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mTORC1 phosphorylation sites encode their sensitivity to starvation and rapamycin
Kang, Seong A; Pacold, Michael E; Cervantes, Christopher L; Lim, Daniel; Lou, Hua Jane; Ottina, Kathleen; Gray, Nathanael S; Turk, Benjamin E; Yaffe, Michael B; Sabatini, David M
The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) protein kinase promotes growth and is the target of rapamycin, a clinically useful drug that also prolongs life span in model organisms. A persistent mystery is why the phosphorylation of many bona fide mTORC1 substrates is resistant to rapamycin. We find that the in vitro kinase activity of mTORC1 toward peptides encompassing established phosphorylation sites varies widely and correlates strongly with the resistance of the sites to rapamycin, as well as to nutrient and growth factor starvation within cells. Slight modifications of the sites were sufficient to alter mTORC1 activity toward them in vitro and to cause concomitant changes within cells in their sensitivity to rapamycin and starvation. Thus, the intrinsic capacity of a phosphorylation site to serve as an mTORC1 substrate, a property we call substrate quality, is a major determinant of its sensitivity to modulators of the pathway. Our results reveal a mechanism through which mTORC1 effectors can respond differentially to the same signals.
PMCID:3771538
PMID: 23888043
ISSN: 1095-9203
CID: 2245242
The bromodomain protein Brd4 insulates chromatin from DNA damage signalling
Floyd, Scott R; Pacold, Michael E; Huang, Qiuying; Clarke, Scott M; Lam, Fred C; Cannell, Ian G; Bryson, Bryan D; Rameseder, Jonathan; Lee, Michael J; Blake, Emily J; Fydrych, Anna; Ho, Richard; Greenberger, Benjamin A; Chen, Grace C; Maffa, Amanda; Del Rosario, Amanda M; Root, David E; Carpenter, Anne E; Hahn, William C; Sabatini, David M; Chen, Clark C; White, Forest M; Bradner, James E; Yaffe, Michael B
DNA damage activates a signalling network that blocks cell-cycle progression, recruits DNA repair factors and/or triggers senescence or programmed cell death. Alterations in chromatin structure are implicated in the initiation and propagation of the DNA damage response. Here we further investigate the role of chromatin structure in the DNA damage response by monitoring ionizing-radiation-induced signalling and response events with a high-content multiplex RNA-mediated interference screen of chromatin-modifying and -interacting genes. We discover that an isoform of Brd4, a bromodomain and extra-terminal (BET) family member, functions as an endogenous inhibitor of DNA damage response signalling by recruiting the condensin II chromatin remodelling complex to acetylated histones through bromodomain interactions. Loss of this isoform results in relaxed chromatin structure, rapid cell-cycle checkpoint recovery and enhanced survival after irradiation, whereas functional gain of this isoform compacted chromatin, attenuated DNA damage response signalling and enhanced radiation-induced lethality. These data implicate Brd4, previously known for its role in transcriptional control, as an insulator of chromatin that can modulate the signalling response to DNA damage.
PMCID:3683358
PMID: 23728299
ISSN: 1476-4687
CID: 2245232
Revival of the abandoned therapeutic wortmannin by nanoparticle drug delivery
Karve, Shrirang; Werner, Michael E; Sukumar, Rohit; Cummings, Natalie D; Copp, Jonathan A; Wang, Edina C; Li, Chenxi; Sethi, Manish; Chen, Ronald C; Pacold, Michael E; Wang, Andrew Z
One of the promises of nanoparticle (NP) carriers is the reformulation of promising therapeutics that have failed clinical development due to pharmacologic challenges. However, current nanomedicine research has been focused on the delivery of established and novel therapeutics. Here we demonstrate proof of the principle of using NPs to revive the clinical potential of abandoned compounds using wortmannin (Wtmn) as a model drug. Wtmn is a potent inhibitor of phosphatidylinositol 3' kinase-related kinases but failed clinical translation due to drug-delivery challenges. We engineered a NP formulation of Wtmn and demonstrated that NP Wtmn has higher solubility and lower toxicity compared with Wtmn. To establish the clinical translation potential of NP Wtmn, we evaluated the therapeutic as a radiosensitizer in vitro and in vivo. NP Wtmn was found to be a potent radiosensitizer and was significantly more effective than the commonly used radiosensitizer cisplatin in vitro in three cancer cell lines. The mechanism of action of NP Wtmn radiosensitization was found to be through the inhibition of DNA-dependent protein kinase phosphorylation. Finally, NP Wtmn was shown to be an effective radiosensitizer in vivo using two murine xenograft models of cancer. Our results demonstrate that NP drug-delivery systems can promote the readoption of abandoned drugs such as Wtmn by overcoming drug-delivery challenges.
