Abstract Protein tyrosine kinase 6 (PTK6) has context-specific, kinase-dependent and -independent functions in normal cells and cancer. In prostate cells, nuclear PTK6 suppresses growth, while active membrane-associated PTK6 promotes oncogenic signaling. Unlike SRC-family kinases, PTK6 is not modified by lipidation to promote membrane association. Although arginine residues in the PTK6 SH2 domain were previously linked to lipid binding, we found they are instead critical for protein stability and arginine methylation. Mutation of R131/R136 or R132 impaired PTK6 ubiquitination and its association with a Cullin-1-RING ubiquitin ligase complex, and its half-life increased from one hour to over 24 h. We show that PTK6 is targeted by PRMT1 and mutation of PTK6 R131/R136 or R132, or treatment with the type 1 PRMT inhibitor GSK3368715, leads to decreased arginine methylation, increased PTK6 protein stability and impaired PTK6 association with its substrates. In prostate cancer cells, ectopic expression of mutant PTK6 R131/R136 or R132 leads to reduced proliferation, anchorage-independent growth, and cell migration and invasion, when compared with wild type PTK6, even though higher levels of mutant protein are expressed. These results indicate that arginine methylation is a key regulatory switch for PTK6 signaling and protein turnover and may be central for the execution of its diverse context-specific functions. Mutation of PTK6 arginine residues has been reported in some cancers, which may contribute to disease-specific PTK6 expression and signaling.

Compartment-specific cellular membrane protein turnover is not well understood. We show that FBXO10, the interchangeable component of the cullin-RING-ligase 1 complex, undergoes lipid modification with geranylgeranyl isoprenoid at cysteine953, facilitating its dynamic trafficking to the outer mitochondrial membrane (OMM). FBXO10 polypeptide lacks a canonical mitochondrial targeting sequence (MTS); instead, its geranylgeranylation at C953 and interaction with two cytosolic factors, cytosolic factor-like δ subunit of type 6 phosphodiesterase (PDE6δ; a prenyl-group-binding protein) and heat shock protein 90 (HSP90; a chaperone), orchestrate specific OMM targeting of prenyl-FBXO10. The FBXO10(C953S) mutant redistributes away from the OMM, impairs mitochondrial ATP production and membrane potential, and increases fragmentation. Phosphoglycerate mutase-5 (PGAM5) was identified as a potential substrate of FBXO10 at the OMM using comparative quantitative proteomics of enriched mitochondria. FBXO10 loss or expression of prenylation-deficient FBXO10(C953S) inhibited PGAM5 degradation, disrupted mitochondrial homeostasis, and impaired myogenic differentiation of human induced pluripotent stem cells (iPSCs) and murine myoblasts. Our studies identify a mechanism for FBXO10-mediated regulation of selective mitochondrial proteostasis potentially amenable to therapeutic intervention.

E3-ubiquitin ligases (E3s) are main components of the ubiquitin-proteasome system (UPS), as they determine substrate specificity in response to internal and external cues to regulate protein homeostasis. However, the regulation of membrane protein ubiquitination by E3s within distinct cell membrane compartments or organelles is not well understood. We show that FBXO10, the interchangeable component of the SKP1/CUL1/F-box ubiquitin ligase complex (SCF-E3), undergoes lipid-modification with geranylgeranyl isoprenoid at Cysteine953 (C953), facilitating its dynamic trafficking to the outer mitochondrial membrane (OMM). FBXO10 polypeptide does not contain a canonical mitochondrial targeting sequence (MTS); instead, its geranylgeranylation at C953 and the interaction with two cytosolic factors, PDE6δ (a prenyl group-binding protein), and HSP90 (a mitochondrial chaperone) orchestrate specific OMM targeting of prenyl-FBXO10 across diverse membrane compartments. The geranylgeranylation-deficient FBXO10(C953S) mutant redistributes away from the OMM, leading to impaired mitochondrial ATP production, decreased mitochondrial membrane potential, and increased mitochondrial fragmentation. Phosphoglycerate mutase 5 (PGAM5) was identified as a potential substrate of FBXO10 at the OMM using comparative quantitative mass spectrometry analyses of enriched mitochondria (LFQ-MS/MS), leveraging the redistribution of FBXO10(C953S). FBXO10, but not FBXO10(C953S), promoted polyubiquitylation and degradation of PGAM5. Examination of the role of this pathway in a physiological context revealed that the loss of FBXO10 or expression of prenylation-deficient-FBXO10(C953S) inhibited PGAM5 degradation, disrupted mitochondrial homeostasis, and impaired myogenic differentiation of human iPSCs and murine myoblasts. Our studies identify a mechanism for selective E3-ligase mediated regulation of mitochondrial membrane proteostasis and metabolic health, potentially amenable to therapeutic intervention.

