Small Ubiquitin-like Modifier 1 Research Articles

Association Between Polymorphisms at Small Ubiquitin-Like Modifier 1 and Nonsyndromic Orofacial Clefts in Western China

Small ubiquitin-like modifier 1 (SUMO1) has been shown to be associated with cleft lip and palate in animal models. However, rarely studies have linked SUMO1 to nonsyndromic cleft lip with or without cleft palate in humans. The purpose of this study was to confirm the contribution of SUMO1 to nonsyndromic orofacial clefts in western Han Chinese. Four single-nucleotide polymorphisms were investigated in 246 case trios in western China using conditional logistic regression, transmission disequilibrium test, and case-parent trio design. Strong evidence of linkage disequilibrium was found between these markers and the disease in both single-nucleotide polymorphism analysis (G allele at rs6761131 [p=0.0008, odds ratio (OR) = 1.92, 95% confidence interval (CI): 1.31-2.81], C allele at rs7580433 [p<0.0001, OR=2.79, 95% CI: 1.93-4.03], and G allele at rs10185956 [p=0.0069, OR=1.61, 95% CI: 1.14-2.27]) and haplotype analysis (A-C for rs6709162-rs7580433 [p=0.00024], C-G for rs7580433-rs10185956 [p=0.00091], and A-C-A for rs6709162-rs7580433-rs10185956 [p=0.004], among others). The risk factors identified in this study may provide a better understanding of the etiological role of SUMO1 in nonsyndromic cleft lip with or without cleft palate incidence.

Sumoylation of ING2 regulates the transcription mediated by Sin3A

ING2 (inhibitor of growth 2) is a candidate tumor-suppressor gene involved in cell cycle control, apoptosis and senescence. Although the functions of ING2 within the chromatin remodeling complex Sin3A/histone deacetylase (HDAC) and in the p53 pathway have been described, how ING2 itself is regulated remains unknown. In this study we report for the first time that ING2 can be sumoylated by small ubiquitin-like modifier 1 (SUMO1) on lysine 195 both in vitro and in vivo. Strikingly, ING2 sumoylation enhances its association with Sin3a. We provide evidences that ING2 can bind to the promoter of genes to mediate their expression and that sumoylation of ING2 is required for this binding to some of these genes. Among them, we identified the gene TMEM71 (transmembrane protein 71), whose expression is regulated by ING2 sumoylation. ING2 must be sumoylated to bind to the promoter of TMEM71 and to recruit the Sin3A chromatin-modifying complex to this promoter, in order to regulate TMEM71 transcription. Hence, sumoylation of ING2 enhances its binding to the Sin3A/HDAC complex and is required to regulate gene transcriptions.

SUMOylation of RIG-I positively regulates the type I interferon signaling

Retinoic acid-inducible gene-I (RIG-I) functions as an intracellular pattern recognition receptor (PRR) that recognizes the 5'-triphosphate moiety of single-stranded RNA viruses to initiate the innate immune response. Previous studies have shown that Lys63-linked ubiquitylation is required for RIG-I activation and the downstream anti-viral type I interferon (IFN-I) induction. Herein we reported that, RIG-I was also modified by small ubiquitin-like modifier-1 (SUMO-1). Functional analysis showed that RIG-I SUMOylation enhanced IFN-I production through increased ubiquitylation and the interaction with its downstream adaptor molecule Cardif. Our results therefore suggested that SUMOylation might serve as an additional regulatory tier for RIG-I activation and IFN-I signaling.

GPS2-dependent corepressor/SUMO pathways govern anti-inflammatory actions of LRH-1 and LXRβ in the hepatic acute phase response

The orphan receptor LRH-1 and the oxysterol receptors LXRalpha and LXRbeta are established transcriptional regulators of lipid metabolism that appear to control inflammatory processes. Here, we investigate the anti-inflammatory actions of these nuclear receptors in the hepatic acute phase response (APR). We report that selective synthetic agonists induce SUMOylation-dependent recruitment of either LRH-1 or LXR to hepatic APR promoters and prevent the clearance of the N-CoR corepressor complex upon cytokine stimulation. Investigations of the APR in vivo, using LXR knockout mice, indicate that the anti-inflammatory actions of LXR agonists are triggered selectively by the LXRbeta subtype. We further find that hepatic APR responses in small ubiquitin-like modifier-1 (SUMO-1) knockout mice are increased, which is due in part to diminished LRH-1 action at APR promoters. Finally, we provide evidence that the metabolically important coregulator GPS2 functions as a hitherto unrecognized transrepression mediator of interactions between SUMOylated nuclear receptors and the N-CoR corepressor complex. Our study extends the knowledge of anti-inflammatory mechanisms and pathways directed by metabolic nuclear receptor-corepressor networks to the control of the hepatic APR, and implies alternative pharmacological strategies for the treatment of human metabolic diseases associated with inflammation.

