K128 ubiquitination constrains RAS activity by expanding its binding interface with GAP proteins.

The RAS pathway is among the most frequently activated signaling nodes in cancer. However, the mechanisms that alter RAS activity in human pathologies are not entirely understood. The most prevalent post-translational modification within the GTPase core domain of NRAS and KRAS is ubiquitination at lysine 128 (K128), which is significantly decreased in cancer samples compared to normal tissue. Here, we found that K128 ubiquitination creates an additional binding interface for RAS GTPase-activating proteins (GAPs), NF1 and RASA1, thus increasing RAS binding to GAP proteins and promoting GAP-mediated GTP hydrolysis. Stimulation of cultured cancer cells with growth factors or cytokines transiently induces K128 ubiquitination and restricts the extent of wild-type RAS activation in a GAP-dependent manner. In KRAS mutant cells, K128 ubiquitination limits tumor growth by restricting RAL/ TBK1 signaling and negatively regulating the autocrine circuit induced by mutant KRAS. Reduction of K128 ubiquitination activates both wild-type and mutant RAS signaling and elicits a senescence-associated secretory phenotype, promoting RAS-driven pancreatic tumorigenesis.

Split-Chloramphenicol Acetyl Transferase Assay to Study Protein-Protein Interactions and Ubiquitylation in Escherichia coli.

Protein-protein interactions and protein modifications play central roles in all living organisms. Of the more than 200 types of post-translational modifications, ubiquitylation is the most abundant, and it profoundly regulates the functionality of the eukaryotic proteome. Various in vitro and in vivo methodologies to study protein interactions and modifications have been developed, each presenting distinctive benefits and caveats. Here, we present a comprehensive protocol for applying a split-Chloramphenicol Acetyl-Transferase (split-CAT) based system, to study protein-protein interactions and ubiquitylation in E. coli . Functional assembly of bait and prey proteins tethered to the split-CAT fragments result in antibiotic resistance and growth on selective media. We demonstrate assays for protein interactions, protein ubiquitylation, and the system response to small compound modulators. To facilitate data collection, we provide an updated Scanner Acquisition Manager Program for Laboratory Experiments (SAMPLE; https://github.com/PragLab/SAMPLE ) that can be employed to monitor the growth of various microorganisms, including E. coli and S. cerevisiae . The advantage posed by this system lies in its sensitivity to a wide range of chloramphenicol concentrations, which allows the detection of a large spectrum of protein-protein interactions, without the need for their purification. The tight linkage between binding or ubiquitylation and growth enables the estimation of apparent relative affinity, and represents the system's quantitative characteristics. Graphical abstract.

Split Chloramphenicol Acetyl-Transferase Assay Reveals Self-Ubiquitylation-Dependent Regulation of UBE3B.

Split reporter protein-based genetic section systems are widely used to identify and characterize protein-protein interactions (PPI). The assembly of split markers that antagonize toxins, rather than required for synthesis of missing metabolites, facilitates the seeding of high density of cells and selective growth. Here we present a newly developed split chloramphenicol acetyltransferase (split-CAT) -based genetic selection system. The N terminus fragment of CAT is fused downstream of the protein of interest and the C terminus fragment is tethered upstream to its postulated partner. We demonstrate the system's advantages for the study of PPIs. Moreover, we show that co-expression of a functional ubiquitylation cascade where the target and ubiquitin are tethered to the split-CAT fragments results in ubiquitylation-dependent selective growth. Since proteins do not have to be purified from the bacteria and due to the high sensitivity of the split-CAT reporter, detection of challenging protein cascades and post-translation modifications is enabled. In addition, we demonstrate that the split-CAT system responds to small molecule inhibitors and molecular glues (GLUTACs). The absence of ubiquitylation-dependent degradation and deubiquitylation in E. coli significantly simplify the interpretation of the results. We harnessed the developed system to demonstrate that like NEDD4, UBE3B also undergoes self-ubiquitylation-dependent inactivation. We show that self-ubiquitylation of UBE3B on K665 induces oligomerization and inactivation in yeast and mammalian cells respectively. Finally, we showcase the advantages of split-CAT in the study of human diseases by demonstrating that mutations in UBE3B that cause Kaufman oculocerebrofacial syndrome exhibit clear E. coli growth phenotypes.