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.

Transmembrane domain-driven PD-1 dimers mediate T cell inhibition.

Programmed cell death-1 (PD-1) is a potent immune checkpoint receptor on T lymphocytes. Upon engagement by its ligands, PD-L1 or PD-L2, PD-1 inhibits T cell activation and can promote immune tolerance. Antagonism of PD-1 signaling has proven effective in cancer immunotherapy, and conversely, agonists of the receptor may have a role in treating autoimmune disease.

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.

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.

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.

The structural features that distinguish PD-L2 from PD-L1 emerged in placental mammals.

Programmed cell death protein 1 (PD-1) is an inhibitory receptor on T lymphocytes that is critical for modulating adaptive immunity. As such, it has been successfully exploited for cancer immunotherapy. Programmed death ligand 1 (PD-L1) and PD-L2 are ligands for PD-1; the former is ubiquitously expressed in inflamed tissues, whereas the latter is restricted to antigen-presenting cells.

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.

Posttranslational Modifications of RAS Proteins.

The three human genes encode four proteins that play central roles in oncogenesis by acting as binary molecular switches that regulate signaling pathways for growth and differentiation. Each is subject to a set of posttranslational modifications (PTMs) that modify their activity or are required for membrane targeting. The enzymes that catalyze the various PTMs are potential targets for anti-RAS drug discovery.

Regulation of NOTCH signaling by RAB7 and RAB8 requires carboxyl methylation by ICMT.

Isoprenylcysteine carboxyl methyltransferase (ICMT) methylesterifies C-terminal prenylcysteine residues of CaaX proteins and some RAB GTPases. Deficiency of either ICMT or NOTCH1 accelerates pancreatic neoplasia in ; mice, suggesting that ICMT is required for NOTCH signaling. We used wing vein and scutellar bristle development to screen Rab proteins predicted to be substrates for ICMT (ste14 in flies).

VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking.

Ras guanosine triphosphatases (GTPases) regulate signaling pathways only when associated with cellular membranes through their C-terminal prenylated regions. Ras proteins move between membrane compartments in part via diffusion-limited, fluid phase transfer through the cytosol, suggesting that chaperones sequester the polyisoprene lipid from the aqueous environment. In this study, we analyze the nature of the pool of endogenous Ras proteins found in the cytosol.

Linear atrophoderma of Moulin.

We present a 40-year-old woman with asymptomatic, linear, hyperpigmented atrophic plaques in a Blaschkoid distribution on the right back and right upper extremity that is consistent with a diagnosis of linear atrophoderma of Moulin. Clinical lesions developed with a biphasic pattern in late adolescence and in adulthood. The pathogenesis of this acquired, progressive Blaschkolinear dermatosis may hold insight into the pathogenesis of this rare dermatologic condition, as well as other dermotoses, which include those resulting from post-zygotic genetic mosaicism.

Primary cutaneous follicle-center lymphoma.

We present a 64-year-old man with a three-year history of pruritic, pink papules and nodules of the face who was found to have a clonal lymphoproliferative B-cell disease that was characterized by a clonal IGH rearrangement. Although morphologic features present in the biopsy specimen were consistent with a reactive process, additional clinicopathologic correlation (anatomic presentation of lesions on the face, the absence of t(14:18) translocation, and bcl-2 and MUM1 expression) reinforced suspicion of a cutaneous B-cell lymphoma. Systemic work-up with CT/PET and a bone marrow biopsy ultimately excluded systemic disease and primary cutaneous follicle-center lymphoma (PCFCL) was a strong diagnostic consideration.

Metabolic labeling of Ras with tritiated palmitate to monitor palmitoylation and depalmitoylation.

Metabolic labeling with tritiated palmitate is a direct method for monitoring posttranslational modification of Ras proteins with this fatty acid. Advances in intensifying screens have allowed for the easy visualization of tritium without the need for extended exposure times. While more energetic radioisotopes are easier to visualize, the lack of commercial source and need for shielding make them more difficult to work with.

