The DNA repair function of the breast cancer susceptibility protein BRCA1 depends in part on its interaction with RAP80, which targets BRCA1 to DNA double strand breaks (DSBs) through recognition of K63-linked polyubiquitin chains. The localization of BRCA1 to DSBs also requires sumoylation. Here, we demonstrated that, in addition to having ubiquitin-interacting motifs, RAP80 also contains a SUMO-interacting motif (SIM) that is critical for recruitment to DSBs. In combination with the ubiquitin-binding activity of RAP80, this SIM enabled RAP80 to bind with nanomolar affinity to hybrid chains consisting of ubiquitin conjugated to SUMO. Furthermore, RNF4, a SUMO-targeted ubiquitin E3 ligase that synthesizes hybrid SUMO-ubiquitin chains, localized to DSBs and was critical for the recruitment of RAP80 and BRCA1 to sites of DNA damage. Our findings, therefore, connect ubiquitin-dependent and SUMO-dependent DSB recognition, revealing that RNF4 synthesized hybrid SUMO-ubiquitin chains are recognized by RAP80 to promote BRCA1 recruitment and DNA repair.

Small ubiquitin-related modifiers (SUMOs) regulate diverse cellular processes through their covalent attachment to target proteins. Vertebrates express three SUMO paralogs: SUMO-1, SUMO-2, and SUMO-3 (SUMO-2 and SUMO-3 are ϳ96% identical and referred to as SUMO-2/3). SUMO-1 and SUMO-2/3 are conjugated, at least in part, to unique subsets of proteins and thus regulate distinct cellular pathways. However, how different proteins are selectively modified by SUMO-1 and SUMO-2/3 is unknown. We demonstrate that BLM, the RecQ DNA helicase mutated in Bloom syndrome, is preferentially modified by SUMO-2/3 both in vitro and in vivo. Our findings indicate that non-covalent interactions between SUMO and BLM are required for modification at non-consensus sites and that preferential SUMO-2/3 modification is determined by preferential SUMO-2/3 binding. We also present evidence that sumoylation of a C-terminal fragment of HIPK2 is dependent on SUMO binding, indicating that non-covalent interactions between SUMO and target proteins provide a general mechanism for SUMO substrate selection and possible paralogselective modification.Post-translational protein modifications play essential roles in regulating all aspects of cell function. Small ubiquitin-related modifiers (SUMOs) 2 are unusual post-translational modifications, because they themselves are proteins of ϳ100 amino acids (1, 2). Through covalent attachment to lysine residues in target proteins, sumoylation regulates a wide range of processes, including transcription activation, DNA synthesis and repair, nucleocytoplasmic transport, and chromosome segregation. The molecular mechanisms by which sumoylation affects individual proteins, and thus this diversity of processes, are in many cases target protein-specific. An emerging theme, however, is that sumoylation often promotes interactions between modified proteins and downstream factors containing SUMO-interacting motifs (SIMs) (1). To date, a single conserved SIM has been identified that consists of a hydrophobic core ((V/I)X(V/I)(V/I)) followed or preceded by a negatively charged cluster of amino acids (3-6). Although only a limited number of SIM-containing proteins has been functionally characterized to date, a large number of proteins contains this motif and is thus predicted to interact non-covalently with SUMO.Invertebrate organisms express only a single SUMO, whereas vertebrates express three paralogs capable of covalent conjugation to other proteins: SUMO-1, SUMO-2, and SUMO-3. SUMO-2 and SUMO-3 are ϳ96% identical to each other (and thus referred to collectively as SUMO-2/3); however, they are only ϳ45% identical to SUMO-1. Increasingly, evidence suggests that SUMO-1 and SUMO-2/3 have distinct cellular functions. Proteomic studies have, for example, shown that SUMO-1 and SUMO-2/3 are conjugated to only partially overlapping subsets of proteins (7,8). In addition, localization studies indicate that SUMO-1 and SUMO-2/3 are conjugated to unique subsets of proteins that localize to different subcellular domains (9, 10)...

SUMO modification of BLM controls the switch between BLM's pro- and anti-recombinogenic roles in homologous recombination following DNA damage during replication.