Modification of Small Hepatitis Delta Virus Antigen by SUMO Protein

Hepatitis delta antigen (HDAg) is a nuclear protein that is intimately involved in hepatitis delta virus (HDV) RNA replication. HDAg consists of two protein species, the small form (S-HDAg) and the large form (L-HDAg). Previous studies have shown that posttranslational modifications of S-HDAg, such as phosphorylation, acetylation, and methylation, can modulate HDV RNA replication. In this study, we show that S-HDAg is a small ubiquitin-like modifier 1 (SUMO1) target protein. Mapping data showed that multiple lysine residues are SUMO1 acceptors within S-HDAg. Using a genetic fusion strategy, we found that conjugation of SUMO1 to S-HDAg selectively enhanced HDV genomic RNA and mRNA synthesis but not antigenomic RNA synthesis. This result supports our previous proposition that the cellular machinery involved in the synthesis of HDV antigenomic RNA is different from that for genomic RNA synthesis and mRNA transcription, requiring different modified forms of S-HDAg. Sumoylation represents a new type of modification for HDAg.

CD43 processing and nuclear translocation of CD43 cytoplasmic tail are required for cell homeostasis

AbstractThe sialomucin CD43 is highly expressed on most hematopoietic cells. In this study, we show that the CD43 ectodomain is shed from murine granulocytes, mast cells, and T cells, but not from macrophages. To study the significance of CD43 shedding, we constructed 2 CD43/34 chimeras in which the CD43 membrane-proximal or transmembrane domain was swapped with the corresponding domain from CD34 that is not shed from cells. Viability of cells that normally shed CD43 was negatively affected when forced to express either of the 2 CD43/34 chimeras, but toxicity was reduced when cells coexpressed wild-type CD43. The CD43 cytoplasmic tail (CD43ct) was found to translocate into the nucleus, and inhibition of either its nuclear translocation or its release by γ-secretase was proapoptotic. Involvement of CD43 in regulation of apoptosis is consistent with our findings that CD43ct was modified by small ubiquitin-like modifier-1 and was colocalized with promyelocytic nuclear bodies. CD43-deficient cells exhibited reduced levels of promyelocytic nuclear bodies and had increased sensitivity to apoptosis induced by growth factor withdrawal or T-regulatory cell suppression. Taken together, our data indicate an essential function of CD43 processing and nuclear localization of CD43ct in cell homeostasis and apoptosis.

Sumoylation of Prox1 controls its ability to induce VEGFR3 expression and lymphatic phenotypes in endothelial cells

Prox1 is a master regulator for the development of lymphatic vasculature and the induction of lymphangiogenesis. In this study, we identified Prox1 as a new target for small ubiquitin-like modifier 1 (SUMO-1). Lysine 556 (K556) was found to be the major sumoylation site for Prox1 in vitro and in vivo. Mutation of this site (from lysine to arginine K556R) reduced DNA binding and the transcriptional activity of Prox1. Overexpression of Prox1 in EA.hy926 endothelial cells induced expression of lymphatic endothelial cell-specific genes including vascular endothelial growth factor receptor 3 (VEGFR3), fibroblast growth factor receptor 3 (FGFR3) and p57 while expression of K556R mutant Prox1 had little effect. The induction of VEGFR3 by Prox1 in EA.hy926 endothelial cells was an indication of their response to VEGF-C-induced lymphangiogenic signals, including the enhancement of proliferation, sprouting and tube formation and the inhibition of apoptosis. This effect is SUMO-dependent because ectopic expression of SUMO-specific protease 2 (SENP2) effectively reduced Prox1 sumoylation and Prox1-induced VEGFR3 expression. In addition, K556R mutant Prox1 could not induce lymphatic phenotypes. Taken together, our results indicate that Prox1 is a target for SUMO-1 and suggest that sumoylation of Prox1 controls its ability to induce VEGFR3 expression and lymphatic phenotypes in endothelial cells.