A high-fat diet (HFD) promotes metastasis through increased uptake of saturated fatty acids (SFAs). The fatty acid transporter CD36 has been implicated in this process, but a detailed understanding of CD36 function is lacking. During matrix detachment, endoplasmic reticulum (ER) stress reduces SCD1 protein, resulting in increased lipid saturation. Subsequently, CD36 is induced in a p38- and AMPK-dependent manner to promote preferential uptake of monounsaturated fatty acids (MUFAs), thereby maintaining a balance between SFAs and MUFAs. In attached cells, CD36 palmitoylation is required for MUFA uptake and protection from palmitate-induced lipotoxicity. In breast cancer mouse models, CD36-deficiency induced ER stress while diminishing the pro-metastatic effect of HFD, and only a palmitoylation-proficient CD36 rescued this effect. Finally, AMPK-deficient tumors have reduced CD36 expression and are metastatically impaired, but ectopic CD36 expression restores their metastatic potential. Our results suggest that, rather than facilitating HFD-driven tumorigenesis, CD36 plays a supportive role by preventing SFA-induced lipotoxicity.

Prenylation and palmitoylation are two major lipid modifications of cellular proteins that anchor proteins to cell membranes. Here, we present a protocol for detecting these modifications in cellular proteins by radioactive metabolic labeling. We describe steps for metabolic labeling of cells, cell harvesting for carrying out immunoprecipitations, subjecting immunocomplexes to SDS-PAGE, and transferring them to polyvinylidine flouride (PVDF) membranes. We then detail detection of labeled target proteins by exposing PVDF membranes to phosphor screens and using a phosphor imager machine. For complete details of this protocol, please refer to Liang et al.1

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.

Gastro-esophageal tumors constitute a big health problem. Treatment options still mainly rely on chemotherapy, and apart from human epidermal growth factor receptor 2 positive and microsatellite instable/Epstein–Barr Virus disease, there are no molecularly guided options. Therefore, despite the large number of identified molecular alterations, precision medicine is still far from the clinic. In this context, the recently developed technology of patient-derived organoids (PDOs) could offer the chance to accelerate drug development and biomarker discovery. Indeed, PDOs are 3D primary cultures that were shown to reproduce patient's tumor characteristics. Moreover, several reports indicated that PDOs can replicate patient's response to a given drug; therefore, they are one of the most promising tools for functional precision medicine.

The ubiquitin proteasome system (UPS) is a proteolytic machinery for the degradation of protein substrates that are post-translationally conjugated with ubiquitin polymers through the enzymatic action of ubiquitin ligases, in a process termed ubiquitylation. Ubiquitylation of substrates precedes their proteolysis via proteasomes, a hierarchical feature of UPS. E3-ubiquitin ligases recruit protein substrates providing specificity for ubiquitylation. Innate and adaptive immune system networks are regulated by ubiquitylation and substrate degradation via E3-ligases/UPS. Deregulation of E3-ligases/UPS components in immune cells is involved in the development of lymphomas, neurodevelopmental abnormalities, and cancers. Targeting E3-ligases for therapeutic intervention provides opportunities to mitigate the unintended broad effects of 26S proteasome inhibition. Recently, bifunctional moieties such as PROTACs and molecular glues have been developed to re-purpose E3-ligases for targeted degradation of unwanted aberrant proteins, with a potential for clinical use. Here, we summarize the involvement of E3-ligases/UPS components in immune-related diseases with perspectives.

