FKBP12-rapamycin Binding Domain Research Articles

Development of a rapamycin-inducible protein-knockdown system in the unicellular red alga Cyanidioschyzon merolae.

An inducible protein-knockdown system is highly effective for investigating the functions of proteins and mechanisms essential for the survival and growth of organisms. However, this technique is not available in photosynthetic eukaryotes. The unicellular red alga Cyanidioschyzon merolae possesses a very simple cellular and genomic architecture and is genetically tractable but lacks RNA interference machinery. In this study, we developed a protein-knockdown system in this alga. The constitutive system utilizes the destabilizing activity of the FK506-binding protein 12 (FKBP12)-rapamycin-binding (FRB) domain of human target of rapamycin kinase or its derivatives to knock down target proteins. In the inducible system, rapamycin treatment induces the heterodimerization of the human FRB domain fused to the target proteins with the human FKBP fused to S-phase kinase-associated protein 1 or Cullin 1, subunits of the SCF E3 ubiquitin ligase. This results in the rapid degradation of the target proteins through the ubiquitin-proteasome pathway. With this system, we successfully degraded endogenous essential proteins such as the chloroplast division protein dynamin-related protein 5B and E2 transcription factor, a regulator of the G1/S transition, within 2 to 3 h after rapamycin administration, enabling the assessment of resulting phenotypes. This rapamycin-inducible protein-knockdown system contributes to the functional analysis of genes whose disruption leads to lethality.

The allosteric mechanism of mTOR activation can inform bitopic inhibitor optimization.

mTOR serine/threonine kinase is a cornerstone in the PI3K/AKT/mTOR pathway. Yet, the detailed mechanism of activation of its catalytic core is still unresolved, likely due to mTOR complexes' complexity. Its dysregulation was implicated in cancer and neurodevelopmental disorders. Using extensive molecular dynamics (MD) simulations and compiled published experimental data, we determine exactly how mTOR's inherent motifs can control the conformational changes in the kinase domain, thus kinase activity. We also chronicle the critical regulation by the unstructured negative regulator domain (NRD). When positioned inside the catalytic cleft (NRD IN state), mTOR tends to adopt a deep and closed catalytic cleft. This is primarily due to the direct interaction with the FKBP-rapamycin binding (FRB) domain which restricts it, preventing substrate access. Conversely, when outside the catalytic cleft (NRD OUT state), mTOR favors an open conformation, exposing the substrate-binding site on the FRB domain. We further show how an oncogenic mutation (L2427R) promotes shifting the mTOR ensemble toward the catalysis-favored state. Collectively, we extend mTOR's "active-site restriction" mechanism and clarify mutation action. In particular, our mechanism suggests that RMC-5552 (RMC-6272) bitopic inhibitors may benefit from adjustment of the (PEG8) linker length when targeting certain mTOR variants. In the cryo-EM mTOR/RMC-5552 structure, the distance between the allosteric and orthosteric inhibitors is ∼22.7 Å. With a closed catalytic cleft, this linker bridges the sites. However, in our activation mechanism, in the open cleft it expands to ∼24.7 Å, offering what we believe to be the first direct example of how discovering an activation mechanism can potentially increase the affinity of inhibitors targeting mutants.

Bright Molecular Strain Probe Templates for Reporting Protein–Protein Interactions

Imaging protein-protein interactions (PPIs) is a hot topic in molecular medicine in the postgenomic sequencing era. In the present study, we report bright and highly sensitive single-chain molecular strain probe templates which embed full-length Renilla luciferase 8.6-535SG (RLuc86SG) or Artificial luciferase 49 (ALuc49) as reporters. These reporters were deployed between FKBP-rapamycin binding domain (FRB) and FK506-binding protein (FKBP) as a PPI model. This unique molecular design was conceptualized to exploit molecular strains of the sandwiched reporters appended by rapamycin-triggered intramolecular PPIs. The ligand-sensing properties of the templates were maximized by interface truncations and substrate modulation. The highest fold intensities, 9.4 and 16.6, of the templates were accomplished with RLuc86SG and ALuc49, respectively. The spectra of the templates, according to substrates, revealed that the colors are tunable to blue, green, and yellow. The putative substrate-binding chemistry and the working mechanisms of the probes were computationally modeled in the presence or absence of rapamycin. Considering that the molecular strain probe templates are applicable to other PPI models, the present approach would broaden the scope of the bioassay toolbox, which harnesses the privilege of luciferase reporters and the unique concept of the molecular strain probes into bioassays and molecular imaging.