The small ubiquitin-like modifier mediates the resistance of prosthesis-loosening fibroblast-like synoviocytes against fas-induced apoptosis

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. 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. 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. 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.

SUMO Conjugation Contributes to Immune Deviation in Nonobese Diabetic Mice by Suppressing c-Maf Transactivation of IL-4

It is not clear why the development of protective Th2 cells is poor in type 1 diabetes (T1D). c-Maf transactivates the IL-4 gene promoting Th2 cell development; therefore, abnormalities in c-Maf may contribute to reduced IL-4 production by CD4 cells from nonobese diabetic (NOD) mice. In this study we demonstrate that despite normal expression, c-Maf binds poorly to the IL-4 promoter (IL-4p) in NOD CD4 cells. Immunoblotting demonstrates that c-Maf can be modified at lysine 33 by SUMO-1 (small ubiquitin-like modifier 1). Sumoylation is facilitated by direct interaction with the E2-conjugating enzyme Ubc9 and increases following T cell stimulation. In transfected cells, sumoylation decreases c-Maf transactivation of IL-4p-driven luciferase reporter activity, reduces c-Maf binding to the IL-4p in chromatin immunoprecipitation assays, and enhances c-Maf localization into promyelocytic leukemia nuclear bodies. Sumoylation of c-Maf is increased in NOD CD4 cells as compared with CD4 cells from diabetes-resistant B10.D2 mice, suggesting that increased c-Maf sumoylation contributes to immune deviation in T1D by reducing c-Maf access to and transactivation of the IL-4 gene.

Adenosine Signaling Mediates SUMO-1 Modification of IκBα during Hypoxia and Reoxygenation

Small ubiquitin-like modifier 1 (SUMO-1) modification of IkappaBalpha has been described to actively participate in NFkappaB regulation. Following proteosomal degradation of IkappaBalpha, an auto-regulatory loop consisting of transcriptional activation of IkappaBalpha gene and SUMO-1 modification of newly synthesized IkappaBalpha proceeds. The SUMOylated IkappaBalpha form is resistant to signal-induced degradation, consequently halting NFkappaB activation. We describe a mechanistic model by which adenosine (Ado) signaling results in significant accumulation of SUMO-1 modified IkappaBalpha with subsequent attenuation of NFkappaB activation. Using models of hypoxia followed by reoxygenation (H/R), we have documented an H/R cycle-dependent increase in extracellular Ado correlating with increases in the cytoplasmic pool of IkappaBalpha/SUMO-1. We demonstrate a dose-dependent increase in IkappaBalpha/SUMO in cells treated with the general Ado receptor agonist NECA and abolished by Ado receptor antagonists. Experiments in cells exposed to cycles of H/R followed by hypoxia demonstrated differential patterns of SUMOylation and phosphorylation of IkappaBalpha, greatly impacting its proteosomal degradation by the 26 S proteasome. Assays targeting knockdown and overexpression of SUMO-1 demonstrated significant regulation of NFkappaB activation and NFkappaB-mediated gene transcription (interleukin-6). These results were confirmed in vivo using wild type and cd73 null mouse lung tissue. In summary, we present an endogenous mechanism by which cells and tissues acquire anti-inflammatory properties by recruiting a nondegradable form of IkappaBalpha, a major control point for NFkappaB activation via Ado signaling.

Puromycin-based vectors promote a ROS-dependent recruitment of PML to nuclear inclusions enriched with HSP70 and Proteasomes

BackgroundPromyelocytic Leukemia (PML) protein can interact with a multitude of cellular factors and has been implicated in the regulation of various processes, including protein sequestration, cell cycle regulation and DNA damage responses. Previous studies reported that misfolded proteins or proteins containing polyglutamine tracts form aggregates with PML, chaperones, and components of the proteasome, supporting a role for PML in misfolded protein degradation.ResultsIn the current study, we have identified a reactive oxygen species (ROS) dependent aggregation of PML, small ubiquitin-like modifier 1 (SUMO-1), heat shock protein 70 (HSP70) and 20S proteasomes in human cell lines that have been transiently transfected with vectors expressing the puromycin resistance gene, puromycin n-acetyl transferase (pac). Immunofluorescent studies demonstrated that PML, SUMO-1, HSP70 and 20S proteasomes aggregated to form nuclear inclusions in multiple cell lines transfected with vectors expressing puromycin (puro) resistance in regions distinct from nucleoli. This effect does not occur in cells transfected with identical vectors expressing other antibiotic resistance genes or with vectors from which the pac sequence has been deleted. Furthermore, ROS scavengers were shown to ablate the effect of puro vectors on protein aggregation in transfected cells demonstrating a dependency of this effect on the redox state of transfected cells.ConclusionTaken together we propose that puromycin vectors may elicit an unexpected misfolded protein response, associated with the formation of nuclear aggresome like structures in human cell lines. This effect has broad implications for cellular behavior and experimental design.