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. In cells, FBXL2 is specifically recognized by GGTase3 despite having a typical carboxy-terminal CaaX prenylation motif that is predicted to be recognized by GGTase1. Our crystal structure analysis of the full-length GGTase3-FBXL2-SKP1 complex reveals an extensive multivalent interface specifically formed between the leucine-rich repeat domain of FBXL2 and PTAR1, which unmasks the structural basis of the substrate-enzyme specificity. By uncovering a missing prenyltransferase and its unique mode of substrate recognition, our findings call for a revision of the 'prenylation code'.

FBXL2 targets IP3R3 for ubiquitin-mediated degradation to limit Ca2+ flux to mitochondria and, consequently, apoptosis. Efficient replication of hepatitis C virus (HCV) requires geranylgeranylation of FBXL2. Here, we show that the viral protein NS5A forms a trimeric complex with IP3R3 and FBXL2, unmasking IP3R3’s degron in the absence of inositol 1,4,5-trisphosphate (IP3) stimulation. FBXL2 knockdown or expression of a stable IP3R3 mutant causes persistent Ca2+ flux and sensitizes cells to apoptosis, resulting in the inhibition of viral replication. Importantly, the effect of FBXL2 silencing is rescued by depleting IP3R3, but not p85β, another established FBXL2 substrate, indicating that the anti-HCV effect of FBXL2 knockdown is largely due to IP3R3 stabilization. Finally, disruption of the FBXL2-NS5A-IP3R3 complex using somatic cell genetics or pharmacologic inhibition results in IP3R3 stabilization and suppression of HCV replication. This study reveals an IP3-independent molecular mechanism through which HCV promotes IP3R3 degradation, thereby inhibiting virus-induced apoptosis and establishing chronic infection.

Carriers of heterozygous germline BAP1 mutations (BAP1(+/-)) develop cancer. We studied plasma from 16 BAP1(+/-) individuals from 2 families carrying different germline BAP1 mutations and 30 BAP1 wild-type (BAP1(WT)) controls from these same families. Plasma samples were analyzed by liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS), ultra-performance liquid chromatography triple quadrupole mass spectrometry (UPLC-TQ-MS), and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). We found a clear separation in the metabolic profile between BAP1(WT) and BAP1(+/-) individuals. We confirmed the specificity of the data in vitro using 12 cell cultures of primary fibroblasts we derived from skin punch biopsies from 12/46 of these same individuals, 6 BAP1(+/-) carriers and 6 controls from both families. BAP1(+/-) fibroblasts displayed increased aerobic glycolysis and lactate secretion, and reduced mitochondrial respiration and ATP production compared with BAP1(WT). siRNA-mediated downregulation of BAP1 in primary BAP1(WT) fibroblasts and in primary human mesothelial cells, led to the same reduced mitochondrial respiration and increased aerobic glycolysis as we detected in primary fibroblasts from carriers of BAP1(+/-) mutations. The plasma and cell culture results were highly reproducible and were specifically and only linked to BAP1 status and not to gender, age or family, or cell type, and required an intact BAP1 catalytic activity. Accordingly, we were able to build a metabolomic model capable of predicting BAP1 status with 100% accuracy using data from human plasma. Our data provide the first experimental evidence supporting the hypothesis that aerobic glycolysis, also known as the ` Warburg effect', does not necessarily occur as an adaptive process that is consequence of carcinogenesis, but rather that it may also predate malignancy by many years and facilitate carcinogenesis.

