Summary Protein kinases are intensely studied mediators of cellular signaling, yet important questions remain regarding their regulation and in vivo properties. Here we use a probe-based chemoprotemics platform to profile several well studied kinase inhibitors against more than 200 kinases in native cell proteomes and reveal new biological targets for some of these inhibitors. Several striking differences were identified between native and recombinant kinase inhibitory profiles, in particular, for the Raf kinases. The native kinase binding profiles presented here closely mirror the cellular activity of these inhibitors, even when the inhibition profiles differ dramatically from recombinant assay results. Additionally, Raf activation events could be detected upon live cell treatment with inhibitors. These studies highlight the complexities of protein kinase behavior in the cellular context and demonstrate that profiling with only recombinant/purified enzymes can be misleading.

A specific small-molecule inhibitor of p97 would provide an important tool to investigate diverse functions of this essential ATPase associated with diverse cellular activities (AAA) ATPase and to evaluate its potential to be a therapeutic target in human disease. We carried out a high-throughput screen to identify inhibitors of p97 ATPase activity. Dual-reporter cell lines that simultaneously express p97-dependent and p97-independent proteasome substrates were used to stratify inhibitors that emerged from the screen. N 2 ,N 4 -dibenzylquinazoline-2,4-diamine (DBeQ) was identified as a selective, potent, reversible, and ATP-competitive p97 inhibitor. DBeQ blocks multiple processes that have been shown by RNAi to depend on p97, including degradation of ubiquitin fusion degradation and endoplasmic reticulum-associated degradation pathway reporters, as well as autophagosome maturation. DBeQ also potently inhibits cancer cell growth and is more rapid than a proteasome inhibitor at mobilizing the executioner caspases-3 and -7. Our results provide a rationale for targeting p97 in cancer therapy.apoptosis | autophagy | unfolded protein response T he AAA (ATPase associated with diverse cellular activities) ATPase p97 is conserved across all eukaryotes and is essential for life in budding yeast (1) and mice (2). p97 was first linked to the ubiquitin-proteasome system (UPS) through its role in the turnover of ubiquitin−β-galactosidase fusion proteins via the "ubiquitin fusion degradation" (UFD) pathway (3). Since then, p97 has been shown to play a critical role in the degradation of misfolded membrane and secretory proteins (4) and has also been linked to a broad array of cellular processes, including Golgi membrane reassembly (5), membrane transport (6), regulation of myofibril assembly (7), cell division (8), formation of protein aggregates (9), and autophagosome maturation (10, 11). The broad range of cellular functions for p97 is thought to derive from its ability to unfold proteins or disassemble protein complexes, but the detailed mechanism of how p97 works and is linked to specific cellular processes remains largely unknown.The structure of p97 comprises three domains: an N-terminal domain that recruits adaptors/substrate specificity factors, followed by two ATPase domains, D1 and D2 (12, 13). p97 monomers assemble to form a homohexamer that is thought to provide a platform for transduction of chemical activity into mechanical force that is applied to substrate proteins. The D1 domain mediates hexamerization (14) and has very low ATPase activity (15). Most of the ATPase activity is contributed by the D2 domain, which is thought to underlie p97's function as a mechanochemical transducer (16).The mechanochemical activity of p97 is linked to substrate proteins by an array of 13 UBX (ubiquitin regulatory X) domain adapters that bind the N-terminal domain of p97 (17), as well as the non-UBX domain adaptors Ufd1 and Npl4 (18). The functions and mechanisms of action of these different p97-adaptor complexes remain poorly u...

Threonylcarbamoyladenosine (t6A) is a universal modification found at position 37 of ANN decoding tRNAs, which imparts a unique structure to the anticodon loop enhancing its binding to ribosomes in vitro. Using a combination of bioinformatic, genetic, structural and biochemical approaches, the universal protein family YrdC/Sua5 (COG0009) was shown to be involved in the biosynthesis of this hypermodified base. Contradictory reports on the essentiality of both the yrdC wild-type gene of Escherichia coli and the SUA5 wild-type gene of Saccharomyces cerevisiae led us to reconstruct null alleles for both genes and prove that yrdC is essential in E. coli, whereas SUA5 is dispensable in yeast but results in severe growth phenotypes. Structural and biochemical analyses revealed that the E. coli YrdC protein binds ATP and preferentially binds RNAThr lacking only the t6A modification. This work lays the foundation for elucidating the function of a protein family found in every sequenced genome to date and understanding the role of t6A in vivo.

Ligand-induced protein degradation has emerged as a compelling approach to promote the targeted elimination of proteins from cells by directing these proteins to the ubiquitin-proteasome machinery. So far, only a limited number of E3 ligases have been found to support ligand-induced protein degradation, reflecting a dearth of E3-binding compounds for proteolysis-targeting chimera (PROTAC) design. Here, we describe a functional screening strategy performed with a focused library of candidate electrophilic PROTACs to discover bifunctional compounds that degrade proteins in human cells by covalently engaging E3 ligases. Mechanistic studies revealed that the electrophilic PROTACs act through modifying specific cysteines in DCAF11, a poorly characterized E3 ligase substrate adaptor. We further show that DCAF11-directed electrophilic PROTACs can degrade multiple endogenous proteins, including FBKP12 and the androgen receptor, in human prostate cancer cells. Our findings designate DCAF11 as an E3 ligase capable of supporting ligand-induced protein degradation via electrophilic PROTACs.