Posttranslational modification by the ubiquitin homologue, small ubiquitin-like modifier 1 (SUMO-1), has been established as an important regulatory mechanism. However, in most cases it is not clear how sumoylation regulates various cellular functions. Emerging evidence suggests that sumoylation may play a general role in regulating protein-protein interactions, as shown in RanBP2͞ Nup358 and RanGAP1 interaction. In this study, we have defined an amino acid sequence motif that binds SUMO. This motif, V͞I-X-V͞ I-V͞I, was identified by NMR spectroscopic characterization of interactions among SUMO-1 and peptides derived from proteins that are known to bind SUMO or sumoylated proteins. This motif binds all SUMO paralogues (SUMO-1-3). Using site-directed mutagenesis, we also show that this SUMO-binding motif in RanBP2͞ Nup358 is responsible for the interaction between RanBP2͞Nup358 and sumoylated RanGAP1. The SUMO-binding motif exists in nearly all proteins known to be involved in SUMO-dependent processes, suggesting its general role in sumoylation-dependent cellular functions.Ubc9 ͉ posttranslational modification ͉ protein-protein interaction ͉ RanBP2 ͉ Nup358 P osttranslational modification by the ubiquitin homologue, small ubiquitin-like modifier 1 (SUMO-1), has been identified as an important mechanism for cellular regulation of transcription, DNA repair, cell cycle progression, protein intracellular trafficking, and nuclear receptor activities (1-5). More than 60 SUMO-1 target proteins and two diseases linked to SUMO-1 modification have been reported (3, 6, 7). Two SUMO paralogues, SUMO-2͞3, are closely related and share 97% amino acid sequence identity (8) but are only 46% and 48% identical to SUMO-1, respectively. The in vivo functions of SUMO-2͞3 modifications appear to be distinct from that of SUMO-1. All three SUMO paralogues are attached to substrate proteins through a biochemical pathway similar to that of ubiquitination (9). Despite the sequence differences between SUMO-1 and SUMO-2͞3, the activation enzyme E1 and conjugation enzyme E2 do not discriminate among the three SUMO molecules (10).Despite increasing information on the importance of SUMO modification in cellular regulation, the mechanism by which SUMO modification regulates these processes is not well understood. Posttranslational modifications, such as phosphorylation and ubiquitination, and now evidently also sumoylation, modulate proteinprotein interactions (11,12). For example, sumoylation of Ran-GAP1 results in its interaction with the nuclear pore protein RanBP2͞Nup358 (13,14). Transcription factors P300 and Elk-1, when modified by SUMO-1, recruit histone deacetylase 6 (HDAC6) (15) and HDAC2 (16), respectively.SUMO modification is likely to provide a new binding site for interactions with other proteins. In principle, SUMO modification could regulate the activity of a protein by altering its conformation. However, this is unlikely to be a general phenomenon, because SUMO modification sites are often located in extended loops, such as in Ra...

Sumoylation has recently been identified as an important mechanism that regulates protein interactions and localization in essential cellular functions, such as gene transcription, subnuclear structure formation, viral infection, and cell cycle progression. A SUMO binding amino acid sequence motif (SBM), which recognizes the SUMO moiety of modified proteins in sumoylation-dependent cellular functions, has been consistently identified by several recent studies. To understand the mechanism of SUMO recognition by the SBM, we have solved the solution structure of SUMO-1 in complex with a peptide containing the SBM derived from the protein PIASX (KVDVIDLTIESSSDEEEDPPAKR). Surprisingly, the structure reveals that the bound orientation of the SBM can reverse depending on the sequence context. The structure also reveals a novel mechanism of recognizing target sequences by a ubiquitin-like module. Unlike ubiquitin binding motifs, which all form helices and bind to the main ␤-sheet of ubiquitin, the SBM forms an extended structure that binds between the ␣-helix and a ␤-strand of SUMO-1. This study provides a clear mechanism of the SBM sequence variations and its recognition of the SUMO moiety in sumoylated proteins.Post-translational modification by the small ubiquitin-like modifiers (SUMO) 2 is an important mechanism that regulates a wide variety of cellular functions such as gene transcription, subnuclear structure formation, viral infection, and cell cycle progression (1-5). In mammalian cells, four SUMO paralogues have been identified (6 -8). SUMO-2, -3, and -4 are closely related and share more than 80% amino acid sequence identity. However, these proteins are less than 50% identical to SUMO-1. The in vivo functions of SUMO-2, -3, and -4 modifications are still not well understood, but it is known that some of their differences are in localization-and tissue-specific expression (6, 9). SUMO modifies a large number of proteins, and recent proteomic studies indicate that as much as 5% of the yeast proteome are SUMO substrates (10 -13).The mechanisms by which sumoylation regulates cellular functions are poorly understood. Current data suggest that the SUMO moiety of SUMO-modified proteins provide a platform for binding other proteins, and thus, SUMO serves as a module in protein interaction networks. Protein-protein interactions, which govern the intricate and dynamic networks of cellular functions and regulation, often involve only a limited number of common modules, such as SH2, SH3, and PDZ domains and short amino acid sequence motifs that bind to these modules (14). The ubiquitin-like structures are special types of modules that can be either part of a protein or covalently attached to other proteins enzymatically (15,16). An obvious advantage of attaching ubiquitin-like modules enzymatically is gaining the ability to turn on and off proteinprotein interactions quickly by conjugation and deconjugation of these modules. There are two lines of evidence which suggest that SUMO is likely to function as a module in med...

SUMMARY Lysyl-tRNA synthetase (LysRS), a component of the translation apparatus, is released from the cytoplasmic multi-tRNA synthetase complex (MSC) to activate the transcription factor MITF in stimulated mast cells through undefined mechanisms. Here we show that Ser207-phosphorylation provokes a new conformer of LysRS that inactivates its translational, but activates its transcriptional function. The crystal structure of an MSC sub-complex established that LysRS is held in the MSC by binding to the N-terminus of the scaffold protein p38/AIMP2. Phosphorylation-created steric clashes at the LysRS domain interface disrupt its binding grooves for p38/AIMP2, releasing LysRS and provoking its nuclear translocation. This alteration also exposes the C-terminal domain of LysRS to bind to MITF and triggers LysRS-directed production of the second messenger Ap4A that activates MITF. Thus our results establish that a single conformational change triggered by phosphorylation leads to multiple effects driving an exclusive switch of LysRS function from translation to transcription.

The conjugation of small ubiquitin-like modifiers SUMO-1, SUMO-2 and SUMO-3 onto target proteins requires the concerted action of the specific E1-activating enzyme SAE1/SAE2, the E2-conjugating enzyme Ubc9, and an E3-like SUMO ligase. NMR chemical shift perturbation was used to identify the surface of Ubc9 that interacts with the SUMO ligase RanBP2. Unlike known ubiquitin E2-E3 interactions, RanBP2 binds to the beta-sheet of Ubc9. Mutational disruption of Ubc9-RanBP2 binding affected SUMO-2 but not SUMO-1 conjugation to Sp100 and to a newly identified RanBP2 substrate, PML. RanBP2 contains a binding site specific for SUMO-1 but not SUMO-2, indicating that a Ubc9-SUMO-1 thioester could be recruited to RanBP2 via SUMO-1 in the absence of strong binding between Ubc9 and RanBP2. Thus we show that E2-E3 interactions are not conserved across the ubiquitin-like protein superfamily and identify a RanBP2-dependent mechanism for SUMO paralog-specific conjugation.

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