COVID-19 liver and gastroenterology findings: An in silico analysis of SARS-CoV-2 interactions with liver molecules.

BACKGROUNDCoronavirus disease 19 (COVID-19) has not only been shown to affect the respiratory system, but has also demonstrated variable clinical presentations including gastrointestinal tract disorders. In addition, abnormalities in liver enzymes have been reported indicating hepatic injury. It is known that severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) might infect cells via the viral receptor angiotensin-converting enzyme 2 (ACE2) which is expressed in several organs including the liver. The viral Spike glycoprotein binds to ACE2 and must be cleaved by Furin and Type 2 Serine Protease to enter the cells. After that, the Akt/mTOR signaling pathway is activated and several COVID-19 changes are triggered.AIMTo analyze liver and gastrointestinal symptoms and cell signaling pathways triggered by SARS-CoV-2 infection due to virus-liver interactions in silico.METHODSIn this in silico study, the three-dimensional structures of the Akt, mTORC1 and Furin (receptors) were selected from the Protein Data Bank (PDB) and the structures of inhibitors (ligands) MK-2206, CC-223 and Naphthofluorescein were selected from PubChem and ZINC databases. Ligand files were downloaded as 2D structures and converted to optimized 3D structures using ViewerLite 4.2 software. Marvin Sketch® software was used to calculate prediction of the protonated form of inhibitors in a physiological environment (pH 7.4). AutoDock Tools (ADT) software was used to calculate and delimit the Grid box used in the molecular docking of each structure selected in the PDB. In addition, protonated ligands were prepared for molecular docking using ADT software. Molecular docking was performed using ADT software tools connected to Vina software. Analysis of the amino acid residues involved in ligand interactions, as well as ligand twists, the atoms involved in interactions, bond type and strength of interactions were performed using PyMol® and Discovery Studio® (BIOVIA) software.RESULTSMolecular docking analysis showed that the mTORC1/CC-223 complex had affinity energy between the receptor and ligand of -7.7 kcal/moL with interactions ranging from 2.7 to 4.99 Å. There were four significant chemical bonds which involved two of five polypeptide chains that formed the FKBP12–Rapamycin-Binding (FRB) domain. The strongest was a hydrogen bond, the only polar interaction, and Van der Waals interactions shown to be present in 12 residues of mTORC1’s FRB domain. With regard to the Akt/MK-2206 complex there were three Van der Waals interactions and 12 chemical bonds in which seven residues of Akt were involved with all five rings of the MK-2206 structure. In this way, both ASP 388 and GLN 391 bind to the same MK-2206 ring, the smaller one. However, LYS 386 had four chemical bonds with the inhibitor, one with each structure ring, while LYS 387 binds two distinct rings. One of the MK-2206 inhibitor's rings which binds to LYS 387 also binds simultaneously to ILE 367 and LEU 385 residues, and the fifth ring of the structure was involved in a bond with the ALA 382 residue. The hydrogen bonds were the shortest bonds in the complex (2.61 and 3.08 Å) and all interactions had an affinity energy of -8.8 kcal/moL. The affinity energy in the Furin/Naphhofluorescein complex was -9.8 kcal/moL and involved six interactions ranging from 2.57 to 4.98 Å. Among them, two were polar and the others were non-polar, in addition to twelve more Van der Waals interactions. Two distinct hydrogen bonds were formed between Furin and its inhibitor involving GLN 388 and ALA 532 residues. ALA 532 also binds to two distinct rings of Naphthofluorescein, while TRP 531 residue has two simultaneous bonds with the inhibitor.CONCLUSIONLiver infection and signaling pathways altered by SARS-CoV-2 can be modulated by inhibitors that demonstrate significant interaction affinity with human proteins, which could prevent the development of infection and symptoms.

A Split Methyl Halide Transferase AND Gate That Reports by Synthesizing an Indicator Gas.

