The differential interactomes of the KRAS splice variants identify BIRC6 as a ubiquitin ligase for KRAS4A.

Transcripts of the KRAS locus are alternatively spliced to generate two proteins, KRAS4A and KRAS4B, which differ in their membrane-targeting sequences. These splice variants have been conserved for more than 450 million years, suggesting non-overlapping functions driven by differential membrane association. Here, we use proximity labeling to map the differential interactomes of the KRAS splice variants.

RAB27B controls palmitoylation-dependent NRAS trafficking and signaling in myeloid leukemia.

RAS mutations are among the most prevalent oncogenic drivers in cancers. RAS proteins propagate signals only when associated with cellular membranes as a consequence of lipid modifications that impact their trafficking. Here, we discovered that RAB27B, a RAB family small GTPase, controlled NRAS palmitoylation and trafficking to the plasma membrane, a localization required for activation.

Differential functions of the KRAS splice variants.

RAS proteins are small GTPases that transduce signals from membrane receptors to signaling pathways that regulate growth and differentiation. Four RAS proteins are encoded by three genes - HRAS, KRAS, NRAS. Among them, KRAS is mutated in human cancer more frequently than any other oncogene.

Origin and Evolution of RAS Membrane Targeting.

KRAS, HRAS and NRAS proto-oncogenes belong to a family of 40 highly homologous genes, which in turn are a subset of a superfamily of >160 genes encoding small GTPases. RAS proteins consist of a globular G-domain (aa1-166) and a 22-23 aa unstructured hypervariable region (HVR) that mediates membrane targeting. The evolutionary origins of the RAS isoforms, their HVRs and alternative splicing of the KRAS locus has not been explored.

Consensus on the RAS dimerization hypothesis: Strong evidence for lipid-mediated clustering but not for G-domain-mediated interactions.

One of the open questions in RAS biology is the existence of RAS dimers and their role in RAF dimerization and activation. The idea of RAS dimers arose from the discovery that RAF kinases function as obligate dimers, which generated the hypothesis that RAF dimer formation might be nucleated by G-domain-mediated RAS dimerization. Here, we review the evidence for RAS dimerization and describe a recent discussion among RAS researchers that led to a consensus that the clustering of two or more RAS proteins is not due to the stable association of G-domains but, instead, is a consequence of RAS C-terminal membrane anchors and the membrane phospholipids with which they interact.

Origin and evolution of RAS oncoprotein membrane targeting.

KRAS, HRAS and NRAS oncogenes belong to a family of 40 highly homologous genes, which in turn are a subset of a superfamily of >160 genes encoding small GTPases. RAS oncoproteins consist of a globular G-domain (aa1-166) and a 22-23aa unstructured hypervariable region (HVR) that mediates membrane targeting. The evolutionarily origins of the RAS isoforms, their HVRs and alternative splicing of the KRAS locus has not been explored.

Palmitoylation and PDE6δ regulate membrane-compartment-specific substrate ubiquitylation and degradation.

Substrate degradation by the ubiquitin proteasome system (UPS) in specific membrane compartments remains elusive. Here, we show that the interplay of two lipid modifications and PDE6δ regulates compartmental substrate targeting via the SCF. FBXL2 is palmitoylated in a prenylation-dependent manner on cysteines 417 and 419 juxtaposed to the CaaX motif.

The role of KRAS splice variants in cancer biology.

The three mammalian genes (, and ) encode four proteins that play central roles in cancer biology. Among them, is mutated more frequently in human cancer than any other oncogene. The pre-mRNA of is alternatively spliced to give rise to two products, KRAS4A and KRAS4B, which differ in the membrane targeting sequences at their respective C-termini.

Spontaneous hydrolysis and spurious metabolic properties of α-ketoglutarate esters.

α-ketoglutarate (KG), also referred to as 2-oxoglutarate, is a key intermediate of cellular metabolism with pleiotropic functions. Cell-permeable esterified analogs are widely used to study how KG fuels bioenergetic and amino acid metabolism and DNA, RNA, and protein hydroxylation reactions, as cellular membranes are thought to be impermeable to KG. Here we show that esterified KG analogs rapidly hydrolyze in aqueous media, yielding KG that, in contrast to prevailing assumptions, imports into many cell lines.

Post-translational modification of RAS proteins.

Mutations of RAS genes drive cancer more frequently than any other oncogene. RAS proteins integrate signals from a wide array of receptors and initiate downstream signaling through pathways that control cellular growth. RAS proteins are fundamentally binary molecular switches in which the off/on state is determined by the binding of GDP or GTP, respectively.

Targeting KRAS4A splicing through the RBM39/DCAF15 pathway inhibits cancer stem cells.

The commonly mutated human KRAS oncogene encodes two distinct KRAS4A and KRAS4B proteins generated by differential splicing. We demonstrate here that coordinated regulation of both isoforms through control of splicing is essential for development of Kras mutant tumors. The minor KRAS4A isoform is enriched in cancer stem-like cells, where it responds to hypoxia, while the major KRAS4B is induced by ER stress.

NRAS is unique among RAS proteins in requiring ICMT for trafficking to the plasma membrane.

