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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.

Polyadenylated RNA was isolated from a mouse pituitary tumor cell line (AtT-20/D16v) which synthesizes and secretes adrenocorticotropic hormone and beta-endorphin. The RNA was used to construct a cDNA library by a double linker technique and the library was screened for pro-opiomelanocortin (POMC) sequences. One recombinant plasmid, pMKSU16, contained a 923-base pair insert comprising the entire POMC-coding sequence as well as 98 bases of 5' noncoding and 5 bases of 3' noncoding sequence. The protein sequence predicted by the cDNA shows mouse POMC to consist of 235 amino acids with seven potential tryptic cleavage sites consisting of pairs of basic amino acid residues. Comparison with the previously published bovine POMC sequence suggests certain regions of POMC are highly conserved between the two species, particularly the regions corresponding to the alpha-, beta-, and gamma-melanocyte-stimulating hormones.

In the anterior pituitary pro-opiomelanocortin (POMC) is the protein precursor to both adrenocorticotropin and beta-lipotropin but in the intermediate pituitary POMC serves as the precursor to alpha-melanocyte-stimulating hormone and beta-endorphin. In addition, POMC expression in the anterior pituitary is inhibited by glucocorticoids but stimulated by corticotropin-releasing factor while POMC expression in the intermediate lobe is not responsive to glucocorticoids but is inhibited by dopamine. In this study we have asked whether tissue-specific processing and regulation of POMC could be related to the presence of more than one POMC gene. We report here that the mouse genome contains two POMC related gene sequences, alpha- and beta-POMC, that alpha-POMC is located on mouse chromosome 12 while beta-POMC is on a different chromosome, probably chromosome 19. Sequencing of phage lambda recombinants containing alpha- and beta-POMC sequences indicated that the alpha-POMC gene in mouse is very similar to the single POMC gene found in human, bovine, and rat genomes. The sequence of the mouse beta-POMC gene is quite different from that of the alpha-POMC gene. One important difference is that the beta-POMC gene has a translation stop signal in place of the first amino acid in beta-endorphin (Tyr). The beta-POMC gene has many features in common with the pseudogene of the beta-globin family.