Monitoring microbial reactions in highly opaque or autofluorescent environments like soils, seawater, and wastewater remains challenging. To develop a simple approach for observing post-translational reactions within microbes situated in environmental matrices, we designed a methyl halide transferase (MHT) fragment complementation assay that reports by synthesizing an indicator gas. We show that backbone fission within regions of high sequence variability in the Rossmann domain yields split MHT (sMHT) AND gates whose fragments cooperatively associate to synthesize CH3Br. Additionally, we identify a sMHT whose fragments require fusion to pairs of interacting partner proteins for maximal activity. We also show that sMHT fragments fused to FKBP12 and the FKBP-rapamycin binding domain of mTOR display significantly enhanced CH3Br production in the presence of rapamycin. This gas production is reversed in the presence of the competitive inhibitor of FKBP12/FKPB dimerization, indicating that sMHT is a reversible reporter of post-translational reactions. This sMHT represents the first genetic AND gate that reports on protein-protein interactions via an indicator gas. Because indicator gases can be measured in the headspaces of complex environmental samples, this assay should be useful for monitoring the dynamics of diverse molecular interactions within microbes situated in hard-to-image marine and terrestrial matrices.

Abstract 662: A discovery of a small molecule mTORC1 allosteric inhibitor targeting cancer cells

Abstract The allosteric inhibition of the mechanistic target of rapamycin complex 1 (mTORC1), the central growth regulator that is highly activated in cancer cells, is unique for rapamycin and rapalogs. Upon binding to 12-kDa FK506-binding protein (FKBP12), the FKBP12-rapamycin specifically complexed with mTORC1 and inhibits the mediated downstream signals incorporated in metabolic processes. Since rapamycin has a large molecular weight and a poor solubility, it does not completely inhibit mTORC1 due to the pharmacokinetic limitations. It just shows a cytostatic effect in the treatment of a few benign cancers which results in tumors re-growth. In order to overcome these limitations, we have looked for a novel potential small molecule mTORC1 allosteric inhibitor by in silico and in vitro methods. Firstly, we carried out the in silico selection using Virtual Ligand Screening (VLS) for 105 compounds to select ligands with the high calculated scores toward FRB/FKBP12 interface as a receptor (PDB ID: 1fap). After further score calculations, we selected the top 5 compounds for the next selection step. We also prepared FKBP-rapamycin binding domain (FRB) of the mTORC1 and FKBP12, by using pET15b expression vector and BL21(DE3) E. coli. For the molecular-level protein: protein interaction (PPI) measurement, we used AlphaLISATM system (PerkinElmer, USA) using the prepared proteins. In addition, we checked the kinase activity using AlphaLISA® p-P70 S6K (T389) assay kit (PerkinElmer, USA), as well as Western blotting against p-S6 (S235/236) and p-4E-BP1 (T37/46). Finally, we performed the cell cytotoxicity assay of the selected compound against cancer and noncancer cell lines. The docking simulations showed that the residues involved in the interaction between FRB/FKBP12 binding site and rapamycin are partially overlapped with those of the screened compounds which stabilized the formation of FRB:Ligand:FKBP12 ternary complex. We also measured the inhibitory effect of the selected compounds against the ternary complex formation by rapamycin and two of them, ZNC1 and ZNC5, showed interactivity by IC50 of 30 nM and 41 nM, respectively. On the other hand, only ZNC5 promoted the formation of the ternary complex by a KD value of 1.7 nM. In addition, ZNC5 inhibited the kinase activity not only in the starvation condition by IC50 of 1.2 nM but also in the feeding-induced mTORC1 activation condition with IC50 of 3.5 nM. Moreover, it has been observed that the ZNC5 inhibited kinase activity of mTORC1 in the cancer cells while no effect on the normal cells. In this study, we discovered the ZNC5, a new allosteric inhibitor to the kinase of mTORC1, through the in silico and in vitro hybrid selection. The smaller size of ZNC5 comparing to rapamycin is probably advantageous for better pharmacokinetics in vivo. Most importantly, ZNC5 specifically inhibited the growth of cancer cells over normal cells, while no such specificity observed by rapamycin. Citation Format: Raef Shams, Yoshihiro Ito, Hideyuki Miyatake. A discovery of a small molecule mTORC1 allosteric inhibitor targeting cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 662.

