TGFbeta signaling is initiated when the type I receptor phosphorylates the MAD-related protein, Smad2, on C-terminal serine residues. This leads to Smad2 association with Smad4, translocation to the nucleus, and regulation of transcriptional responses. Here we demonstrate that Smad7 is an inhibitor of TGFbeta signaling. Smad7 prevents TGFbeta-dependent formation of Smad2/Smad4 complexes and inhibits the nuclear accumulation of Smad2. Smad7 interacts stably with the activated TGFbeta type I receptor, thereby blocking the association, phosphorylation, and activation of Smad2. Furthermore, mutations in Smad7 that interfere with receptor binding disrupt its inhibitory activity. These studies thus define a novel function for MAD-related proteins as intracellular antagonists of the type I kinase domain of TGFbeta family receptors.

C/EBP homologous protein (CHOP) is a stress-inducible nuclear protein that is crucial for the development of programmed cell death and regeneration; however, the regulation of its function has not been well characterized. Slbo, a Drosophila homolog of C/EBP (CCAAT/enhancer binding protein), was shown to be unstabilized by tribbles. Here, we identified TRB3 as a tribbles ortholog in humans, which associated with CHOP to suppress the CHOP-dependent transactivation. TRB3 is induced by various forms endoplasmic reticulum (ER) stress later than CHOP. Tunicamycin treatment enhanced the TRB3 promoter activity, while dominant-negative forms of CHOP suppressed the tunicamycin-induced activation. In addition, the tunicamycin response region in the TRB3 promoter contains amino-acid response elements overlapping the CHOP-binding site, and CHOP and ATF4 cooperated to activate this promoter activity. Knockdown of endogenous ATF4 or CHOP expression dramatically repressed tunicamycin-induced TRB3 induction. Furthermore, knockdown of TRB3 expression decreased ER stress-dependent cell death. These results indicate that TRB3 is a novel target of CHOP/ATF4 and downregulates its own induction by repression of CHOP/ATF4 functions, and that it is involved in CHOP-dependent cell death during ER stress.

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Smad proteins are crucial for the intracellular signaling of transforming growth factor-b (TGF-b). Upon their receptor-induced activation, Smad proteins are phosphorylated and translocated to the nucleus to activate the transcription of a select set of target genes. Here, we show that the co-activator p300/CBP bound and acetylated Smad3 as well as Smad2 in vivo, and that the acetylation was stimulated by TGF-b. A major acetylation site of Smad3 by p300/CBP is Lys-378 in the MH2 domain (Smad3C) known to be critical for the regulation of transcriptional activity. Replacement of Lys-378 with Arg decreased the transcriptional activity of GAL4-Smad3C in a luciferase assay. Moreover, p300/CBP potentiated the transcriptional activity of GAL4-Smad3C, but not the acetylation-resistant GAL4-Smad3C(K378R) mutant. These results suggest that acetylation of Smad3 by p300/CBP regulates positively its transcriptional activity.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor ␤ (TGF␤) family of growth factors performing multiple essential functions in embryos and adult metazoans (19,20). The BMP receptors initiate a cascade of Ser/Thr phosphorylation upon binding of BMPs, starting with the BMP type II receptor (BMRPII) kinase phosphorylating and activating the BMPRI kinase, which phosphorylates and activates downstream signal transducers, the Smad proteins, and thus modulates gene expression (2,3,7,30,31). Several proteins are known as specific negative regulators of the BMP signaling pathway: secreted BMP antagonists (chordin and noggin), antagonistic Smads (Smad6 and Smad7), and the E3 ubiquitin ligase for BMP-specific Smad proteins (Smurf1) (3,7,30).Heterozygous mutations of the gene encoding BMPRII have been identified among patients with idiopathic pulmonary artery hypertension (IPAH), a disease characterized by elevated pulmonary artery (PA) pressure and eventually right ventricular failure (1,10,28,36,46). PAs from IPAH patients are characterized by increased muscularization of small arteries and thickening or fibrosis of the intima, resulting in obstruction of PAs and increased pressure. It was also reported that a mutant form of BMPRII found in IPAH, when overexpressed in a smooth muscle cell (SMC)-restricted fashion in transgenic mice, causes an increase in PA pressure and pulmonary arterial muscularization, suggesting that BMPRII mutation in SMCs is sufficient to produce pulmonary hypertension (48). Several BMPRII mutations are found within the extracellular and the kinase domains as well as the long carboxyl (C)-terminal "tail" domain (BMPRII-TD), which has neither enzymatic activity nor homology to known domains (28,46). The finding of BMPRII-TD mutations in IPAH suggests that BMPRII-TD might have a regulatory role in the BMP pathway. It has been shown that BMPRII-TD participates in mediating BMP-induced apoptosis in PA SMCs (PASMCs) (24, 37). Several proteins have been shown to interact with BMPRII-TD, including LIM kinase 1 (LIMK1) and 25,29), but little is known about the role of these proteins in the regulation of the BMP signaling pathway. Recently, it has been found that BMPRII-TD interacts with Eps15R, a component of clathrin-coated pits (CCPs) that regulates endocytosis of the BMP receptor complex and affects downstream signaling (15). It is unclear whether endocytosis of the BMP receptors via CCPs plays a role in the regulation of the BMP pathway in vascular cells.In a search for novel BMPRII-TD-interacting proteins, we isolated the mammalian homolog of Drosophila Tribbles, Trb3 (5, 17, 33). Trb3 contains a variant Ser/Thr kinase domain which lacks the key residues for catalytic activity and ATP binding domain (e.g., DLKLRK in Trb3 instead of DLKPEN) (33). Thus, it is believed to be a nonfunctional kinase. In Drosophila, tribbles appears to play a role in the regulation of the cell cycle and cell migration by inhibiting string and slbo, the homologs of CDC25 and C/EBP, respectively ...