Transforming growth factor-β (TGF-β)/Smads regulate a wide variety of biological responses through transcriptional regulation of target genes. Smad3 plays a key role in TGF-β/Smad-mediated transcriptional responses. Here, we show that the proline-rich linker region of Smad3 contains a transcriptional activation domain. When the linker region is fused to a heterologous DNA-binding domain, it activates transcription. We show that the linker region physically interacts with p300. The adenovirus E1a protein, which binds to p300, inhibits the transcriptional activity of the linker region, and overexpression of p300 can rescue the linker-mediated transcriptional activation. In contrast, an adenovirus E1a mutant, which cannot bind to p300, does not inhibit the linker-mediated transcription. The native Smad3 protein lacking the linker region is unable to mediate TGF-β transcriptional activation responses, although it can be phosphorylated by the TGF-β receptor at the C-terminal tail and has a significantly increased ability to form a heteromeric complex with Smad4. We show further that the linker region and the C-terminal domain of Smad3 synergize for transcriptional activation in the presence of TGF-β. Thus our findings uncover an important function of the Smad3 linker region in Smad-mediated transcriptional control.
Smad4 plays a key role in TGF-β (transforming growth factor β)/Smad-mediated transcriptional responses. We show that Smad4 is sumoylated both in vivo and in vitro . Recent studies showed that sumoylation of Smad4 regulated its stability, but the effect of sumoylation on the intrinsic transcriptional activity of Smad4 was not defined. We show that overexpression of SUMO (small ubiquitin-related modifier)-1 and Ubc9 can inhibit a TGF-β-responsive reporter gene, whereas co-transfection with SUMO-1 protease-1 (SuPr-1) can increase the TGF-β response. We show further that mutation of the Smad4 sumoylation sites or co-transfection with SuPr-1 greatly increases Smad4 transcriptional activity. Moreover, direct fusion of SUMO-1 to the sumoylation mutant Smad4 potently inhibits its transcriptional activity. Thus, as it is being rapidly discovered that sumoylation inhibits the activities of many transcription factors, sumoylation also represses Smad4 transcriptional activity. The net effect of sumoylation of Smad4 can therefore be either stimulatory or inhibitory, depending on the target promoter that is analysed.
ArgRS (arginyl-tRNA synthetase) belongs to the class I aaRSs (aminoacyl-tRNA synthetases), though the majority of ArgRS species lack the canonical KMSK sequence characteristic of class I aaRSs. A DNA fragment of the ArgRS gene from Bacillus stearothermophilus was amplified using primers designed according to the conserved regions of known ArgRSs. Through analysis of the amplified DNA sequence and known tRNA Arg s with a published genomic sequence of B. stearothermophilus , the gene encoding ArgRS ( argS ´) was amplified by PCR and the gene encoding tRNA Arg (ACG) was synthesized. ArgRS contained 557 amino acid residues including the canonical KMKS sequence. Recombinant ArgRS and tRNA Arg (ACG) were expressed in Escherichia coli . ArgRS purified by nickel-affinity chromatography had no ATPase activity. The kinetics of ArgRS and cross-recognition between ArgRSs and tRNA Arg s from B. stearothermophilus and E. coli were studied. The activities of B. stearothermophilus ArgRS mutated at Lys 382 and Lys 385 of the KMSK sequence and at Gly 136 upstream of the HIGH loop were determined. From the mutation results, we concluded that there was mutual compensation of Lys 385 and Gly 136 for the amino acid-activation activity of B. stearothermophilus ArgRS.