The product of the KRAS oncogene, KRAS4B, promotes tumor growth when associated with the plasma membrane (PM). PM association is mediated, in part, by farnesylation of KRAS4B, but trafficking of nascent KRAS4B to the PM is incompletely understood. We performed a genome-wide screen to identify genes required for KRAS4B membrane association and identified a G protein-coupled receptor, GPR31. GPR31 associated with KRAS4B on cellular membranes in a farnesylation-dependent fashion, and retention of GPR31 on the endoplasmic reticulum inhibited delivery of KRAS4B to the PM. Silencing of GPR31 expression partially mislocalized KRAS4B, slowed the growth of KRAS-dependent tumor cells, and blocked KRAS-stimulated macropinocytosis. Our data suggest that GPR31 acts as a secretory pathway chaperone for KRAS4B.

BRCA1-associated protein 1 (BAP1) regulates calcium flux in the endoplasmic reticulum to facilitate the execution of apoptosis, unveiling a new facet of the role of BAP1 as an environmental tumour suppressor. BAP1 is a tumour suppressor associated with germline mutations in several malignancies including uveal melanoma and mesothelioma. BAP1 tumour suppressor activity has previously been linked to its nuclear role in maintaining genome integrity. Here, the authors reveal a new role for BAP1 in the endoplasmic reticulum, where it regulates calcium flux to facilitate the execution of apoptosis. Loss of BAP1 function prevents apoptosis in transformed cells with accumulated DNA damage. The results unveil a new facet of the role of BAP1 as an environmental tumour suppressor. BRCA1-associated protein 1 (BAP1) is a potent tumour suppressor gene that modulates environmental carcinogenesis1,2,3. All carriers of inherited heterozygous germline BAP1-inactivating mutations (BAP1+/−) developed one and often several BAP1−/− malignancies in their lifetime4, mostly malignant mesothelioma, uveal melanoma2,5, and so on6,7,8,9,10. Moreover, BAP1-acquired biallelic mutations are frequent in human cancers8,11,12,13,14. BAP1 tumour suppressor activity has been attributed to its nuclear localization, where it helps to maintain genome integrity15,16,17. The possible activity of BAP1 in the cytoplasm is unknown. Cells with reduced levels of BAP1 exhibit chromosomal abnormalities and decreased DNA repair by homologous recombination18, indicating that BAP1 dosage is critical. Cells with extensive DNA damage should die and not grow into malignancies. Here we discover that BAP1 localizes at the endoplasmic reticulum. Here, it binds, deubiquitylates, and stabilizes type 3 inositol-1,4,5-trisphosphate receptor (IP3R3), modulating calcium (Ca2+) release from the endoplasmic reticulum into the cytosol and mitochondria, promoting apoptosis. Reduced levels of BAP1 in BAP1+/− carriers cause reduction both of IP3R3 levels and of Ca2+ flux, preventing BAP1+/− cells that accumulate DNA damage from executing apoptosis. A higher fraction of cells exposed to either ionizing or ultraviolet radiation, or to asbestos, survive genotoxic stress, resulting in a higher rate of cellular transformation. We propose that the high incidence of cancers in BAP1+/− carriers results from the combined reduced nuclear and cytoplasmic activities of BAP1. Our data provide a mechanistic rationale for the powerful ability of BAP1 to regulate gene–environment interaction in human carcinogenesis.