Abstract IA05: Dimerization is critical for the functions of wild-type and mutant KRAS

Abstract Background: Mutations in KRAS are among the most frequent RAS alterations in human cancers and the prevalent driver event in lung adenocarcinoma (LUAD). There are no effective targeted therapies for KRAS-driven LUAD and chemotherapy remains the standard of care. Small-molecule inhibitors of the MAPK pathway, one of the prominent downstream KRAS mediators, show minimal clinical activity either as single agents or in combination with chemotherapy. Recent data demonstrated that loss of wild-type KRAS enhances tumor fitness in KRAS mutant cancer cells while concomitantly increasing sensitivity to MEK inhibition. We hypothesized that dimerization/oligomerization between KRAS proteins could be a key regulator for lung adenocarcinoma biology and determinant of treatment response. Methods: To study the role of KRAS dimerization, we selected and characterized a dimerization-impaired mutant KRAS (KRASD154Q). KRAS-driven murine and human LUAD cell lines lacking the wild-type KRAS allele were reconstituted with either KRASWT or KRASD154Q to evaluate their impact on tumor progression and response to MEK inhibition. In parallel, we generated an inducible system to force either wild-type/mutant or mutant/mutant KRAS dimerization by taking advantage of the rapamycin-analog mediated heterodimerization of FK506 binding protein (FKBP) and FKBP-rapamycin binding domain (FRB). The rapamycin-analog AP21967 induces FKBP/FRB dimerization, hence allowing a tunable control of wild-type/mutant versus mutant/mutant KRAS dimers formation. Results: We demonstrated, using FRET (Fluorescence Resonance Energy Transfer) assays, that KRASD154Q mutant disrupts KRAS dimerization without affecting membrane localization or GTPase activity. Introduction of KRASWT into LUAD cells that solely express mutant KRAS decreased their growth both in vitro and in vivo. In the presence of KRASWT both the IC50 and drug concentration required to inhibit pERK1/2 by MEK inhibitors increased in vivo and in vitro. These findings were not observed in the presence of the dimerization-impaired wild-type KRAS protein (KRASD154Q). Similarly, forced dimerization between wild-type/mutant KRAS resulted in impaired cell growth as compared to forced mutant/mutant KRAS dimerization. Interestingly, introduction of the D154Q in cis with an oncogenic KRAS mutation (G12C or G12D), in the absence of the wild-type KRAS allele led to a significant reduction of growth both in vitro and in vivo without impacting GTPase activity. Mechanistically, impaired mutant KRAS dimerization resulted in inefficient CRAF/BRAF heterodimers formation. Conclusions: Dimerization of wild-type KRAS with mutant KRAS results in growth inhibition and changes the therapeutic index for MEK inhibitors. Mutant-mutant KRAS dimerization is critical for the full oncogenic properties of mutant KRAS. Collectively, these observations suggest that strategies designed to interfere with KRAS dimerization should be evaluated as a therapeutic approach in KRAS-mutant cancers. Citation Format: Chiara Ambrogio. Dimerization is critical for the functions of wild-type and mutant KRAS [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA05.

Expanding the Chemogenetic Toolbox by Circular Permutation

To expand the repertoire of chemogenetic tools tailored for molecular and cellular engineering, we describe herein the design of cpRAPID as a circularly permuted rapamycin-inducible dimerization system composed of the canonical FK506-binding protein (FKBP) and circular permutants of FKBP12–rapamycin binding domain (cpFRB). By permuting the topology of the four helices within FRB, we have created cpFRB–FKBP pairs that respond to ligand with varying activation kinetics and dynamics. The cpRAPID system enables chemical-controllable subcellular redistribution of proteins, as well as inducible transcriptional activation when coupled with the CRISPR activation (CRISPRa) technology to induce a GFP reporter and endogenous gene expression. We have further demonstrated the use of cpRAPID to generate chemically switchable split nanobody (designated Chessbody) for ligand-gated antigen recognition in living cells. Collectively, the circular permutation approach offers a powerful means for diversifying the chemogenetics toolbox to benefit the burgeoning synthetic biology field.

Protein-Protein Interaction Mapping by 2C-BioID.

Protein-protein interactions (PPIs) add an essential layer of complexity to the information encoded by the genome. Modulation of such interactions is a key feature of most, if not all, cellular activities and allows cells to respond rapidly to both internal and external signals and stimuli. In this respect, the development of the BioID assay to interrogate PPIs within a cellular context represents an important adjunct to the range of tools currently at researchers' disposal. To address some of its current limitations, we devised 2C-BioID, in which biotin ligase and the protein of interest remain as separate entities until induced to associate. This is accomplished using the well-established FKBP-FRB dimerization system (based on the rapamycin-induced binding of FK506 binding protein and FKBP12-rapamycin binding domain.). The design of 2C-BioID ensures that biotin ligase association with the protein of interest occurs only after addition of the rapamycin analogue AP21967. As such, 2C-BioID alleviates potential targeting issues and improves the ability to exclude false positives, thereby refining the specificity of BioID-generated interactomes. © 2019 by John Wiley & Sons, Inc.

