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.
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 PDE6Î´ also required ICMT and promoted anterograde trafficking from the Golgi. We conclude that carboxyl methylation of NRAS is required for efficient palmitoylation, PDE6Î´ binding, and homeostatic flux through the Golgi, processes that direct delivery to the plasma membrane.
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
A small-molecule ICMT inhibitor delays senescence of Hutchinson-Gilford progeria syndrome cells
Chen, Xue; Yao, Haidong; Kashif, Muhammad; RevÃªchon, Gwladys; Eriksson, Maria; Hu, Jianjiang; Wang, Ting; Liu, Yiran; TÃ¼ksammel, Elin; Strömblad, Staffan; Ahearn, Ian M; Philips, Mark R; Wiel, Clotilde; Ibrahim, Mohamed X; Bergo, Martin O
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 Zmpste24-deficient mice. However, we don't know whether Icmt inactivation improves phenotypes in an authentic HGPS mouse model. Moreover, it is unknown whether pharmacologic targeting of ICMT would be tolerated by cells and produce similar cellular effects as genetic inactivation. Here, we show that knockout of Icmt improves survival of HGPS mice and restores vascular smooth muscle cell numbers in the aorta. We also synthesized a potent ICMT inhibitor called C75 and found that it delays senescence and stimulates proliferation of late-passage HGPS cells and Zmpste24-deficient mouse fibroblasts. Importantly, C75 did not influence proliferation of wild-type human cells or Zmpste24-deficient mouse cells lacking Icmt, indicating drug specificity. These results raise hopes that ICMT inhibitors could be useful for treating children with HGPS.
Scaffold association factor B (SAFB) is required for expression of prenyltransferases and RAS membrane association
Zhou, Mo; Kuruvilla, Leena; Shi, Xiarong; Viviano, Stephen; Ahearn, Ian M; Amendola, Caroline R; Su, Wenjuan; Badri, Sana; Mahaffey, James; Fehrenbacher, Nicole; Skok, Jane; Schlessinger, Joseph; Turk, Benjamin E; Calderwood, David A; Philips, Mark R
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 KRAS-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. We identified five enzymes in the prenylation pathway and SAFB, a nuclear protein with both DNA and RNA binding domains. Silencing SAFB led to marked mislocalization of all RAS isoforms as well as RAP1A but not RAB7A, a pattern that phenocopied silencing FNTA, the prenyltransferase Î± subunit shared by farnesyltransferase and geranylgeranyltransferase type I. We found that SAFB promoted RAS membrane association by controlling FNTA expression. SAFB knockdown decreased GTP loading of RAS, abrogated alternative prenylation, and sensitized RAS-mutant cells to growth inhibition by FTI. Our work establishes the prenylation pathway as paramount in KRAS membrane association, reveals a regulator of prenyltransferase expression, and suggests that reduction in FNTA expression may enhance the efficacy of FTIs.
The structural features that distinguish PD-L2 from PD-L1 emerged in placental mammals
Philips, Elliot A; Garcia-España, Antonio; Tocheva, Anna S; Ahearn, Ian M; Adam, Kieran R; Pan, Ruimin; Mor, Adam; Kong, Xiang-Peng
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 (APCs). PD-L2 binds to PD-1 with 3-fold stronger affinity compared to PD-L1. To date, this affinity discrepancy has been attributed to a tryptophan (W110PD-L2) that is unique to PD-L2 and has been assumed to fit snuggly into a pocket on the PD-1 surface. Contrary to this model, using surface plasmon resonance (SPR) to monitor real-time binding of recombinantly expressed and purified proteins, we found that W110PD-L2 acts as an "elbow" that helps shorten PD-L2 engagement with PD-1 and therefore lower affinity. Further, we identified a "latch" between the C and D Î² strands of the binding face as the source of the PD-L2 affinity advantage. We show that the 3-fold affinity advantage of PD-L2 is the consequence of these two opposing features, the W110PD-L2 "elbow" and a C-D region "latch." Interestingly, using phylogenetic analysis, we found that these features evolved simultaneously upon the emergence of placental mammals, suggesting that PD-L2-affinity tuning was part of the alterations to the adaptive immune system required for placental gestation.
