smads Gal4 DBD PIASs expression plasmid Gal4 Luc Smads Gal4 DBD

Gal4 Luc

cotransfected

Cell treated TGF-β or not

測Luciferase活性 Result : 1.ability : Smad3 > Smad2 > Smad4 2 .PIAS3 can directly avtivate TGF-β/Smad-dependent transcription。 6 Sumoylation of Smad4 is not required in PIAS3-mediated activation

1.PIAS family proteins possess SUMO E3 ligase activities 2.Smad4 has been shown to be sumoylated . a.K159 in the linker region SUMO b.K113 the N-terminal domain (small ubiquitin-related modifier) Luciferase Reporter Gene Assay

Smads Gal4 DBD

Smad4 (267–552) : no sumoylation sites Smad3 (199–424) Smad2 (231–424) C-terminal domain linker region (part) Result: 1.Sumoylation of Smad4 is not required in PIAS3-mediated activation. 2.Smad2.3.4 ( C-terminal domain and part of the linker region) are activated by PIAS3 . 7 PIAS3 can interact with Smads

Immunoprecipitation and Immunoblot： Result

Smads-Flag Cell Flag-tagged Smads

PIAS3-Myc Myc-tagged PIAS3

treated withTGF-β or not Immunoprecipitation 1.PIAS3 can interact with Smads. beads Myc antibody 2.interection： Smad3 > Smad2 > Smad4 (weak) 3.TGF-βtreatment had little effect. Immunoblot Flag antibody 8 PIAS3 Interacts with Smad3 and other Smads in vivo and in vitro 1.GST Pull-Down Assay in vitro translation 1.PIAS3 binds to all three GST-Smad proteins. (strongest with GST-Smad3 in vitro) 35S 2. PIAS3 also binds well to GST-Smad4 PIAS3 (1) certain inhibitory factors or modifications may prevent Smad4 from binding to PIAS3 in vivo. GST (2) weak interaction between Smad4 and PIAS3 Smads in vivo is mediated through Smad3 and Smad2.

2.Immunoprecipitation and Immunoblot (endogenous)： treated withTGF-β or not Cell Immunoprecipitation Smads goat antibody (Smad3 and Smad2) PIAS3 Immunoblot rabbit PIAS3 antibody 1.TGF-β treatment had little effect on the interaction. 2.endogenous PIAS3 and Smad3/2 interacted with each other at basal state.. 9 Smad3 can interact with PIAS3 by C-terminal domain Immunoprecipitation and Immunoblot 1.Modify Smad3-Flag tag plasmid FL：full length N：N-terminal domain LC：linker and C-terminal domain C：C-terminal domain PIAS3 interaction with the C-terminal domain of Smad3. ( necessary for transcriptional activation)

2.Modify PIAS3-Myc tag plasmid

1 273 392 416 584 SAP domain Ring domain Acidic Serine-rich

1.274–584 and 274–392 that contain the RING domain, associated with Smad3. 2. TGF-β treatment had almost no effect on the interactions with all these deletion mutants (data not show). 10 PIAS3 can interact with the general coactivators p300/CBP PIAS3 activate Smad-dependent transcription by binding to general transcriptional coactivators p300/CBP. General transcriptional coactivators p300/CBP 1.p300/CBP have intrinsic histone acetyltransferase (HAT) activity. 2.Smad3 or Smad2 (C-terminal domains)interaction with p300/CBP. 3.Smad4 interaction with p300/CBP by SAD domain (linker region). Immunoprecipitation and Immunoblot Cell treated withTGF-β or not Myc-tagged PIAS3 Immunoprecipitation + HA-beads HA-tagged P300 or Immunoblot HA-tagged CBP Myc-antibody blot Result: 1.PIAS3 can interact with p300 and CBP. 2.TGF-β treatment has little effect on their interactions. 11 PIAS3 can interact with the general coactivators p300/CBP Immunoprecipitation and Immunoblot 1.deletion mutant 274–392 ( RING domain ) 2.mutant PIAS3 (C299S, H301A) (No function RING domain)

