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Results: 7

1.
FIG. 7.

FIG. 7. From: A Transforming Growth Factor ?-Induced Smad3/Smad4 Complex Directly Activates Protein Kinase A.

Model of how a TGFβ-induced Smad3/Smad4 complex directly activates PKA. TGFβ directly binds to TGFβ RII, which leads to the phosphorylation of TGFβ RI. This phosphorylation activates the RI protein kinase, which then phosphorylates Smad3. Phosphorylated Smad3 binds to Smad4, and this complex binds to the regulatory subunits of PKA (R), leading to the release of catalytic subunits (C) and resulting in the activation of downstream target genes.

Lizhi Zhang, et al. Mol Cell Biol. 2004 March;24(5):2169-2180.
2.
FIG.4.

FIG.4. From: A Transforming Growth Factor ?-Induced Smad3/Smad4 Complex Directly Activates Protein Kinase A.

TGFβ-mediated activation of PKA requires AKAP. (A) An AKAP inhibitor blocks PKA activation by TGFβ. Mv1Lu cells were pretreated with the AKAP inhibitor Ht31 or its control peptide Ht31P, each at a concentration of 25 μM for 30 min, and then 100 pM TGFβ was added for 15 min. PKA assays were performed. Results are expressed as increases over the control from three separate experiments (*, P < 0.05 versus the control). (B) An AKAP inhibitor blocks the formation of a Smad/PKA regulatory subunit complex. Mv1Lu cells were pretreated with the AKAP inhibitor Ht31 or its control peptide Ht31P at a concentration of 25 μM for 30 min, and then 100 pM TGFβ was added for 15 min. Coimmunoprecipitations (IP) were performed as described. IB, immunoblot. (C) A Smad3/Smad4 complex can dissociate PKA holoenzyme in vitro. RIIa2Ca2 PKA holoenzymes were formed and purified as described (10). The activity of RIIa2Ca2 was measured in the presence of 100 nM cAMP, Smad3D protein (1 μM), Smad4 protein (1 μM), or both Smad3D and Smad4 proteins (1 μM concentration of each protein). Results are expressed as increases over the control from three separate experiments (*, P < 0.01 versus the control).

Lizhi Zhang, et al. Mol Cell Biol. 2004 March;24(5):2169-2180.
3.
FIG. 3.

FIG. 3. From: A Transforming Growth Factor ?-Induced Smad3/Smad4 Complex Directly Activates Protein Kinase A.

An activated Smad3/Smad4 complex interacts with the PKA regulatory subunits to activate PKA. (A) Smad3A does not bind to the PKA regulatory subunit in TGFβ-treated cells. Mv1Lu cells were transfected with the vector pCMV5Flag-Smad3 or pCMV5Flag-Smad3A, and cells were treated with 100 pM TGFβ for 15 min. Coimmunoprecipitations (IP) were performed as described. IB, immunoblot. (B) TGFβ does not activate PKA in Smad3 null mouse pancreatic acinar cells. Pancreatic acinar cells were isolated from wild-type and Smad3 null mice and treated with TGFβ (100 pM) for 15 min. In some experiments, acinar cells from Smad3 null mice were infected with an adenovirus expressing wild-type Smad3. PKA assays were performed. Results are expressed as increases over the control from three separate experiments (*, P < 0.05 versus the control). (C) Constitutively active Smad3 (Smad3D) can activate PKA. Mv1Lu cells were either treated with TGFβ (100 pM) for 15 min or transfected with the vector pCMV5Flag-Smad3 or pCMV5Flag-Smad3D for 16 h, and PKA assays were performed. Results are expressed as increases over the control from three separate experiments (*, P < 0.05 versus the control). (D) A Smad3/Smad4 protein complex can bind with RIIα in vitro. One microgram of purified RIIα protein was incubated with 1 μg of purified GST, GSTSmad3D, Smad3D, or GSTSmad4 protein. GST pull-down assays were performed. Immunoblotting was performed by using anti-RIIα, anti-Smad3, and anti-Smad4 antibodies.

Lizhi Zhang, et al. Mol Cell Biol. 2004 March;24(5):2169-2180.
4.
FIG. 6.

FIG. 6. From: A Transforming Growth Factor ?-Induced Smad3/Smad4 Complex Directly Activates Protein Kinase A.

TGFβ-induced p21Cip1 expression and growth inhibition is mediated through PKA activation (A) TGFβ-induced p21Cip1 expression can be blocked by H89 and PKI. Mv1Lu cells were either transfected with a PKI expression vector or pretreated with 3 μM H89 for 30 min. TGFβ (100 pM) was added for 16 h. Western blotting was performed with anti-p21Cip1 antibody. The membrane was stripped and reblotted with anti-β-actin antibody as a loading control. The lower panel represents relative density from three experiments (*, P < 0.05 versus the control). (B) TGFβ-induced p21Cip1 expression can also be blocked by an AKAP inhibitor. Mv1Lu cells were pretreated with 25 μM of either Ht31 or Ht31P for 30 min, and then 100 pM TGFβ was added for 16 h. Western blotting was performed with anti-p21Cip1 antibody. (C) TGFβ-mediated growth inhibition was determined by MTS assay. Mv1Lu cells were grown in 96-well plates at a concentration of 3,000 cells/well in the absence or presence of TGFβ (100 pM) for up to 5 days. Cells were also treated with 3 μM H89 or transfected with a plasmid expressing PKI. A total of 20 μl of MTS-phenozine methosulfate solution (Promega) was added daily, and absorbance was measured by a universal microplate spectrophotometer. Results are expressed as a percentage of the control from three separate experiments.

