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. 2004 Mar;24(5):2169–2180. doi: 10.1128/MCB.24.5.2169-2180.2004

FIG. 2.

FIG. 2.

FIG. 2.

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.