In response to environmental cues that promote IP3 (inositol 1,4,5-trisphosphate) generation, IP3 receptors (IP3Rs) located on the endoplasmic reticulum allow the 'quasisynaptical' feeding of calcium to the mitochondria to promote oxidative phosphorylation. However, persistent Ca2+ release results in mitochondrial Ca2+ overload and consequent apoptosis. Among the three mammalian IP3Rs, IP3R3 appears to be the major player in Ca2+-dependent apoptosis. Here we show that the F-box protein FBXL2 (the receptor subunit of one of 69 human SCF (SKP1, CUL1, F-box protein) ubiquitin ligase complexes) binds IP3R3 and targets it for ubiquitin-, p97- and proteasome-mediated degradation to limit Ca2+ influx into mitochondria. FBXL2-knockdown cells and FBXL2-insensitive IP3R3 mutant knock-in clones display increased cytosolic Ca2+ release from the endoplasmic reticulum and sensitization to Ca2+-dependent apoptotic stimuli. The phosphatase and tensin homologue (PTEN) gene is frequently mutated or lost in human tumours and syndromes that predispose individuals to cancer. We found that PTEN competes with FBXL2 for IP3R3 binding, and the FBXL2-dependent degradation of IP3R3 is accelerated in Pten-/- mouse embryonic fibroblasts and PTEN-null cancer cells. Reconstitution of PTEN-null cells with either wild-type PTEN or a catalytically dead mutant stabilizes IP3R3 and induces persistent Ca2+ mobilization and apoptosis. IP3R3 and PTEN protein levels directly correlate in human prostate cancer. Both in cell culture and xenograft models, a non-degradable IP3R3 mutant sensitizes tumour cells with low or no PTEN expression to photodynamic therapy, which is based on the ability of photosensitizer drugs to cause Ca2+-dependent cytotoxicity after irradiation with visible light. Similarly, disruption of FBXL2 localization with GGTi-2418, a geranylgeranyl transferase inhibitor, sensitizes xenotransplanted tumours to photodynamic therapy. In summary, we identify a novel molecular mechanism that limits mitochondrial Ca2+ overload to prevent cell death. Notably, we provide proof-of-principle that inhibiting IP3R3 degradation in PTEN-deregulated cancers represents a valid therapeutic strategy.

F-box proteins are the substrate-recognition subunits of SCF (Skp1/Cul1/F-box protein) ubiquitin ligase complexes. Purification of the F-box protein FBXL2 identified the PI(3)K regulatory subunit p85β and tyrosine phosphatase PTPL1 as interacting proteins. FBXL2 interacts with the pool of p85β that is free of p110 PI(3)K catalytic subunits and targets this pool for ubiquitylation and subsequent proteasomal degradation. FBXL2-mediated degradation of p85β is dependent on the integrity of its CaaX motif. Whereas most SCF substrates require phosphorylation to interact with their F-box proteins, phosphorylation of p85β on Tyr 655, which is adjacent to the degron, inhibits p85β binding to FBXL2. Dephosphorylation of phospho-Tyr-655 by PTPL1 stimulates p85β binding to and degradation through FBXL2. Finally, defects in the FBXL2-mediated degradation of p85β inhibit the binding of p110 subunits to IRS1, attenuate the PI(3)K signalling cascade and promote autophagy. We propose that FBXL2 and PTPL1 suppress p85β levels, preventing the inhibition of PI(3)K by an excess of free p85 that could compete with p85–p110 heterodimers for IRS1. Phosphatidylinositol-3-OH kinase (PI(3)K) signalling regulates many cellular events such as cell growth and survival. Pagano and colleagues show that the E3 ligase SCF-FBXL2 promotes PI(3)K signalling and inhibits autophagy by targeting the free p85β subunit of the p110–p85 PI(3)K complex for degradation.

DEPTOR is a recently identified inhibitor of the mTOR kinase that is highly regulated at the posttranslational level. In response to mitogens, we found that DEPTOR was rapidly phosphorylated on three serines in a conserved degron, facilitating binding and ubiquitylation by the F box protein βTrCP, with consequent proteasomal degradation of DEPTOR. Phosphorylation of the βTrCP degron in DEPTOR is executed by CK1α after a priming phosphorylation event mediated by either the mTORC1 or mTORC2 complexes. Blocking the βTrCP-dependent degradation of DEPTOR via βTrCP knockdown or expression of a stable DEPTOR mutant that is unable to bind βTrCP results in mTOR inhibition. Our findings reveal that mTOR cooperates with CK1α and βTrCP to generate an auto-amplification loop to promote its own full activation. Moreover, our results suggest that pharmacologic inhibition of CK1 may be a viable therapeutic option for the treatment of cancers characterized by activation of mTOR-signaling pathways.