Search

Demonstrates that mutating the RasGAP binding sites in the EphB2 JM region is not sufficient to switch the EphB2 signal from down-regulation to up-regulation of the mitogen-activated kinase (MAPK) signaling. Finding that endogenous RasGAP is required for down-regulation of the erk MAPK pathway by Eph receptor signaling; Mutation of the EphB2 juxtamenbrane region revealing a sequence dependence for receptor regulation and phosphorylation.

The transcription factor c-MAF could be polyubiquitinated and subsequently degraded in the proteasomes. Theoretically, any lysine residues in c-MAF could be ubiquitinated. In the present study, we tried to find out the specific lysine residue(s) mediating c-MAF ubiquitination. Through a series of mutational screens from lysine (K) to arginine (R), we found that any single lysine mutation (K to R) failed to prevent c-MAF ubiquitination, and any single lysine residue alone could not mediate c-MAF ubiquitination, which indicated that multiple lysine residues were required for c-MAF ubiquitination. Bioinformatics and computing analyses revealed that K85 and K350 could mediate c-MAF ubiquitination, which was confirmed by the cell-based assays. However, this duo was not the only pair because the K85R/K350R mutant could also be ubiquitinated. Functionally, both M12 (K85/K350) and W12 (K85R/K350R) mutants increased cyclin D2 promoter-driven luciferase activity, but they were less potent than the lysine-free counterpart (M14). In addition, M14 induced a higher level of expression of cyclin D2 at both mRNA and protein levels. Therefore, we demonstrated that c-MAF ubiquitination is mediated by multiple lysine residues, of which K85 and K350 were sufficient but not the only residues in mediating c-MAF ubiquitination. Moreover, c-MAF was found to be degraded by lysosomes. This study added a novel insight for c-MAF ubiquitination and degradation, suggesting that c-MAF stability is strictly regulated. [ABSTRACT FROM AUTHOR]

Summary The multi-domain deubiquitinase USP15 regulates diverse eukaryotic processes and has been implicated in numerous diseases. We developed ubiquitin variants (UbVs) that targeted either the catalytic domain or each of three adaptor domains in USP15, including the N-terminal DUSP domain. We also designed a linear dimer (diUbV), which targeted the DUSP and catalytic domains, and exhibited enhanced specificity and more potent inhibition of catalytic activity than either UbV alone. In cells, the UbVs inhibited the deubiquitination of two USP15 substrates, SMURF2 and TRIM25, and the diUbV inhibited the effects of USP15 on the transforming growth factor β pathway. Structural analyses revealed that three distinct UbVs bound to the catalytic domain and locked the active site in a closed, inactive conformation, and one UbV formed an unusual strand-swapped dimer and bound two DUSP domains simultaneously. These inhibitors will enable the study of USP15 function in oncology, neurology, immunology, and inflammation. Graphical Abstract Highlights • Tight and selective UbVs target USP15 catalytic and adaptor domains • UbV inhibitors lock the USP15 active site in an inactive conformation • A strand-swapped UbV dimer binds two DUSP domains simultaneously • Linear UbV dimers are potent and specific USP15 inhibitors in cells Teyra et al. developed ubiquitin variants (UbVs) that targeted distinct domains of the deubiquitinase USP15 and revealed alternative inhibition and dimerization strategies. Linear UbV dimers exhibited improved potency and specificity of USP15 inhibition. UbVs can be used to study the catalytic mechanism and biological functions of USP15. [ABSTRACT FROM AUTHOR]