KRAS4A directly regulates hexokinase 1
Amendola, Caroline R; Mahaffey, James P; Parker, Seth J; Ahearn, Ian M; Chen, Wei-Ching; Zhou, Mo; Court, Helen; Shi, Jie; Mendoza, Sebastian L; Morten, Michael J; Rothenberg, Eli; Gottlieb, Eyal; Wadghiri, Youssef Z; Possemato, Richard; Hubbard, Stevan R; Balmain, Allan; Kimmelman, Alec C; Philips, Mark R
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 region1. 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 mutation2. No functional distinctions among the splice variants have so far been established. Oncogenic KRAS alters the metabolism of tumour cells3 in several ways, including increased glucose uptake and glycolysis even in the presence of abundant oxygen4 (the Warburg effect). Whereas these metabolic effects of oncogenic KRAS have been explained by transcriptional upregulation of glucose transporters and glycolytic enzymes3-5, it is not known whether there is direct regulation of metabolic enzymes. Here we report a direct, GTP-dependent interaction between KRAS4A and hexokinase 1 (HK1) that alters the activity of the kinase, and thereby establish that HK1 is an effector of KRAS4A. This interaction is unique to KRAS4A because the palmitoylation-depalmitoylation cycle of this RAS isoform enables colocalization with HK1 on the outer mitochondrial membrane. The expression of KRAS4A in cancer may drive unique metabolic vulnerabilities that can be exploited therapeutically.
Posttranslational Modifications of RAS Proteins
Ahearn, Ian; Zhou, Mo; Philips, Mark R
The three human RAS 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. The PTMs of RAS proteins are the focus of this review.
Regulation of NOTCH signaling by RAB7 and RAB8 requires carboxyl methylation by ICMT
Court, Helen; Ahearn, Ian M; Amoyel, Marc; Bach, Erika A; Philips, Mark R
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 Pdx1-Cre;LSL-KrasG12D mice, suggesting that ICMT is required for NOTCH signaling. We used Drosophila melanogaster wing vein and scutellar bristle development to screen Rab proteins predicted to be substrates for ICMT (ste14 in flies). We identified Rab7 and Rab8 as ICMT substrates that when silenced phenocopy ste14 deficiency. ICMT, RAB7, and RAB8 were all required for efficient NOTCH1 signaling in mammalian cells. Overexpression of RAB8 rescued NOTCH activation after ICMT knockdown both in U2OS cells expressing NOTCH1 and in fly wing vein development. ICMT deficiency induced mislocalization of GFP-RAB7 and GFP-RAB8 from endomembrane to cytosol, enhanced binding to RABGDI, and decreased GTP loading of RAB7 and RAB8. Deficiency of ICMT, RAB7, or RAB8 led to mislocalization and diminished processing of NOTCH1-GFP. Thus, NOTCH signaling requires ICMT in part because it requires methylated RAB7 and RAB8.
VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking
Zhou, Mo; Wiener, Heidi; Su, Wenjuan; Zhou, Yong; Liot, Caroline; Ahearn, Ian; Hancock, John F; Philips, Mark R
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. The majority of the pool consists of farnesylated, but not palmitoylated, N-Ras that is associated with a high molecular weight (HMW) complex. Affinity purification and mass spectrographic identification revealed that among the proteins found in the HMW fraction is VPS35, a latent cytosolic component of the retromer coat. VPS35 bound to N-Ras in a farnesyl-dependent, but neither palmitoyl- nor guanosine triphosphate (GTP)-dependent, fashion. Silencing VPS35 increased N-Ras's association with cytoplasmic vesicles, diminished GTP loading of Ras, and inhibited mitogen-activated protein kinase signaling and growth of N-Ras-dependent melanoma cells.