Isoprenylcysteine carboxyl methyltransferase (ICMT) is the third of three enzymes that sequentially modify the C-terminus of CaaX proteins, including RAS. Although all four RAS proteins are substrates for ICMT, each traffics to membranes differently by virtue of their hypervariable regions that are differentially palmitoylated. We found that among RAS proteins, NRAS was unique in requiring ICMT for delivery to the PM, a consequence of having only a single palmitoylation site as its secondary affinity module.

A small-molecule ICMT inhibitor delays senescence of Hutchinson-Gilford progeria syndrome cells.

A farnesylated and methylated form of prelamin A called progerin causes Hutchinson-Gilford progeria syndrome (HGPS). Inhibiting progerin methylation by inactivating the isoprenylcysteine carboxylmethyltransferase (ICMT) gene stimulates proliferation of HGPS cells and improves survival of -deficient mice. However, we don't know whether inactivation improves phenotypes in an authentic HGPS mouse model.

Breaking Tradition to Bridge Bench and Bedside: Accelerating the MD-PhD-Residency Pathway.

Problem: Physician-scientists are individuals trained in both clinical practice and scientific research. Often, the goal of physician-scientist training is to address pressing questions in biomedical research. The established pathways to formally train such individuals are mainly MD-PhD programs and physician-scientist track residencies.

Scaffold association factor B (SAFB) is required for expression of prenyltransferases and RAS membrane association.

Inhibiting membrane association of RAS has long been considered a rational approach to anticancer therapy, which led to the development of farnesyltransferase inhibitors (FTIs). However, FTIs proved ineffective against -driven tumors. To reveal alternative therapeutic strategies, we carried out a genome-wide CRISPR-Cas9 screen designed to identify genes required for KRAS4B membrane association.

SAP interacts with CD28 to inhibit PD-1 signaling in T lymphocytes.

T cell co-stimulation is important for the maintenance of immunologic tolerance. Co-inhibitory receptors including programmed cell death-1 (PD-1) confer peripheral tolerance to prevent autoimmunity. SAP (SH2D1A) is an adaptor molecule that is important in T cell signaling and has been shown to interact with signaling lymphocytic activation molecule (SLAM) family receptors also in the context of self-tolerance.

Selective Alanine Transporter Utilization Creates a Targetable Metabolic Niche in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) evolves a complex microenvironment comprised of multiple cell types, including pancreatic stellate cells (PSC). Previous studies have demonstrated that stromal supply of alanine, lipids, and nucleotides supports the metabolism, growth, and therapeutic resistance of PDAC. Here we demonstrate that alanine cross-talk between PSCs and PDAC is orchestrated by the utilization of specific transporters.

KRAS4A directly regulates hexokinase 1.

The most frequently mutated oncogene in cancer is KRAS, which uses alternative fourth exons to generate two gene products (KRAS4A and KRAS4B) that differ only in their C-terminal membrane-targeting region. Because oncogenic mutations occur in exons 2 or 3, two constitutively active KRAS proteins-each capable of transforming cells-are encoded when KRAS is activated by mutation. No functional distinctions among the splice variants have so far been established.

GGTase3 is a newly identified geranylgeranyltransferase targeting a ubiquitin ligase.

Protein prenylation is believed to be catalyzed by three heterodimeric enzymes: FTase, GGTase1 and GGTase2. Here we report the identification of a previously unknown human prenyltransferase complex consisting of an orphan prenyltransferase α-subunit, PTAR1, and the catalytic β-subunit of GGTase2, RabGGTB. This enzyme, which we named GGTase3, geranylgeranylates FBXL2 to allow its localization at cell membranes, where this ubiquitin ligase mediates the polyubiquitylation of membrane-anchored proteins.

A Potent Isoprenylcysteine Carboxylmethyltransferase (ICMT) Inhibitor Improves Survival in Ras-Driven Acute Myeloid Leukemia.

Blockade of Ras activity by inhibiting its post-translational methylation catalyzed by isoprenylcysteine carboxylmethyltransferase (ICMT) has been suggested as a promising antitumor strategy. However, the paucity of inhibitors has precluded the clinical validation of this approach. In this work we report a potent ICMT inhibitor, compound [UCM-1336, IC = 2 μM], which is selective against the other enzymes involved in the post-translational modifications of Ras.

Molecular basis for autoinhibition of RIAM regulated by FAK in integrin activation.

RAP1-interacting adapter molecule (RIAM) mediates RAP1-induced integrin activation. The RAS-association (RA) segment of the RA-PH module of RIAM interacts with GTP-bound RAP1 and phosphoinositol 4,5 bisphosphate but this interaction is inhibited by the N-terminal segment of RIAM. Here we report the structural basis for the autoinhibition of RIAM by an intramolecular interaction between the IN region (aa 27-93) and the RA-PH module.

K-Ras Lys-42 is crucial for its signaling, cell migration, and invasion.

Ras proteins participate in multiple signal cascades, regulating crucial cellular processes, including cell survival, proliferation, and differentiation. We have previously reported that Ras proteins are modified by sumoylation and that Lys-42 plays an important role in mediating the modification. In the current study, we further investigated the role of Lys-42 in regulating cellular activities of K-Ras.