PMCID:3361429
PMID: 22547809
ISSN: 1091-6490
CID: 2245222
Functional genomics reveal that the serine synthesis pathway is essential in breast cancer
Possemato, Richard; Marks, Kevin M; Shaul, Yoav D; Pacold, Michael E; Kim, Dohoon; Birsoy, Kivanc; Sethumadhavan, Shalini; Woo, Hin-Koon; Jang, Hyun G; Jha, Abhishek K; Chen, Walter W; Barrett, Francesca G; Stransky, Nicolas; Tsun, Zhi-Yang; Cowley, Glenn S; Barretina, Jordi; Kalaany, Nada Y; Hsu, Peggy P; Ottina, Kathleen; Chan, Albert M; Yuan, Bingbing; Garraway, Levi A; Root, David E; Mino-Kenudson, Mari; Brachtel, Elena F; Driggers, Edward M; Sabatini, David M
Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell growth and proliferation. RNA interference (RNAi)-based loss-of-function screening has proven powerful for the identification of new and interesting cancer targets, and recent studies have used this technology in vivo to identify novel tumour suppressor genes. Here we developed a method for identifying novel cancer targets via negative-selection RNAi screening using a human breast cancer xenograft model at an orthotopic site in the mouse. Using this method, we screened a set of metabolic genes associated with aggressive breast cancer and stemness to identify those required for in vivo tumorigenesis. Among the genes identified, phosphoglycerate dehydrogenase (PHGDH) is in a genomic region of recurrent copy number gain in breast cancer and PHGDH protein levels are elevated in 70% of oestrogen receptor (ER)-negative breast cancers. PHGDH catalyses the first step in the serine biosynthesis pathway, and breast cancer cells with high PHGDH expression have increased serine synthesis flux. Suppression of PHGDH in cell lines with elevated PHGDH expression, but not in those without, causes a strong decrease in cell proliferation and a reduction in serine synthesis. We find that PHGDH suppression does not affect intracellular serine levels, but causes a drop in the levels of alpha-ketoglutarate, another output of the pathway and a tricarboxylic acid (TCA) cycle intermediate. In cells with high PHGDH expression, the serine synthesis pathway contributes approximately 50% of the total anaplerotic flux of glutamine into the TCA cycle. These results reveal that certain breast cancers are dependent upon increased serine pathway flux caused by PHGDH overexpression and demonstrate the utility of in vivo negative-selection RNAi screens for finding potential anticancer targets.
PMCID:3353325
PMID: 21760589
ISSN: 0028-0836
CID: 1086622
Slow self-activation enhances the potency of viridin prodrugs
Blois, Joseph; Yuan, Hushan; Smith, Adam; Pacold, Michael E; Weissleder, Ralph; Cantley, Lewis C; Josephson, Lee
When the viridin wortmannin (Wm) is modified by reaction with certain nucleophiles at the C20 position, the compounds obtained exhibit an improved antiproliferative activity even though a covalent reaction between C20 and a lysine in the active site of PI3 kinase is essential to Wm's ability to inhibit this enzyme. Here we show that this improved potency results from an intramolecular attack by the C6 hydroxyl group that slowly converts these inactive prodrugs to the active species Wm over the 48 h duration of the antiproliferative assay. Our results provide a guide for selecting Wm-like compounds to maximize kinase inhibition with the variety of protocols used to assess the role of PI3 kinase in biological systems, or for achieving optimal therapeutic effects in vivo . In addition, the slow self-activation of WmC20 derivatives provides a mechanism that can be exploited to obtain kinase inhibitors endowed with physical and pharmacokinetic properties far different from man-made kinase inhibitors because they do not bind to kinase active sites.
