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

Simanshu, Dhirendra K; Philips, Mark R; Hancock, John F
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.
PMID: 36990093
ISSN: 1097-4164
CID: 5463312

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

Liang, David; Jiang, Liping; Bhat, Sameer Ahmed; Missiroli, Sonia; Perrone, Mariasole; Lauriola, Angela; Adhikari, Ritika; Gudur, Anish; Vasi, Zahra; Ahearn, Ian; Guardavaccaro, Daniele; Giorgi, Carlotta; Philips, Mark; Kuchay, Shafi
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 SCFFBXL2. FBXL2 is palmitoylated in a prenylation-dependent manner on cysteines 417 and 419 juxtaposed to the CaaX motif. Palmitoylation/depalmitoylation regulates its subcellular trafficking for substrate engagement and degradation. To control its subcellular distribution, lipid-modified FBXL2 interacts with PDE6δ. Perturbing the equilibrium between FBXL2 and PDE6δ disrupts the delivery of FBXL2 to all membrane compartments, whereas depalmitoylated FBXL2 is enriched on the endoplasmic reticulum (ER). Depalmitoylated FBXL2(C417S/C419S) promotes the degradation of IP3R3 at the ER, inhibits IP3R3-dependent mitochondrial calcium overload, and counteracts calcium-dependent cell death upon oxidative stress. In contrast, disrupting the PDE6δ-FBXL2 equilibrium has the opposite effect. These findings describe a mechanism underlying spatially-restricted substrate degradation and suggest that inhibition of FBXL2 palmitoylation and/or binding to PDE6δ may offer therapeutic benefits.
PMID: 36662618
ISSN: 2211-1247
CID: 5426422

Origin and Evolution of RAS Membrane Targeting

Garcia-España, Antonio; Philips, Mark R.
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. We found that KRAS is basal to the RAS proto-oncogene family and its duplication generated HRAS in the common ancestor of vertebrates. In a second round of duplication HRAS generated NRAS and KRAS generated an additional RAS gene we have designated KRASBL, absent in mammals and birds. KRAS4A arose through a duplication and insertion of the 4th exon of NRAS into the 3rd intron of KRAS. We found evolutionary conservation of a short polybasic region (PBR1) in HRAS, NRAS and KRAS4A, a second polybasic region (PBR2) in KRAS4A, two neutralized basic residues (NB) and a serine in KRAS4B and KRASBL, and a modification of the CaaX motif in vertebrates with farnesyl rather than geranylgeranyl polyisoprene lipids, suggesting that a less hydrophobic membrane anchor is critical to RAS protein function. The persistence of four RAS isoforms through >400 million years of evolution argues strongly for differential function.
ISSN: 0950-9232
CID: 5460762

The role of KRAS splice variants in cancer biology

Nuevo-Tapioles, Cristina; Philips, Mark R
The three mammalian RAS genes (HRAS, NRAS and KRAS) encode four proteins that play central roles in cancer biology. Among them, KRAS is mutated more frequently in human cancer than any other oncogene. The pre-mRNA of KRAS is alternatively spliced to give rise to two products, KRAS4A and KRAS4B, which differ in the membrane targeting sequences at their respective C-termini. Notably, both KRAS4A and KRAS4B are oncogenic when KRAS is constitutively activated by mutation in exon 2 or 3. Whereas KRAS4B is the most studied oncoprotein, KRAS4A is understudied and until recently considered relatively unimportant. Emerging work has confirmed expression of KRAS4A in cancer and found non-overlapping functions of the splice variants. The most clearly demonstrated of these is direct regulation of hexokinase 1 by KRAS4A, suggesting that the metabolic vulnerabilities of KRAS-mutant tumors may be determined in part by the relative expression of the splice variants. The aim of this review is to address the most relevant characteristics and differential functions of the KRAS splice variants as they relate to cancer onset and progression.
PMID: 36393833
ISSN: 2296-634x
CID: 5384892

Spontaneous hydrolysis and spurious metabolic properties of α-ketoglutarate esters

Parker, Seth J; Encarnación-Rosado, Joel; Hollinshead, Kate E R; Hollinshead, David M; Ash, Leonard J; Rossi, Juan A K; Lin, Elaine Y; Sohn, Albert S W; Philips, Mark R; Jones, Drew R; Kimmelman, Alec C
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. Esterified KG analogs exhibit spurious KG-independent effects on cellular metabolism, including extracellular acidification, arising from rapid hydrolysis and de-protonation of α-ketoesters, and significant analog-specific inhibitory effects on glycolysis or mitochondrial respiration. We observe that imported KG decarboxylates to succinate in the cytosol and contributes minimally to mitochondrial metabolism in many cell lines cultured in normal conditions. These findings demonstrate that nuclear and cytosolic KG-dependent reactions may derive KG from functionally distinct subcellular pools and sources.
PMID: 34385458
ISSN: 2041-1723
CID: 4972642