Result: 1.The PIAS3 deletion mutant 274–392(RING domain) can bind to p300 on its own. 2.The RING domain mutant PIAS3 cannot bind to p300. 3.Similar results were also obtained for CBP (data not shown). 4.The RING domain mutant PIAS3 has a reduced ability to bind Smad3. 5. RING domain of PIAS3 is essential for interaction with p300/CBP. 12 RING domain of PIAS3 is important for PIAS3-mediated activation

Luciferase Reporter Gene Assay - PIAS3 (WT and Mut-RING domain )

Result: 1.wild-type PIAS3 increased SBE4-luc transcription in a dose-dependent manner. 2.PIAS3 (Mut-RING domain)lost the ability to activate SBE4-Luc transcription. 13 RING domain of PIAS3 is important for PIAS3-mediated activation Luciferase Reporter Gene Assay

Result: 1.PIAS3 (274–392) inhibited the activation function of PIAS3(dose-dependent manner) 2.the PIAS3 (274–392) can function as a dominant- negative mutant.

Binding p300 activation WT PIAS3

PIAS3(274–392) Only RING domain Mut PIAS3 Mut RING domain 14 PIAS3, Smad3, and p300 Can Form a Ternary Complex

Immunoprecipitation and Immunoblot Smad3 PIAS3 P300 Cell P300 Myc-tagged PIAS3 + PIAS3 Smad3 PIAS3 HA-tagged P300 Ternary Complex ? Result: Flag-tagged Smads 1.PIAS3, Smad3, and p300 can form a treated withTGF-β or not ternary complex. 2.ternary complex Immunoprecipitation formation is increased Flag-antibody in the presence of TGF-β treatment. Immunoprecipitation 3. Explain that PIAS3 HA-antibody stimulation of Smad Immunoblot transcriptional activity Myc-antibody blot in a TGF-β-dependent manner. 15 Comparison of the Abilities of PIAS3 and PIASy to Interact with p300 and HDAC1 1.PIAS3 interaction with p300 is important for its activation function. 2.PIASy can inhibit Smad transcriptional activity through recruitment of HDAC1 (histone deacetylase) Coimmunoprecipitation-immunoblot assay Result:

1.PIASy can’t binding to p300. 2. both PIAS3 and PIASy can bind to HDAC1.

The highly specific interaction of PIAS3 with p300 may correlate with its activation function. 16 Summary

1.PIAS3 can activate Smad transcriptional activity. 2.PIAS3 interacts with Smad3 and other Smads in vivo and in vitro. 3.PIAS3 can interact with the general coactivators p300/CBP. 4. RING domain of PIAS3 a.SUMO E3 ligase activity b.important for interaction with p300/CBP and activation of Smad transcriptional activity. 5.PIAS3, Smad3, and p300 can form a ternary complex, and TGF-β treatment increases this complex formation. 6.PIAS3 interacts with p300 and HDAC1, but PIASy can’t interact with p300.

17 Discussion

1.TGF-β a little affected : PIAS3–Smad3 association and the PIAS3–p300 interaction. b.significantly increased : PIAS3–Smad3–p300 ternary complex c. a.and b.results from inducible interaction between Smad3 and p300.

Suggest that PIAS3 stimulates Smad transcriptional activity through formation of a complex with Smad proteins and p300/CBP. 2.Sumoylation Smad3 - not required in PIAS3-mediated activation. p300 - not be a substrate for sumoylation by PIAS3 in our system. others - ????

Whether sumoylation of Smad3 or other targets is necessary for the stimulatory effect of PIAS3 on Smad transcriptional activity remains to be determined.

3.PIAS3 can use multiple modes to regulate transcription. 18 Discussion

4.model

activator repressor ZEB-1/δef1(PIAS3) ZEB-2/SIP1(PIASy) coactivator p300 or P/CAF(p300/CBP) V V corepressor CtBP(HDAC) V X

It remains to be determined whether the displacement model is also used by PIAS3 for its activation function.