Regulating the regulator: post-translational modification of RAS.

RAS proteins are monomeric GTPases that act as binary molecular switches to regulate a wide range of cellular processes. The exchange of GTP for GDP on RAS is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), which regulate the activation state of RAS without covalently modifying it. By contrast, post-translational modifications (PTMs) of RAS proteins direct them to various cellular membranes and, in some cases, modulate GTP-GDP exchange.

Single-walled carbon nanotubes deliver peptide antigen into dendritic cells and enhance IgG responses to tumor-associated antigens.

We studied the feasibility of using single-wall carbon nanotubes (SWNTs) as antigen carriers to improve immune responses to peptides that are weak immunogens, a characteristic typical of human tumor antigens. Binding and presentation of peptide antigens by the MHC molecules of antigen presenting cells (APCs) is essential to mounting an effective immune response. The Wilm's tumor protein (WT1) is upregulated in many human leukemias and cancers and several vaccines directed at this protein are in human clinical trials.

FKBP12 binds to acylated H-ras and promotes depalmitoylation.

A cycle of palmitoylation/depalmitoylation of H-Ras mediates bidirectional trafficking between the Golgi apparatus and the plasma membrane, but nothing is known about how this cycle is regulated. We show that the prolyl isomerase (PI) FKBP12 binds to H-Ras in a palmitoylation-dependent fashion and promotes depalmitoylation. A variety of inhibitors of the PI activity of FKBP12, including FK506, rapamycin, and cycloheximide, increase steady-state palmitoylation.

Phospholipase D1 regulates lymphocyte adhesion via upregulation of Rap1 at the plasma membrane.

Rap1 is a small GTPase that modulates adhesion of T cells by regulating inside-out signaling through LFA-1. The bulk of Rap1 is expressed in a GDP-bound state on intracellular vesicles. Exocytosis of these vesicles delivers Rap1 to the plasma membrane, where it becomes activated.

Topology of mammalian isoprenylcysteine carboxyl methyltransferase determined in live cells with a fluorescent probe.

Isoprenylcysteine carboxyl methyltransferase (Icmt) is a highly conserved enzyme that methyl esterifies the alpha carboxyl group of prenylated proteins including Ras and related GTPases. Methyl esterification neutralizes the negative charge of the prenylcysteine and thereby increases membrane affinity. Icmt is an integral membrane protein restricted to the endoplasmic reticulum (ER).

Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division.

Rac1 regulates a wide variety of cellular processes. The polybasic region of the Rac1 C terminus functions both as a plasma membrane-targeting motif and a nuclear localization sequence (NLS). We show that a triproline N-terminal to the polybasic region contributes to the NLS, which is cryptic in the sense that it is strongly inhibited by geranylgeranylation of the adjacent cysteine.

Analysis of K-Ras phosphorylation, translocation, and induction of apoptosis.

K-Ras is a member of a family of proteins that associate with the plasma membrane by virtue of a lipid modification that inserts into the membrane and a polybasic region that associates with the anionic head groups of inner leaflet phospholipids. In the case of K-Ras, the lipid is a C-terminal farnesyl isoprenoid adjacent to a polylysine sequence. The affinity of K-Ras for the plasma membrane can be modulated by diminishing the net charge of the polybasic region.

PKC regulates a farnesyl-electrostatic switch on K-Ras that promotes its association with Bcl-XL on mitochondria and induces apoptosis.

K-Ras associates with the plasma membrane (PM) through farnesylation that functions in conjunction with an adjacent polybasic sequence. We show that phosphorylation by protein kinase C (PKC) of S181 within the polybasic region promotes rapid dissociation of K-Ras from the PM and association with intracellular membranes, including the outer membrane of mitochondria where phospho-K-Ras interacts with Bcl-XL. PKC agonists promote apoptosis of cells transformed with oncogenic K-Ras in a S181-dependent manner.