Abstract
Transforming growth factor β (TGFβ) signaling normally functions to regulate embryonic development and cellular homeostasis. It is increasingly recognized that TGFβ signaling is regulated by cross-talk with other signaling pathways. We previously reported that TGFβ activates protein kinase A (PKA) independent of cAMP through an interaction of an activated Smad3-Smad4 complex and the regulatory subunit of the PKA holoenzyme (PKA-R). Here we define the interaction domains of Smad4 and PKA-R and the functional consequences of this interaction. Using a series of Smad4 and PKA-R truncation mutants, we identified amino acids 290-300 of the Smad4 linker region as critical for the specific interaction of Smad4 and PKA-R. Co-immunoprecipitation assays showed that the B cAMP binding domain of PKA-R was sufficient for interaction with Smad4. Targeting of B domain regions conserved among all PKA-R isoforms and exposed on the molecular surface demonstrated that amino acids 281-285 and 320-329 were required for complex formation with Smad4. Interactions of these specific regions of Smad4 and PKA-R were necessary for TGFβ-mediated increases in PKA activity, CREB (cAMP-response element-binding protein) phosphorylation, induction of p21, and growth inhibition. Moreover, this Smad4-PKA interaction was required for TGFβ-induced epithelial mesenchymal transition, invasion of pancreatic tumor cells, and regulation of tumor growth in vivo.
Highlights
TGF induces a Smad3-Smad4 complex and protein kinase A (PKA)-R interaction
Identification of the Smad4 Region That Interacts with PKA-R—We previously reported that TGF activates PKA through a direct interaction between an activated Smad3Smad4 complex and the PKA regulatory subunit (PKA-R)
We have previously reported that TGF activates PKA in a cAMP-independent manner by direct binding of an activated Smad3-Smad4 complex to the regulatory subunit of the PKA holoenzyme, forming a trimeric complex and resulting in activation of PKA [24]
Summary
TGF induces a Smad3-Smad complex and PKA-R interaction. Results: We define interaction domains between Smad and PKA-R required for TGF-mediated cell growth and EMT regulation. Targeting of B domain regions conserved among all PKA-R isoforms and exposed on the molecular surface demonstrated that amino acids 281–285 and 320 –329 were required for complex formation with Smad4 Interactions of these specific regions of Smad and PKA-R were necessary for TGFmediated increases in PKA activity, CREB (cAMP-response element-binding protein) phosphorylation, induction of p21, and growth inhibition. We demonstrate that the interaction of these defined regions of Smad and the regulatory subunit of PKA was responsible for TGF-mediated increases in PKA activity, CREB phosphorylation, induction of the cell cycle regulatory protein p21Cip, growth inhibition, EMT, and pancreatic cancer cell invasion. This interaction mediated tumor growth in vivo. These findings have significant implications for understanding how TGF signaling may be regulated during normal cellular function and in disease states
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