PMCID:2663427
PMID: 18630894
ISSN: 1520-4804
CID: 2245212
Using a small molecule inhibitor of peptide: N-glycanase to probe its role in glycoprotein turnover
Misaghi, Shahram; Pacold, Michael E; Blom, Daniël; Ploegh, Hidde L; Korbel, Gregory Alan
Peptide:N-glycanase (PNGase) is ostensibly the sole enzyme responsible for deglycosylation of unfolded N-linked glycoproteins dislocated from the ER to the cytosol. Here we show the pan-caspase inhibitor, Z-VAD-fmk, to be an active site-directed irreversible inhibitor of yeast and mammalian PNGase at concentrations below those used to inhibit caspases in vivo. Through chemical synthesis we determined that the P1 residue, electrophile position, and leaving group are important structural parameters for PNGase inhibition. We show that Z-VAD-fmk inhibits PNGase in living cells and that degradation of class I MHC heavy chains and TCRalpha, in an identical cellular setting, is markedly different. Remarkably, proteasome-mediated turnover of class I MHC heavy chains proceeds even when PNGase is completely inhibited, suggesting that the function of PNGase may be to facilitate more efficient proteasomal proteolysis of N-linked glycoproteins through glycan removal.
PMID: 15610852
ISSN: 1074-5521
CID: 2981762
Yeast N-glycanase distinguishes between native and non-native glycoproteins
Hirsch, Christian; Misaghi, Shahram; Blom, Daniël; Pacold, Michael E; Ploegh, Hidde L
N-glycanase from Saccharomyces cerevisiae (Png1) preferentially removes N-glycans from misfolded proteins. The ability of Png1 to distinguish between folded and misfolded glycoproteins is reminiscent of substrate recognition by UDP-glucose glycoprotein glucosyl transferase, an enzyme that possesses this trait. The only known in vivo substrates of Png1 are aberrant glycoproteins that originate in the endoplasmic reticulum, and arrive in the cytoplasm for proteasomal degradation. The substrate specificity of Png1 is admirably suited for this task.
PMCID:1298977
PMID: 14726951
ISSN: 1469-221x
CID: 2981442
The role of phosphoinositides and phosphorylation in regulation of NADPH oxidase
Perisic, Olga; Wilson, Michael I; Karathanassis, Dimitrios; Bravo, Jerónimo; Pacold, Michael E; Ellson, Chris D; Hawkins, Phillip T; Stephens, Len; Williams, Roger L
PMID: 15581496
ISSN: 0065-2571
CID: 2981712
Identification of domain required for catalytic activity of auxilin in supporting clathrin uncoating by Hsc70
Ma, Yuchen; Greener, Tsvika; Pacold, Michael E; Kaushal, Shivani; Greene, Lois E; Eisenberg, Evan
During clathrin-mediated endocytosis Hsc70, supported by the J-domain protein auxilin, uncoats clathrin-coated vesicles. Auxilin contains both a clathrin-binding domain and a J-domain that binds Hsc70, and it has been suggested that these two domains are both necessary and sufficient for auxilin activity. To test this hypothesis, we created a chimeric protein consisting of the J-domain of auxilin linked to the clathrin-binding domain of the assembly protein AP180. This chimera supported uncoating, but unlike auxilin it acted stoichiometrically rather than catalytically because, like Hsc70, it remained associated with the uncoated clathrin. This observation supports our proposal that Hsc70 chaperones uncoated clathrin by inducing formation of a stable Hsc70-clathrin-AP complex. It also shows that Hsc70 acts by dissociating individual clathrin triskelions rather than cooperatively destabilizing clathrin-coated vesicles. Because the chimera lacks the C-terminal subdomain of the auxilin clathrin-binding domain, it seemed possible that this subdomain is required for auxilin to act catalytically, and indeed its deletion caused auxilin to act stoichiometrically. In contrast, deletion of the N-terminal subdomain weakened auxilin-clathrin binding and prevented auxilin from polymerizing clathrin. Therefore the C-terminal subdomain of the clathrin-binding domain of auxilin is required for auxilin to act catalytically, whereas the N-terminal subdomain strengthens auxilin-clathrin binding.
PMID: 12377777
ISSN: 0021-9258
CID: 2980902