Programming Post-Translational Control over the Metabolic Labeling of Cellular Proteins with a Noncanonical Amino Acid.

Transcriptional control can be used to program cells to label proteins with noncanonical amino acids by regulating the expression of orthogonal aminoacyl tRNA synthetases (aaRSs). However, we cannot yet program cells to control labeling in response to aaRS and ligand binding. To identify aaRSs whose activities can be regulated by interactions with ligands, we used a combinatorial approach to discover fragmented variants of Escherichia coli methionyl tRNA synthetase (MetRS) that require fusion to associating proteins for maximal activity. We found that these split proteins could be leveraged to create ligand-dependent MetRS using two approaches. When a pair of MetRS fragments was fused to FKBP12 and the FKBP-rapamycin binding domain (FRB) of mTOR and mutations were introduced that direct substrate specificity toward azidonorleucine (Anl), Anl metabolic labeling was significantly enhanced in growth medium containing rapamycin, which stabilizes the FKBP12-FRB complex. In addition, fusion of MetRS fragments to the termini of the ligand-binding domain of the estrogen receptor yielded proteins whose Anl metabolic labeling was significantly enhanced when 4-hydroxytamoxifen (4-HT) was added to the growth medium. These findings suggest that split MetRS can be fused to a range of ligand-binding proteins to create aaRSs whose metabolic labeling activities depend upon post-translational interactions with ligands.

Blockade of Y177 and Nuclear Translocation of Bcr-Abl Inhibits Proliferation and Promotes Apoptosis in Chronic Myeloid Leukemia Cells.

The gradual emerging of resistance to imatinib urgently calls for the development of new therapy for chronic myeloid leukemia (CML). The fusion protein Bcr-Abl, which promotes the malignant transformation of CML cells, is mainly located in the cytoplasm, while the c-Abl protein which is expressed in the nucleus can induce apoptosis. Based on the hetero-dimerization of FKBP (the 12-kDa FK506- and rapamycin-binding protein) and FRB (the FKBP-rapamycin binding domain of the protein kinase, mTOR) mediated by AP21967, we constructed a nuclear transport system to induce cytoplasmic Bcr-Abl into nuclear. In this study, we reported the construction of the nuclear transport system, and we demonstrated that FN3R (three nuclear localization signals were fused to FRBT2098L with a FLAG tag), HF2S (two FKBP domains were in tandem and fused to the SH2 domain of Grb2 with an HA tag) and Bcr-Abl form a complexus upon AP21967. Bcr-Abl was imported into the nucleus successfully by the nuclear transport system. The nuclear transport system inhibited CML cell proliferation through mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 5 (STAT5) pathways mainly by HF2S. It was proven that nuclear located Bcr-Abl induced CML cell (including imatinib-resistant K562G01 cells) apoptosis by activation of p73 and its downstream molecules. In summary, our study provides a new targeted therapy for the CML patients even with Tyrosine Kinase Inhibitor (TKI)-resistance.

Anterograde Transport of Rab4-Associated Vesicles Regulates Synapse Organization in Drosophila

Local endosomal recycling at synapses is essential to maintain neurotransmission. Rab4GTPase, found on sorting endosomes, is proposed to balance the flow of vesicles among endocytic, recycling, and degradative pathways in the presynaptic compartment. Here, we report that Rab4-associated vesicles move bidirectionally in Drosophila axons but with ananterograde bias, resulting in their moderate enrichment at the synaptic region of the larval ventralganglion. Results from FK506 binding protein (FKBP) and FKBP-Rapamycin binding domain (FRB) conjugation assays in rat embryonic fibroblasts together with genetic analyses in Drosophila indicate that an association with Kinesin-2 (mediated by the tail domain of Kinesin-2α/KIF3A/KLP64D subunit) moves Rab4-associated vesicles toward the synapse. Reduction in the anterograde traffic of Rab4 causes an expansion of the volume of the synapse-bearing region in the ventral ganglion and increases the motility of Drosophila larvae. These results suggest that Rab4-dependent vesicular traffic toward the synapse plays a vital role in maintaining synaptic balance in this neuronal network.

