The NEDD8-activating enzyme (NAE) initiates a protein homeostatic pathway essential for cancer cell growth and survival. MLN4924 is a selective inhibitor of NAE currently in clinical trials for the treatment of cancer. Here, we show that MLN4924 is a mechanism-based inhibitor of NAE and creates a covalent NEDD8-MLN4924 adduct catalyzed by the enzyme. The NEDD8-MLN4924 adduct resembles NEDD8 adenylate, the first intermediate in the NAE reaction cycle, but cannot be further utilized in subsequent intraenzyme reactions. The stability of the NEDD8-MLN4924 adduct within the NAE active site blocks enzyme activity, thereby accounting for the potent inhibition of the NEDD8 pathway by MLN4924. Importantly, we have determined that compounds resembling MLN4924 demonstrate the ability to form analogous adducts with other ubiquitin-like proteins (UBLs) catalyzed by their cognate-activating enzymes. These findings reveal insights into the mechanism of E1s and suggest a general strategy for selective inhibition of UBL conjugation pathways.

An Epstein-Barr virus-encoded protein, LMP2, blocks the effects of surface immunoglobulin (slg) cross-linking on calcium mobilization and on lytic reactivation of EBV in latently infected and growth-transformed primary human B lymphocytes. In wild-type EBV-transformed cells, LMP2 is constitutively tyrosine phosphorylated and is associated with Lyn and Syk protein-tyrosine kinases (PTKs). Baseline Lyn PTK activity is substantially reduced, and slg cross-linking fails to activate Lyn, Syk, Pl3-K, PLC gamma 2, Vav, Shc, and MAPK. Syk, Pl3-K, PLC gamma 2, and Vav are constitutively tyrosine phosphorylated, and their tyrosine phosphorylation does not change following slg cross-linking. In contrast, cross-linking slg on cells transformed by LMP2 null mutant EBV recombinants triggers the same protein tyrosine kinase cascade as in noninfected B lymphocytes. These data are consistent with a model in which LMP2 is a constitutive dominant negative modulator of slg receptor signaling through its effects on Lyn, Syk, or regulators of these kinases.

Stimulation of resting B lymphocytes with antibodies to surface unoglobulin (sIgD or sIgM) induces protein tyrosine phosphorylation, implicating one or more Bcell protein-tyrosine kinases (PTKs) in sIg signal transduction.We have evaluated whether members of the src family of PTKs are involved in this process. (12)(13)(14)(15). Of the enzymes that may be involved in B-cell tyrosine phosphorylation signaling, members of the src family of protein-tyrosine kinases (PTKs) need to be considered as direct or indirect slg signaling components, based on previous evidence implicating p56lck and p60tf" in T-cell proliferation and activation events (refs. 16-18; reviewed in refs. 19 and 20). Moreover, the recent description of a B-cell-specific member of the src family, blk (21), suggests that this group of PTKs may be enzymes recruited in the B-cell activation cascade.

No abstract

The 44-amino-acid E5 protein of bovine papillomavirus type 1 is the shortest known protein with transforming activity. To identify the specific amino acids required for in vitro focus formation in mouse C127 cells, we used oligonucleotide-directed saturation mutagenesis to construct an extensive collection of mutants with missense mutations in the ES gene. Characterization of mutants with amino acid substitutions in the hydrophobic middle third of the ES protein indicated that efficient transformation requires a stretch of hydrophobic amino acids but not a specific amino acid sequence in this portion of the protein. Many amino acids in the carboxyl-terminal third of the protein can also undergo substitution without impairment of focus-forming activity, but the amino acids at seven positions, including two cysteine residues that mediate dimer formation, appear essential for efficient transforming activity. These essential amino acids are the most well conserved among related fibropapillomaviruses. The small size of the ES protein, its lack of similarity to other transforming proteins, and its ability to tolerate many amino acid substitutions implies that it transforms cells via a novel mechanism.The E5 gene of bovine papillomavirus type 1 (BPV1) encodes a short hydrophobic protein required for efficient in vitro transformation of established mouse cell lines. Frameshift mutations or the insertion of a translation termination codon in open reading frame (ORF) ES markedly decreases the ability of the viral DNA to induce foci in mouse C127 cells (5,9,16,20,24). Detailed mutational and biochemical analysis of this gene indicates that the portion of ORF E5 downstream of the first methionine codon in the reading frame is translated to produce a 7-kilodalton polypeptide required for efficient focus formation (5, 9, 29). In addition, expression of this portion of the E5 gene from a strong heterologous promoter is sufficient to induce focus formation (27,28,32). The ES protein is predicted to be only 44 amino acids long (Fig. 1). Its amino-terminal two-thirds consists almost exclusively of strongly hydrophobic amino acid residues, and the carboxyl-terminal third contains several charged and polar amino acid residues. There is no apparent similarity between the papillomavirus ES proteins and other sequenced proteins including known viral or cellular transforming proteins.An antiserum generated against the carboxyl-terminal third of the BPV1 E5 protein has been used to immunoprecipitate the 7-kilodalton E5 protein from BPV1-transformed mouse and hamster cells (5,29,33). In accordance with its predicted hydrophobic composition, the E5 protein is recovered predominantly in the membrane fraction of mechanically disrupted cells (29). Moreover, isolation of the E5 protein under nonreducing conditions results in the detection of E5 dimers and higher-order oligomers that can be converted to monomers by treatment with dithiothreitol (DTT) (5). We have proposed that the E5 protein oligomerizes via disulfide bonds at conserved cystei...