SUMO1 polymorphisms are associated with non-syndromic cleft lip with or without cleft palate

Small ubiquitin-like modifier 1 (SUMO1) haploinsufficiency results in cleft lip and palate in animal models. However, no studies have linked SUMO1 to non-syndromic cleft lip with or without cleft palate (NSCLP) in humans. In the present study, we investigated the potential association between SUMO1 single nucleotide polymorphisms (SNPs) and risk for human NSCLP. From 181 patients and 162 healthy controls, we found statistically significant correlations between a 4-SNP SUMO1 haplotype and NSCLP. These data are the first to suggest a role for SUMO1 gene variation in human NSCLP development.

Abstract 5276: Sumoylation Of Klf5 Is A Molecular Switch Regulating Ppar-δ-containing Transcriptional Programs Of Lipid Metabolism

Metabolic syndrome is increasingly recognized as a major risk factor for cardiovascular disease. We have previously shown that a zinc finger transcription factor, Krüppel-like factor 5 (KLF5), plays an important role in cardiovascular diseases, such as atherosclerosis and cardiac hypertrophy. Interestingly, KLF5 is also expressed in metabolic tissues, such as adipose tissue, skeletal muscle and pancreatic β-cells. Moreover, we found that KLF5 is crucial for adipocyte differentiation. Therefore, it is very likely that KLF5 plays multiple roles in development and progression of metabolic syndrome and its cardiovascular and metabolic consequences including atherosclerotic cardiovascular disease. Indeed, KLF5 heterozygous knockout ( KLF5 +/− ) mice were resistant to high-fat-induced obesity and metabolic syndrome, despite consuming more food than wild-type mice. This appears to in part reflect their enhanced energy expenditure. Expression of the genes involved in lipid oxidation and energy uncoupling, including uncoupling protein (UCP) and carnitine-palmitoyl transferase 1b (CPT1b) was upregulated in the soleus muscles of KLF5 +/− mice. KLF5 could be reversibly modified by small ubiquitin-like modifier 1 (SUMO1), after which SUMOylated KLF5 strongly inhibited CPT1b , UCP3 and UCP2 promoter activity. Results of chromatin immunoprecipitation, two-hybrid, and reporter assays showed that under basal conditions SUMOylated KLF5 associated with transcriptionally repressive regulatory complexes containing unliganded PPARδ and corepressors. However, upon agonist stimulation of PPARδ, the deSUMOylating enzyme was recruited and KLF5 was deSUMOylated. The unSUMOylated KLF5 now formed transactivating complexes with liganded PPARδ and CBP. Thus, SUMOylation appears to be a molecular switch affecting function of KLF5 and the transcriptional regulatory programs governing lipid metabolism. Moreover, KLF5 is essential for the PPARδ agonist-dependent transcriptional control. Results of the present study have established KLF5 as a novel key molecule in lipid metabolism and suggest that the posttranscriptional modification of KLF5 is an attractive novel therapeutic target for both metabolic and cardiovascular diseases.

Regulation of Mitotic Spindle Asymmetry by SUMO and the Spindle-Assembly Checkpoint in Yeast