Post-translational modification of RAS proteins

Campbell, Sharon L; Philips, Mark R
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. As such, the intrinsic and regulated nucleotide-binding and hydrolytic properties of the RAS GTPase were historically believed to account for the entirety of the regulation of RAS signaling. However, it is increasingly clear that RAS proteins are also regulated by a vast array of post-translational modifications (PTMs). The current challenge is to understand what are the functional consequences of these modifications and which are physiologically relevant. Because PTMs are catalyzed by enzymes that may offer targets for drug discovery, the study of RAS PTMs has been a high priority for RAS biologists.
PMID: 34365229
ISSN: 1879-033x
CID: 5006062

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

Chen, Wei-Ching; To, Minh D; Westcott, Peter M K; Delrosario, Reyno; Kim, Il-Jin; Philips, Mark; Tran, Quan; Bollam, Saumya R; Goodarzi, Hani; Bayani, Nora; Mirzoeva, Olga; Balmain, Allan
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. KRAS4A splicing is controlled by the DCAF15/RBM39 pathway, and deletion of KRAS4A or pharmacological inhibition of RBM39 using Indisulam leads to inhibition of cancer stem cells. Our data identify existing clinical drugs that target KRAS4A splicing, and suggest that levels of the minor KRAS4A isoform in human tumors can be a biomarker of sensitivity to some existing cancer therapeutics.
PMID: 34257283
ISSN: 2041-1723
CID: 4965302

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

Ahearn, Ian M; Court, Helen R; Siddiqui, Farid; Abankwa, Daniel; Philips, Mark R
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. Although not absolutely required for palmitoylation, acylation was diminished in the absence of ICMT. Photoactivation and FRAP of GFP-NRAS revealed increase flux at the Golgi, independent of palmitoylation, in the absence of ICMT. Association of NRAS with the prenyl-protein chaperone PDE6δ also required ICMT and promoted anterograde trafficking from the Golgi. We conclude that carboxyl methylation of NRAS is required for efficient palmitoylation, PDE6δ binding, and homeostatic flux through the Golgi, processes that direct delivery to the plasma membrane.
PMID: 33579760
ISSN: 2575-1077
CID: 4780552

ULK1 inhibition overcomes compromised antigen presentation and restores antitumor immunity in LKB1 mutant lung cancer

Deng, Jiehui; Thennavan, Aatish; Dolgalev, Igor; Chen, Ting; Li, Jie; Marzio, Antonio; Poirier, John T; Peng, David; Bulatovic, Mirna; Mukhopadhyay, Subhadip; Silver, Heather; Papadopoulos, Eleni; Pyon, Val; Thakurdin, Cassandra; Han, Han; Li, Fei; Li, Shuai; Ding, Hailin; Hu, Hai; Pan, Yuanwang; Weerasekara, Vajira; Jiang, Baishan; Wang, Eric S; Ahearn, Ian; Philips, Mark; Papagiannakopoulos, Thales; Tsirigos, Aristotelis; Rothenberg, Eli; Gainor, Justin; Freeman, Gordon J; Rudin, Charles M; Gray, Nathanael S; Hammerman, Peter S; Pagano, Michele; Heymach, John V; Perou, Charles M; Bardeesy, Nabeel; Wong, Kwok-Kin
PMID: 34142094
ISSN: 2662-1347
CID: 4917722

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

Modrek, Aram S; Tanese, Naoko; Placantonakis, Dimitris G; Sulman, Erik P; Rivera, Rafael; Du, Kevin L; Gerber, Naamit K; David, Gregory; Chesler, Mitchell; Philips, Mark R; Cangiarella, Joan
PROBLEM/OBJECTIVE: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. Although graduates of these pathways are well equipped to be physician-scientists, numerous factors, including funding and length of training, discourage application to such programs and impede success rates. APPROACH/METHODS:To address some of the pressing challenges in training and retaining burgeoning physician-scientists, New York University Grossman School of Medicine formed the Accelerated MD-PhD-Residency Pathway in 2016. This pathway builds on the previously established accelerated three-year MD pathway to residency at the same institution. The Accelerated MD-PhD-Residency Pathway conditionally accepts MD-PhD trainees to a residency position at the same institution through the National Resident Matching Program. OUTCOMES/RESULTS:Since its inception, 2 students have joined the Accelerated MD-PhD-Residency Pathway, which provides protected research time in their chosen residency. The pathway reduces the time to earn an MD and PhD by one year and reduces the MD training phase to three years, reducing the cost and lowering socioeconomic barriers. Remaining at the same institution for residency allows for the growth of strong research collaborations and mentoring opportunities, which foster success. NEXT STEPS/UNASSIGNED:The authors and institutional leaders plan to increase the number of trainees that are accepted into the Accelerated MD-PhD-Residency Pathway and track the success of these students through residency and into practice to determine if the pathway is meeting its goal of increasing the number of practicing physician-scientists. The authors hope this model can serve as an example to leaders at other institutions who may wish to adopt this pathway for the training of their MD-PhD students.
PMID: 33464738
ISSN: 1938-808x
CID: 4760452