PIAS1 regulates CP2c localization and active promoter complex formation in erythroid cell-specific α-globin expression

Data presented here extends our previous observations on α-globin transcriptional regulation by the CP2 and PIAS1 proteins. Using RNAi knockdown, we have now shown that CP2b, CP2c and PIAS1 are each necessary for synergistic activation of endogenous α-globin gene expression in differentiating MEL cells. In this system, truncated PIAS1 mutants lacking the ring finger domain recruited CP2c to the nucleus, as did wild-type PIAS1, demonstrating that this is a sumoylation-independent process. In vitro, recombinant CP2c, CP2b and PIAS1 bound DNA as a stable CBP (CP2c/CP2b/PIAS1) complex. Following PIAS1 knockdown in MEL cells, however, the association of endogenous CP2c and CP2b with the α-globin promoter simultaneously decreased. By mapping the CP2b- and CP2c-binding domains on PIAS1, and the PIAS1-binding domains on CP2b and CP2c, we found that two regions of PIAS1 that interact with CP2c/CP2b are required for its co-activator function. We propose that CP2c, CP2b, and PIAS1 form a hexametric complex with two units each of CP2c, CP2b, and PIAS1, in which PIAS1 serves as a clamp between two CP2 proteins, while CP2c binds directly to the target DNA and CP2b mediates strong transactivation

Small ubiquitin-like modifier 1 [corrected] mediates the resistance of prosthesis-loosening fibroblast-like synoviocytes against Fas-induced apoptosis

OBJECTIVE: To study the expression of small ubiquitin-like modifier 1 (SUMO-1) in aseptic loosening of prosthesis implants and to investigate its role in regulating the susceptibility of prosthesis-loosening fibroblast-like synoviocytes (FLS) to Fas-induced apoptosis. METHODS: Specimens of aseptically loosened tissue were obtained at revision surgery, and the expression of SUMO-1 was analyzed by in situ hybridization. SUMO-1 levels in FLS were determined by quantitative polymerase chain reaction and Western blot analysis. Immunohistochemistry and confocal microscopy were used to study the subcellular localization of SUMO-1. The functional role of SUMO-1 in Fas-induced apoptosis of prosthesis-loosening FLS was investigated by small interfering RNA-mediated knockdown of SUMO-1 and by gene transfer of the nuclear SUMO-specific protease SENP1. RESULTS: SUMO-1 was expressed strongly in aseptically loosened tissue and was found prominently at sites adjacent to bone. Prosthesis-loosening FLS expressed levels of SUMO-1 similar to the levels expressed by rheumatoid arthritis (RA) FLS, with SUMO-1 being found mainly in promyelocytic leukemia protein nuclear bodies. Knockdown of SUMO-1 had no effect on spontaneous apoptosis but significantly increased the susceptibility of prosthesis-loosening FLS to Fas-induced apoptosis. Gene transfer of the nuclear SUMO-specific protease SENP1 reverted the apoptosis-inhibiting effects of SUMO-1. CONCLUSION: These data suggest that SUMO-1 is involved in the activation of both RA FLS and prosthesis-loosening FLS by preventing these cells from undergoing apoptosis. Modification of nuclear proteins by SUMO-1 contributes to the antiapoptotic effects of SUMO-1 in prosthesis-loosening FLS, providing evidence for the specific activation of sumoylation during their differentiation. Therefore, SUMO-1 may be an interesting target for novel strategies to prevent aseptic prosthesis loosening

Sumoylation of nucleophosmin/B23 regulates its subcellular localization, mediating cell proliferation and survival

Nucleophosmin/B23 is a major multifunctional nucleolar phosphoprotein that plays a critical role in ribosome biogenesis and cell proliferation. Arf tumor suppressor binds B23 and enhances its sumoylation. However, the biological effects of this event remain unknown. Here we show that B23 is sumoylated on both Lysine 230 and 263 residues, but the latter is the major one. Mutation of K263, but not K230, into R abolishes its centrosomal and nucleolar residency. Moreover, Rb binds to wild-type B23, but fails to interact with K263R. Sumoylation enhances B23 binding to Rb. Consequently, B23 potently stimulates E2F1-mediated transcriptional activity, which is abolished in B23 K263R. Further, K263R mutation makes B23 vulnerable to caspase-3 cleavage and sensitizes cells to apoptosis. Surprisingly, K230R mutant strongly binds to phosphatidylinositol-3,4,5-trisphosphate and suppresses DNA fragmentation. Thus, B23 sumoylation regulates its subcellular localization, cell proliferation, and survival activities

Regulation of Neuronal Protein Trafficking and Translocation by SUMOylation

Post-translational modifications of proteins are essential for cell function. Covalent modification by SUMO (small ubiquitin-like modifier) plays a role in multiple cell processes, including transcriptional regulation, DNA damage repair, protein localization and trafficking. Factors affecting protein localization and trafficking are particularly crucial in neurons because of their polarization, morphological complexity and functional specialization. SUMOylation has emerged as a major mediator of intranuclear and nucleo-cytoplasmic translocations of proteins involved in critical pathways such as circadian rhythm, apoptosis and protein degradation. In addition, SUMO-regulated re-localization of extranuclear proteins is required to sustain neuronal excitability and synaptic transmission. Thus, SUMOylation is a key arbiter of neuronal viability and function. Here, we provide an overview of recent advances in our understanding of regulation of neuronal protein localization and translocation by SUMO and highlight exciting areas of ongoing research