During mitosis, the kinetochore microtubules capture and segregate chromosomes, and the astral microtubules position the spindle within the cell. Although the spindle is symmetric, proper positioning of the spindle in asymmetrically dividing cells generally correlates with the formation of morphologically and structurally distinct asters [1]. In budding yeast, the spindle-orientation proteins Kar9 and dynein decorate only one aster of the metaphase spindle and direct it toward the bud [2, 3]. The mechanisms controlling the distribution of Kar9 and dynein remain unclear. Here, we show that SUMO regulates astral-microtubule function in at least two ways. First, Kar9 was sumoylated in vivo. Sumoylation and Cdk1-dependent phosphorylation of Kar9 independently promoted Kar9 asymmetry on the spindle. Second, proper regulation of kinetochore function by SUMO was also required for Kar9 asymmetry. Indeed, activation of the spindle-assembly checkpoint (SAC) due to SUMO and kinetochore defects promoted symmetric redistribution of Kar9 in a Mad2-dependent manner. The control of Kar9 distribution by the SAC was independent of Kar9 sumoylation and phosphorylation. Together, our data reveal that three independent mechanisms contribute to Kar9 asymmetry: Cdk1-dependent phosphorylation, sumoylation, and SAC signaling. Hence, the two seemingly independent spindle domains, kinetochores and astral microtubules, function in a tightly coordinated fashion.

SUMOylation of the MAGUK protein CASK regulates dendritic spinogenesis

Membrane-associated guanylate kinase (MAGUK) proteins interact with several synaptogenesis-triggering adhesion molecules. However, direct evidence for the involvement of MAGUK proteins in synapse formation is lacking. In this study, we investigate the function of calcium/calmodulin-dependent serine protein kinase (CASK), a MAGUK protein, in dendritic spine formation by RNA interference. Knockdown of CASK in cultured hippocampal neurons reduces spine density and shrinks dendritic spines. Our analysis of the time course of RNA interference and CASK overexpression experiments further suggests that CASK stabilizes or maintains spine morphology. Experiments using only the CASK PDZ domain or a mutant lacking the protein 4.1–binding site indicate an involvement of CASK in linking transmembrane adhesion molecules and the actin cytoskeleton. We also find that CASK is SUMOylated. Conjugation of small ubiquitin-like modifier 1 (SUMO1) to CASK reduces the interaction between CASK and protein 4.1. Overexpression of a CASK–SUMO1 fusion construct, which mimicks CASK SUMOylation, impairs spine formation. Our study suggests that CASK contributes to spinogenesis and that this is controlled by SUMOylation.

The mobility of Bach2 nuclear foci is regulated by SUMO-1 modification

The small ubiquitin-like modifier-1 (SUMO-1) modulates the functions of nuclear proteins by changing their structure and/or subnuclear localization. Several nuclear proteins form dynamic higher order nuclear structures, termed non-chromatin nuclear domains, which are involved in the regulation of nuclear function. However, the role that SUMO modification of the component proteins plays in the regulation of the activity and function of nuclear domains is unclear. Here we demonstrate that nuclear domains formed by Bach2, a transcription repressor, show restricted movement and undergo fusion events upon oxidative stress. Mutation of the SUMO-acceptor lysines in Bach2 alters the behavior of these nuclear foci and results in a decreased frequency of fusion events. We propose that SUMO modification is an important regulatory system for the mobility of the nuclear domains formed by Bach2.

The Cyclophilin-like Domain of Ran-binding Protein-2 Modulates Selectively the Activity of the Ubiquitin-Proteasome System and Protein Biogenesis

The ubiquitin-proteasome system (UPS) plays a critical role in protein degradation. The 19S regulatory particle (RP) of the 26S proteasome mediates the recognition, deubiquitylation, unfolding, and channeling of ubiquitylated substrates to the 20S proteasome. Several subunits of the 19S RP interact with a growing number of factors. The cyclophilin-like domain (CLD) of Ran-binding protein-2 (RanBP2/Nup358) associates specifically with at least one subunit, S1, of the base subcomplex of the 19S RP, but the functional implications of this interaction on the UPS activity are elusive. This study shows the CLD of RanBP2 promotes selectively the accumulation of a subset of reporter substrates of the UPS, such as the ubiquitin (Ub)-fusion yellow fluorescent protein (YFP) degradation substrate, Ub(G76V)-YFP, and the N-end rule substrate, Ub-R-YFP. Conversely, the degradation of endoplasmic reticulum and misfolded proteins, and of those linked to UPS-independent degradation, is not affected by CLD. The selective effect of CLD on the UPS in vivo is independent of, and synergistic with, proteasome inhibitors, and CLD does not affect the intrinsic proteolytic activity of the 20S proteasome. The inhibitory activity of CLD on the UPS resides in a purported SUMO binding motif. We also found two RanBP2 substrates, RanGTPase-activating protein and retinitis pigmentosa GTPase regulator interacting protein-1alpha1, whose steady-state levels are selectively modulated by CLD. Hence, the CLD of RanBP2 acts as a novel auxiliary modulator of the UPS activity; it may contribute to the molecular and subcellular compartmentation of the turnover of properly folded proteins and modulation of the expressivity of several neurological diseases.

