Protein Kinase A Modulates Transforming Growth Factor-  Signaling through a Direct Interaction with Smad4 Protein (original) (raw)

2013, Journal of Biological Chemistry

Background: TGF␤ induces a Smad3-Smad4 complex and PKA-R interaction. Results: We define interaction domains between Smad4 and PKA-R required for TGF␤-mediated cell growth and EMT regulation. Conclusion: An interaction between Smad4 and PKA-R regulates TGF␤ signaling. Significance: A novel cross-talk mechanism between TGF␤ and PKA signaling is identified that is critical for execution of TGF␤-mediated cellular responses. Transforming growth factor ␤ (TGF␤) signaling normally functions to regulate embryonic development and cellular homeostasis. It is increasingly recognized that TGF␤ signaling is regulated by cross-talk with other signaling pathways. We previously reported that TGF␤ 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 TGF␤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 TGF␤-induced epithelial mesenchymal transition, invasion of pancreatic tumor cells, and regulation of tumor growth in vivo. Transforming growth factor ␤ (TGF␤) is a member of a large family of structurally related proteins that normally functions to regulate embryonic development and cellular homeostasis, including regulation of proliferation, differentiation, matrix production, and apoptosis in a cell and context-specific manner (1-4). Alterations in the TGF␤ signaling pathway may result in human diseases such as developmental disorders, vascular diseases, and cancer. Although TGF␤ acts as a tumor suppressor in the early stage of epithelial carcinogenesis, TGF␤ promotes tumor progression in advanced stages by inducing tumor growth, epithelial mesenchymal transition (EMT), 2 invasion, evasion of immune surveillance, and metastasis. TGF␤ signaling is mediated by two types of transmembrane serine/threonine kinase receptors (type I and II) that bind ligand and transmit signals from the cell surface to the cell interior by activating a signaling cascade that involves Smad proteins, a family of highly conserved intracellular proteins. Smads can be subdivided into three classes based on their functional properties: receptor-regulated Smads (Smad1,-2,-3,-5,-8), a common mediator Smad (Smad4), and the inhibitory Smads (Smad6 and-7) (5-7). Although each Smad has a distinct function, all are composed of highly conserved N-terminal (MH1) and C-terminal (MH2) domains with a variable prolinerich linker region, with exception of the inhibitory Smads, which lack a MH1 domain. The MH1 domain is involved in DNA binding and maintaining the Smad in an inactive conformation. The linker region that connects the MH1 and MH2 domains contains important peptide motifs with binding sites for Smurf (Smad ubiquitination-related factor) ubiquitin ligases, phosphorylation sites for several classes of protein kinases, and specific to Smad4, a nuclear export signal. The MH2 domain participates in the formation of homo-and hetero-oligomers and mediates interactions with other proteins during transcriptional regulation (8-11). Among the R-Smads, Smad2 and Smad3 respond specifically to TGF␤ and activin (12). Upon ligand binding, Smad2 and Smad3 bind directly to the type I receptor and are phosphorylated by TGF␤ receptor complexes at the SSXS motif at the C terminus of the proteins, which results in release from the receptor. The phosphorylated Smads form heteromeric complexes with Smad4 and translocate into the nucleus to regulate gene expression (13, 14). Inside the nucleus, the activated * This work was supported, in whole or in part, by National Institutes of Health Grant DK-061507 (to D. M. S.). □ S This article contains supplemental Figs. S1-S4.