A Novel Assay to Identify the Trafficking Proteins that Bind to Specific Vesicle Populations.

Here we describe a method capable of identifying interactions between candidate trafficking proteins and a defined vesicle population in intact cells. The assay involves the expression of an FKBP12-rapamycin binding domain (FRB)-tagged candidate vesicle-binding protein that can be inducibly linked to an FKBP-tagged molecular motor. If the FRB-tagged candidate protein binds the labeled vesicles, then linking the FRB and FKBP domains recruits motors to the vesicles and causes a predictable, highly distinctive change in vesicle trafficking. We describe two versions of the assay: a general protocol for use in cells with a typical microtubule-organizing center and a specialized protocol designed to detect protein-vesicle interactions in cultured neurons. We have successfully used this assay to identify kinesins and Rabs that bind to a variety of different vesicle populations. In principle, this assay could be used to investigate interactions between any category of vesicle trafficking proteins and any vesicle population that can be specifically labeled.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract 1724: Genomic mechanisms of exquisite sensitivity and acquired resistance to everolimus in a patient with anaplastic thyroid carcinoma

Abstract Understanding genetic mechanisms of sensitivity and resistance to targeted anticancer therapies may improve patient selection and rational treatment designs. One approach to increase this understanding involves the study of exceptional responders: rare patients with unexpected exquisite sensitivity or durable responses to therapy. We identified an exceptional responder on a study of the allosteric mTOR inhibitor everolimus in thyroid cancer: a 57-yr-old woman with refractory metastatic anaplastic thyroid carcinoma (ATC), a highly aggressive neoplasm with no adequate therapies and a median survival of 5 months. After beginning treatment with everolimus, the patient experienced a near complete response that lasted for 18 months, followed by progressive disease, which was then re-biopsied. To date, mechanisms of clinical resistance to mTOR inhibition have not been described. We performed whole exome sequencing (WES) of both pre-treatment and drug resistant tumor tissue to look for the underlying mechanisms of exquisite sensitivity and acquired resistance to everolimus. WES of the pre-treatment tumor revealed a somatic nonsense mutation in TSC2, a tumor suppressor gene whose inactivation is known to activate the mTOR pathway and result in sensitivity to mTOR inhibition in some cancers. WES of the drug resistant tumor additionally revealed a mutation in mTOR (mTOR-F2108L) not detected in the pre-treatment tumor. Structural modeling demonstrated that this mutation occurs in the FKBP12-rapamycin binding domain of mTOR and is predicted to prevent binding of the drug to the protein. Overexpressing mTOR-F2108L in HEK-293T cells resulted in significant resistance to rapamycin compared to cells expressing wild type (wt) mTOR. In cells expressing the mutant mTOR, rapamycin did not decrease phosphorylation of S6K1, a downstream target of mTOR, compared with cells expressing wt mTOR. Notably, cells expressing mTOR-F2108L remained sensitive to the direct TOR inhibitor torin, suggesting a therapeutic approach to overcome resistance in this patient. In summary, we add ATC to the growing list of cancers found to be exquisitely sensitive to everolimus when activating mTOR pathway mutations are present. Moreover, we present the first reported, to our knowledge, mechanism of acquired resistance to everolimus identified in patients. The fact that this occurs via a binding domain mutation that blocks allosteric mTOR inhibition suggests that followup therapy with direct TOR inhibitors may still have benefit in some patients who develop resistance to everolimus. The use of precision medicine approaches in ATC to screen for alterations in the mTOR pathway may help identify subsets of patients who would benefit from targeted therapies directed against mTOR. Moreover, the use of serial biopsies to profile patients who develop resistance to everolimus could dictate optimal followup treatment in ATC and other cancers. Citation Format: Nikhil Wagle, Brian C. Grabiner, Eliezer M. Van Allen, Ali Amin-Mansour, Scott C. Carter, Nathanael Gray, Justine A. Barletta, Scott J. Swanson, Daniel Ruan, David J. Kwiatkowski, Glenn J. Hanna, Robert I. Haddad, David Sabatini, Pasi A. Janne, Levi A. Garraway, Jochen H. Lorch. Genomic mechanisms of exquisite sensitivity and acquired resistance to everolimus in a patient with anaplastic thyroid carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1724. doi:10.1158/1538-7445.AM2014-1724

Catalytic mTOR inhibitors can overcome intrinsic and acquired resistance to allosteric mTOR inhibitors.