Identification of a novel autoantibody directed against small ubiquitin‐like modifier activating enzyme in dermatomyositis

Myositis-specific autoantibodies (MSAs) are directed against cell machinery proteins such as aminoacyl-transfer RNA synthetases, signal recognition particle, Mi-2, and CADM-140. Because serologic subsets can define patients with specific clinical manifestations, the identification of further MSAs may help to identify additional disease subsets within the myositis spectrum. Sera from 20 adult patients with dermatomyositis (DM) were screened for autoantibodies. Two patients were further characterized due to the presence of the same novel immunoprecipitation (IP) pattern on sodium docecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and similar clinical manifestations. Both patients presented with cutaneous disease, followed by proximal myositis 6 months later. Both patients had associated nonspecific interstitial pneumonia but no signs of malignancy. The novel targets were identified using a combination of IP, SDS-PAGE, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Indirect HEp-2 immunofluorescence on sera from both patients displayed a diffuse, coarse, speckled, nucleolar-sparing pattern. IP revealed the presence of previously uncharacterized bands at approximately 40 kd and approximately 90 kd in both patients. The novel targets were identified as the small ubiquitin-like modifier 1 (SUMO-1) activating enzyme A subunit and SUMO-1 activating enzyme B subunit. These findings reveal previously uncharacterized autoantibodies directed against a protein involved in posttranslational modification, the SUMO activating enzyme, in 2 patients with DM who had similar clinical features, including severe skin disease and interstitial pneumonia.

Bax/Bak promote sumoylation of DRP1 and its stable association with mitochondria during apoptotic cell death

Dynamin-related protein 1 (DRP1) plays an important role in mitochondrial fission at steady state and during apoptosis. Using fluorescence recovery after photobleaching, we demonstrate that in healthy cells, yellow fluorescent protein (YFP)–DRP1 recycles between the cytoplasm and mitochondria with a half-time of 50 s. Strikingly, during apoptotic cell death, YFP-DRP1 undergoes a transition from rapid recycling to stable membrane association. The rapid cycling phase that characterizes the early stages of apoptosis is independent of Bax/Bak. However, after Bax recruitment to the mitochondrial membranes but before the loss of mitochondrial membrane potential, YFP-DRP1 becomes locked on the membrane, resulting in undetectable fluorescence recovery. This second phase in DRP1 cycling is dependent on the presence of Bax/Bak but independent of hFis1 and mitochondrial fragmentation. Coincident with Bax activation, we detect a Bax/Bak-dependent stimulation of small ubiquitin-like modifier-1 conjugation to DRP1, a modification that correlates with the stable association of DRP1 with mitochondrial membranes. Altogether, these data demonstrate that the apoptotic machinery regulates the biochemical properties of DRP1 during cell death.

Transforming Growth Factor-β-independent Regulation of Myogenesis by SnoN Sumoylation

Recent progress has been made on the role of oncoproteins c-Ski and related SnoN in the control of cellular transformation. c-Ski/SnoN potently repress transforming growth factor-beta (TGF-beta) antiproliferative signaling through physical interaction with signal transducers called Smads. Overexpression of c-Ski/SnoN also induces skeletal muscle differentiation, but how c-Ski/SnoN function in myogenesis is largely unknown. During our investigation on the role of sumoylation in TGF-beta signaling, we inadvertently found that SnoN is modified by small ubiquitin-like modifier-1 (SUMO-1). Here, we biochemically characterize SnoN sumoylation in detail and report the physiological function of the modification. Sumoylation occurs primarily at lysine 50 (Lys-50). PIAS1 and PIASx proteins physically interact with SnoN to stimulate its sumoylation, thus serving as SUMO-protein isopeptide ligases (E3) for SnoN sumoylation. SnoN sumoylation does not alter its metabolic stability or its ability to repress TGF-beta signaling. Notably, loss of sumoylation in the Lys-50 site (via a Lys-to-Arg point mutation) potently activates muscle-specific gene expression and enhances myotube formation. Our study suggests a novel role for SUMO modification in the regulation of myogenic differentiation.