Lizhi Zhang, et al. Mol Cell Biol. 2004 March;24(5):2169-2180.
5.

FIG. 2. From: A Transforming Growth Factor ?-Induced Smad3/Smad4 Complex Directly Activates Protein Kinase A.

TGFβ activation of PKA is dependent on TGFβ-induced interaction of a Smad3/Smad4 complex with the regulatory subunits of PKA. (A) dnSmad4 blocks TGFβ-induced PKA activation. Mv1Lu cells were transfected with the vector pCMV5dnSmad4 or empty vector for 16 h. Either 100 pM TGFβ or 10 μM forskolin was added for 15 min, and PKA assays were performed. Results are expressed as increases over the control from three separate experiments (*, P < 0.05 versus the control). (B) TGFβ does not activate PKA in Smad4 null cells. EF7(Smad4−/−) cells were transfected with the vector pCMV5Smad4 for 16 h. TGFβ (100 pM) was added to nontransfected and transfected cells for 15 min, and PKA assays were performed. Results are expressed as increases over the control from three separate experiments (*, P < 0.05 versus the control). (C) Smad4 interacts with PKA regulatory subunits but not catalytic subunits. Mv1Lu cells were treated with TGFβ (100 pM) for the indicated times. Cell lysate (500 μg) was used for immunoprecipitation (IP) with 1 μg of anti-PKA RIβ, anti-PKA RIIα, or anti-PKA Cα subunit antibodies, and then the immunoblot (IB) was detected with the indicated antibodies. One microgram of anti-His antibody from the same species and 25 μg of cell lysate served as controls. (D) Smad3, but not Smad2, interacts with PKA regulatory subunits upon TGFβ treatment. Mv1Lu cells were transfected with the vector pCMV5Flag-Smad2 or pCMV5Flag-Smad3, and cells were treated with 100 pM TGFβ for the indicated times. Coimmunoprecipitations were performed as described. (E) PKA regulatory subunits interact with endogenous Smad3. Mv1Lu cells were treated with 100 pM TGFβ for 15 min. Coimmunoprecipitations were performed with anti-PKA RIIα antibody, and the blot was detected with an anti-Smad3 antibody.

Lizhi Zhang, et al. Mol Cell Biol. 2004 March;24(5):2169-2180.
6.
FIG. 1.

FIG. 1. From: A Transforming Growth Factor ?-Induced Smad3/Smad4 Complex Directly Activates Protein Kinase A.

TGFβ activates PKA without increasing intracellular cAMP levels. (A and B) Mv1Lu cells were serum starved (24 h) and were then treated with TGFβ (100 pM) for the indicated time periods (A) or at the indicated doses for 15 min (B). In vitro kinase assays for PKA activity were performed with a biotinylated PKA peptide substrate (Kemptide [LRRASLG]; Promega). A specific PKA inhibitor H89 (3 μM) was used to pretreat some cells for 30 min. PKA activity was also measured in cells transfected with a pcDNA3.0 plasmid which expresses the specific PKA molecular inhibitor PKI. Results are expressed as increases over the control from three separate experiments (*, P < 0.05 versus the control). (C) TGFβ does not increase cAMP. In the presence of the phosphodiesterase inhibitor IBMX (100 μM), cAMP levels were measured in Mv1Lu cells after treatment with TGFβ (100 pM) or forskolin (10 μM) for 15 min by using a Biotrak enzyme immunoassay assay kit (Amersham). Results are from three separate experiments (*, P < 0.05 versus the control).

Lizhi Zhang, et al. Mol Cell Biol. 2004 March;24(5):2169-2180.
7.
FIG. 5.

FIG. 5. From: A Transforming Growth Factor ?-Induced Smad3/Smad4 Complex Directly Activates Protein Kinase A.

TGFβ's ability to activate CREB is dependent on Smads and PKA. (A to C) TGFβ induces CREB DNA binding. Mv1Lu cells were serum starved (24 h) and were treated with TGFβ at the indicated doses for 1 h (A) or with TGFβ (100 pM) for the indicated time periods (B). Nuclear extracts were prepared, and 5 μg of nuclear protein was used to perform EMSAs. Unlabeled cold probe was used as a control. Nuclear extracts were also preincubated with anti-CREB antibody for the supershift assay (C). Results are representative of three different experiments. (D) The phosphorylation of CREB by TGFβ was detected by using an antibody directed against pSer133-CREB. Ten micrograms of nuclear protein was used to perform Western blotting. TGFβ had no effect on total levels of CREB protein. (E) Mv1Lu cells were cotransfected with the CRE-luciferase and LacZ reporter genes for 8 h. The cells were also transfected with a dnSmad4 expression vector or pretreated with 3 μM H89. TGFβ (100 pM) was added for 8 h. Luciferase activity was measured and normalized to LacZ activity. The results are expressed as increases over the control and are from three separate experiments (*, P < 0.05 versus the control). (F) TGFβ does not activate CREB in Smad4 null cells. EF7(Smad4−/−) cells were serum starved for 24 h and 100 pM TGFβ was added for 60 min. In some experiments, EF7 cells were transfected with wild-type Smad4 [EF7(Smad4+/+)]. Western blotting with anti-phospho-CREB antibody was performed. Results are representative of three separate experiments. (G) Dominant negative Smad3 (Smad3A) blocks the CREB activation by TGFβ. Mv1Lu cells were transfected with vector pCMV5Flag-Smad3A for 16 h. TGFβ (100 pM) was added for 60 min. Ten micrograms of nuclear protein was used to perform Western blotting with anti-phospho-CREB antibody.

Lizhi Zhang, et al. Mol Cell Biol. 2004 March;24(5):2169-2180.

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