We tested the antitumor efficacy of mTOR catalytic site inhibitor MLN0128 in models with intrinsic or acquired rapamycin-resistance. Cell lines that were intrinsically rapamycin-resistant as well as those that were intrinsically rapamycin-sensitive were sensitive to MLN0128 in vitro. MLN0128 inhibited both mTORC1 and mTORC2 signaling, with more robust inhibition of downstream 4E-BP1 phosphorylation and cap-dependent translation compared to rapamycin in vitro. Rapamycin-sensitive BT474 cell line acquired rapamycin resistance (BT474 RR) with prolonged rapamycin treatment in vitro. This cell line acquired an mTOR mutation (S2035F) in the FKBP12-rapamycin binding domain; mTORC1 signaling was not inhibited by rapalogs but was inhibited by MLN0128. In BT474 RR cells, MLN0128 had significantly higher growth inhibition compared to rapamycin in vitro and in vivo. Our results demonstrate that MLN0128 may be effective in tumors with intrinsic as well as acquired rapalog resistance. mTOR mutations are a mechanism of acquired resistance in vitro; the clinical relevance of this observation needs to be further evaluated.

Split focal adhesion kinase for probing protein–protein interactions

Since protein–protein interactions (PPIs) regulate a variety of cellular processes, the detection of PPIs is crucial for elucidating the underlying molecular mechanisms as well as developing therapeutics. In this study, we propose a novel system to detect PPIs using the distinct domains of focal adhesion kinase (FAK). In this system named “split FAK”, the linker and kinase domains in native FAK are tethered separately to two target proteins of interest. The interaction between the target proteins brings the linker and kinase domains into proximity, which leads to phosphorylation at Y397 of the linker domain, recruitment of another tyrosine kinase Src, and phosphorylation at Y576 of the kinase domain. PPIs are readily detected by probing phosphorylation at Y397 and Y576 of these domains. To demonstrate this system, we designed a series of split FAK chimeras with different domain structures. Consequently, dimerizer-induced interaction between FK506-binding protein 12 (FKBP) and the T2098L mutant of FKBP12-rapamycin binding domain (FRB) was clearly detected by probing phosphorylation at the specific tyrosine residues of most of the split FAK chimeras. This is a novel PPI detection system based on a mechanism-inspired design of a trans-activated split kinase.

MTOR kinase structure, mechanism and regulation

The mammalian target of rapamycin (mTOR), a phosphoinositide 3-kinase related protein kinase, controls cell growth in response to nutrients and growth factors and is frequently deregulated in cancer. Here we report co-crystal structures of a truncated mTOR-mLST8 complex with an ATP transition state mimic and with ATP-site inhibitors. The structures reveal an intrinsically active kinase conformation, with catalytic residues and mechanism remarkably similar to canonical protein kinases. The active site is highly recessed due to the FKBP12-Rapamycin binding (FRB) domain and an inhibitory helix protruding from the catalytic cleft. mTOR activating mutations map to the structural framework that holds these elements in place, indicating the kinase is controlled by restricted access. In vitro biochemistry indicates that the FRB domain acts as a gatekeeper, with its rapamycin-binding site interacting with substrates to grant them access to the restricted active site. FKBP12-rapamycin inhibits by directly blocking substrate recruitment and by further restricting active site access. The structures also reveal active site residues and conformational changes that underlie inhibitor potency and specificity.

