Serum response factor is modulated by the SUMO-1 conjugation system.

Serum stimulation leads to activation of the serum response factor (SRF)-mediated transcription of immediate-early genes such as c-fos via various signal transduction pathways. We have previously reported that promyelocytic leukemia protein (PML) is involved in the transcriptional regulation by SRF. PML is one of the well-known substrates for modification by small ubiquitin-related modifier-1 (SUMO-1) and several SUMO-1-modified proteins associate with PML. Here, we report that SRF is modified by SUMO-1 chiefly at lysine(147) within the DNA-binding domain. Substitution of this target lysine for alanine did not affect the translocation of SRF to PML-nuclear bodies. The SRF mutant augmented the transcriptional activity under Rho A-stimulated condition but not under serum-starved condition, suggesting that activated SRF is suppressed by its sumoylation. These data support the transcriptional role of SUMO-1 conjugating system in cellular serum response.

PML-nuclear bodies are involved in cellular serum response.

BACKGROUND: Serum stimulation leads to the activation of various signal transduction pathways in cells, and the resultant signals are integrated into the serum response factor (SRF)-dependent transcription of immediate-early genes such as c-fos. RESULTS: To further characterize this response, we investigated the mechanism which controls serum response transcription in cultured human cells. Frequency of PML (promyelocytic leukaemia)-nuclear bodies (NBs) formation increases shortly after serum stimulation, probably facilitating the interaction of SRF and CBP acetyltransferase at the NBs. PML modulates SRF-mediated c-fos promoter activities upon addition of serum to cells or expression of constitutively active Rho family GTPases. We mapped the region in the SRF that interacts with PML to the C-terminal transactivation domain. An SRF mutant deleted of the transactivation domain neither co-localizes with CBP in NBs nor fulfills its transcriptional role. Under conditions of serum stimulation, the formation of NBs coincides with the immediate-early expression of the endogenous c-fos gene in fibroblasts and in all-trans retinoic acid-treated acute promyelocytic leukaemia NB4 cells. CONCLUSION: These data provide an insight into the involvement of NBs in modulating the transcription of serum-induced immediate-early genes.

The aryl hydrocarbon receptor nuclear transporter is modulated by the SUMO-1 conjugation system.

The aryl hydrocarbon receptor nuclear transporter (ARNT) is a member of the basic helix-loop-helix/PAS (Per-ARNT-Sim) family of transcription factors, which are important for cell regulation in response to environmental conditions. ARNT is an indispensable partner of the aryl hydrocarbon receptor (AHR) or hypoxia-inducible factor-1alpha. This protein is also able to form homodimers such as ARNT/ARNT. However, the molecular mechanism that regulates the transcriptional activity of ARNT remains to be elucidated. Here, we report that ARNT is modified by SUMO-1 chiefly at Lys(245) within the PAS domain of this protein, both in vivo and in vitro. Substitution of the target lysine with alanine enhanced the transcriptional potential of ARNT per se. Furthermore, green fluorescent protein-fused ARNT tended to form nuclear foci in approximately 20% of the transfected cells, and the foci partly colocalized with PML nuclear bodies. PML, one of the well known substrates for sumoylation, was found to augment the transcriptional activities of ARNT. ARNT bound AHR or PML, whereas the sumoylated form of ARNT associated with AHR, but not with PML, resulting in a reduced effect of PML on transactivation by ARNT. Our data suggest that the sumoylation of ARNT modulates its transcriptional role through affecting the ability of ARNT to interact with cooperative molecules such as PML. This exemplifies a crucial role of protein sumoylation in modulating protein-protein interactions.

Cooperation of HECT-domain ubiquitin ligase hHYD and DNA topoisomerase II-binding protein for DNA damage response.

Ubiquitin ligases define the substrate specificity of protein ubiquitination and subsequent proteosomal degradation. The catalytic sequence was first characterized in the C terminus of E6-associated protein (E6AP) and referred to as the HECT (homologous to E6AP C terminus) domain. The human homologue of the regulator of cell proliferation hyperplastic discs in Drosophila, designated hHYD, is a HECT-domain ubiquitin ligase. Here we show that hHYD provides a ubiquitin system for a cellular response to DNA damage. A yeast two-hybrid screen showed that DNA topoisomerase IIbeta-binding protein 1 (TopBP1) interacted with hHYD. Endogenous hHYD bound the BRCA1 C-terminus domains of TopBP1 that are highlighted in DNA damage checkpoint proteins and cell cycle regulators. Using an in vitro reconstitution, specific E2 (ubiquitin-conjugating) enzymes (human UbcH4, UbcH5B, and UbcH5C) transferred ubiquitin molecules to hHYD, leading to the ubiquitination of TopBP1. TopBP1 was usually ubiquitinated and degraded by the proteosome, whereas X-irradiation diminished the ubiquitination of TopBP1 probably via the phosphorylation, resulting in the stable colocalization of up-regulated TopBP1 with gamma-H2AX nuclear foci in DNA breaks. These results demonstrated that hHYD coordinated TopBP1 in the DNA damage response.