No abstract

Interferon-gamma (IFN-γ

Opiate abuse has been shown to cause adaptive changes in presynaptic release and protein phosphorylation-mediated synaptic plasticity, but the underlying mechanisms remain unclear. Neuronal SNARE proteins serve as important regulatory molecules underlying neural plasticity in view of their major role in the process of neurotransmitter release. In the present study, the expression of SNAP-25, a t-SNARE protein essential for vesicle release, was found to be dramatically regulated in hippocampus after chronic morphine treatment, which was visualized with two-dimensional gel electrophoresis. The spots of SNAP-25 in the gel were shifted along the dimension of isoelectric point, indicating a likely change of the post-transcriptional modification. Immunoblotting analysis with specific antibody to Ser 187 , a protein kinase C (PKC) phosphorylation site of SNAP-25, revealed that the specific phosphorylation was correspondingly decreased, which was correlated with morphine-induced inhibition of PKC activity. Moreover, the level of ternary complex of SNARE proteins in either synaptosomes or PC12 cells was significantly reduced after chronic morphine treatment. This suggests a causal relationship between the inhibition of PKC-dependent SNAP-25 phosphorylation and the down-regulation of SNARE complex formation after chronic morphine treatment. Further analysis of SNARE complex formed by transfection of the wild-type or Ser 187 mutants of SNAP-25 showed that only wildtype-formed complex was inhibited by morphine treatment. Thus, these results indicate that chronic morphine treatment inhibits phosphorylation of SNAP-25 at Ser 187 and leads to a down-regulation of SNARE complex formation, which presents a potential molecular mechanism for the alteration of exocytotic process and neural plasticity during opiate abuse.Opiate abuse causes extensive neural adaptive changes in the brain (1-3), which may be involved in a formation of aberrant learning (2). Accumulating evidences demonstrate that opiates significantly alter synaptic transmission and neural plasticity in the hippocampus, a center of learning and memory (4 -8). Importantly, presynaptic neurotransmitter release, which can be modulated by phosphorylation, serves as a critical element in the regulation of synaptic transmission during opiate abuse. Many protein kinases involved in modulation of transmitter release and synaptic plasticity, such as cAMP-dependent protein kinase (PKA), 1 protein kinase C (PKC), and Ca 2ϩ /calmodulin-dependent protein kinase II (CaMKII), also participate in cellular and synaptic adaptation mediating opiate dependence (3, 9). However, the protein substrates of these kinases involved in opiate abuse still need to be further elucidated.Regulated membrane fusion of synaptic vesicles and subsequent transmitter release involves the assembly of ternary complexes from soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins (10). The ternary complex is formed by the plasma membrane proteins synaptosomal-associated protein ...

Sumoylation is an important enhancer of responses to DNA replication stress and the SUMO-targeted ubiquitin E3 ligase RNF4 regulates these responses by ubiquitylation of sumoylated DNA damage response factors. The specific targets and functional consequences of RNF4 regulation in response to replication stress, however, have not been fully characterized. Here we demonstrated that RNF4 is required for the restart of DNA replication following prolonged hydroxyurea (HU)-induced replication stress. Contrary to its role in repair of γ-irradiation-induced DNA double-strand breaks (DSBs), our analysis revealed that RNF4 does not significantly impact recognition or repair of replication stress-associated DSBs. Rather, using DNA fiber assays, we found that the firing of new DNA replication origins, which is required for replication restart following prolonged stress, was inhibited in cells depleted of RNF4. We also provided evidence that RNF4 recognizes and ubiquitylates sumoylated Bloom syndrome DNA helicase BLM and thereby promotes its proteosome-mediated turnover at damaged DNA replication forks. Consistent with it being a functionally important RNF4 substrate, co-depletion of BLM rescued defects in the firing of new replication origins observed in cells depleted of RNF4 alone. We concluded that RNF4 acts to remove sumoylated BLM from collapsed DNA replication forks, which is required to facilitate normal resumption of DNA synthesis after prolonged replication fork stalling and collapse.

The DNA repair function of the breast cancer susceptibility protein BRCA1 depends in part on its interaction with RAP80, which targets BRCA1 to DNA double strand breaks (DSBs) through recognition of K63‐linked polyubiquitin chains. The localization of BRCA1 to DSBs also requires sumoylation. Here, we demonstrated that, in addition to having ubiquitin‐interacting motifs, RAP80 also contains a SUMO‐interacting motif (SIM) that is critical for recruitment to DSBs. In combination with the ubiquitin‐binding activity of RAP80, this SIM enabled RAP80 to bind with nanomolar affinity to hybrid chains consisting of ubiquitin conjugated to SUMO. Furthermore, RNF4, a SUMO‐targeted ubiquitin E3 ligase that synthesizes hybrid SUMO‐ubiquitin chains, localized to DSBs and was critical for the recruitment of RAP80 and BRCA1 to sites of DNA damage. Our findings, therefore, connect ubiquitin‐dependent and SUMO‐dependent DSB recognition, revealing that RNF4 synthesized hybrid SUMO‐ubiquitin chains are recognized by RAP80 to promote BRCA1 recruitment and DNA repair.