Signal transduction and signaling networks

Cellular signaling can be complex and dynamic. In the past several decades, it has become clear that many signal transduction pathways are not simple one-way transmissions of upstream stimuli, but are mediated through extremely complicated networks. Our next challenge is to move beyond the static biochemical description of networks into developing a functional quantitative understanding of their behavior. In this Minisymposium, we presented diverse approaches for beginning to achieve this goal. Galit Lahav (Harvard Medical School) opened the session and demonstrated how the temporal dynamics of the tumor suppressor protein p53 in single cells affect cell fate decisions. Cells that naturally show a series of p53 pulses in response to irradiation and recover from the damage were perturbed to instead show sustained p53. This perturbation led to activation of different target genes and pushed cells toward permanent cell cycle arrest. p53 dynamics therefore contribute to transferring information in cells and provide a new axis for pushing cells toward a specific cellular outcome. Andrei V. Karginov (University of Illinois at Chicago) focused on the spatiotemporal regulation of the Src signaling pathway by engineering a chimera of the Src kinase domain and an insertable FKBP12 protein, iFKBP. By fusing the FKBP12-rapamycin binding domain (FRB) to the specific downstream effectors, he restricted the Src activation to the complex it forms with FRB-bearing downstream targets. He presented evidence that a specific complex of Src and FAK leads to focal adhesion, while a complex of Src and p130Cas leads to filopodia formation. Robin E. Lee (Dana-Farber Cancer Institute) demonstrated that, upon tumor necrosis factor-α (TNF-α) stimulation, the early-response genes of NF-κB use memory to assess NF-κB activation. He analyzed the nuclear translocation dynamics of p65, a major NF-κB transcription factor, and then the number of mRNA transcripts for target genes in the same cells. The gene transcriptions of three early-response genes, IL8, A20, and IκB α, were regulated by the fold changes of nuclear NF-κB. His results, supported by computational modeling, suggest that competitive transcription factor–DNA interactions can provide memory of pre-ligand NF-κB states, allowing fold-change signal detection. The NF-κB signaling pathway has been also shown to involve ubiquitin modification of important molecules. Fumiyo Ikeda (Institute of Molecular Biotechnology) reported that an atypical type of ubiquitin chain, linearly linked chains, plays a critical role in the regulation of a Sharpin-dependent, anti-apoptotic pathway downstream of TNF. She presented a critical apoptosis signaling molecule, FADD, as a novel substrate of the Sharpin-containing E3 ligase that plays a role in the regulation of the apoptotic cascade. Jeffery A. Nickerson (University of Massachusetts Medical School) focused on the regulation of mRNA export by phosphatidylinositol 3-kinase/protein kinase B (PI3 kinase/Akt) signal transduction. His group implemented a fluorescence recovery after photobleaching system for screening signal transduction pathways that regulate mRNA nuclear export and the binding of the exon junction complex core proteins. By combining the analysis of mRNA export to the cytoplasm, they showed that the PI3 kinase/AKT pathway regulates not only mRNA export complex formation, but also the rate of mRNA nuclear export. Anna Podgornaia (Massachusetts Institute of Technology) tackled a challenging question to understand the transient protein–protein interaction surface that determines protein pairing by using a systematic mutagenesis approach. She focused on the histidine kinase PhoQ and its cognate response regulator PhoP, which together regulate bacterial signaling. Out of 160,000 PhoQ variants completely randomized at four residues, 4600 variants were signal-responsive. She is exploring why all these 4600 sequences are not used by extant PhoQ orthologues, which only have 260 different interface sequences.

Engineered Manipulation of Signaling Networks: Control of Kinase Activation and Interactions Dissects Parallel Src Pathways

Activation of a kinase in a specific protein complex or subcellular location determines what signaling pathway will be initiated. Current methods typically cannot activate a specific kinase and simultaneously cause it to interact with one specific downstream target. Here we provide such a tool, and use it to determine the role of individual Src-mediated signaling pathways in stimulation of different cell morphological changes. Focal adhesion kinase (FAK) and p130Cas can bind and activate Src kinase, propagating signals through parallel pathways. Localization of Src in the cytoplasm or at the plasma membrane also determines the function of Src. We therefore focused on activation of Src acting through FAK, p130Cas, or at the cytoplasm versus the plasma membrane. An engineered, “insertable” FKBP12 protein (iFKBP) was introduced into the catalytic domain of Src, while FKBP12-rapamycin binding domain (FRB) was attached to the specific downstream effector. This rendered the kinase inactive until rapamycin induced heterodimerization with FRB. Using this technology we restricted Src activation to the complex it formed with FRB-bearing downstream targets. Activation of Src, without targeting specific downstream molecules, led to cell spreading, reorganization of focal adhesions, and the production of filopodia and lamellipodial protrusions. Using the new approach (RapR-TAP), we showed that activation of Src specifically in complex with FAK led to focal adhesion rearrangement and only slow cell spreading, while activation in complex with p130Cas led to rapid spreading and filopodia formation, but no apparent effects on focal adhesions. Comparing Src activation in the cytosol versus at the plasma membrane showed that membrane localization is necessary to stabilize protrusions, whereas cytoplasmic Src drives rearrangement of focal adhesions. This demonstrates a broadly generalizable strategy to activate specific kinase-mediated signaling pathways in living cells.