Showing papers by "Sally Kornbluth published in 2008"

Inhibition of Apoptosome Formation by Suppression of Hsp90β Phosphorylation in Tyrosine Kinase-Induced Leukemias

Abstract: Constitutively active tyrosine kinases promote leukemogenesis by increasing cell proliferation and inhibiting apoptosis. However, mechanisms underlying apoptotic inhibition have not been fully elucidated. In many settings, apoptosis occurs by mitochondrial cytochrome c release, which nucleates the Apaf-1/caspase-9 apoptosome. Here we report that the leukemogenic kinases, Bcr-Abl, FLT3/D835Y, and Tel-PDGFRbeta, all can inhibit apoptosome function. In cells expressing these kinases, the previously reported apoptosome inhibitor, Hsp90beta, bound strongly to Apaf-1, preventing cytochrome c-induced Apaf-1 oligomerization and caspase-9 recruitment. Hsp90beta interacted weakly with the apoptosome in untransformed cells. While Hsp90beta was phosphorylated at Ser 226/Ser 255 in untransformed cells, phosphorylation was absent in leukemic cells. Expression of mutant Hsp90beta (S226A/S255A), which mimics the hypophosphorylated form in leukemic cells, conferred resistance to cytochrome c-induced apoptosome activation in normal cells, reflecting enhanced binding of nonphosphorylatable Hsp90beta to Apaf-1. In Bcr-Abl-positive mouse bone marrow cells, nonphosphorylatable Hsp90beta expression conferred imatinib (Gleevec) resistance. These data provide an explanation for apoptosome inhibition by activated leukemogenic tyrosine kinases and suggest that alterations in Hsp90beta-apoptosome interactions may contribute to chemoresistance in leukemias.

Across the meiotic divide - CSF activity in the post-Emi2/XErp1 era.

Abstract: Vertebrate eggs are arrested at the metaphase stage of meiosis II. Only upon fertilization will the metaphase-II-arrested eggs exit meiosis II and enter interphase. In 1971, Masui and Markert injected egg extracts into a two-cell-stage embryo and found that the injected blastomere arrested at the next mitosis. On the basis of these observations, they proposed the existence of an activity present in the eggs that is responsible for meiosis-II arrest and can induce mitotic arrest, and named this activity cytostatic factor (CSF). Although the existence of CSF was hypothesized more than 35 years ago, its precise identity remained unclear until recently. The discovery of the Mos-MAPK pathway and characterization of the anaphase-promoting complex/cyclosome (APC/C) as a central regulator of M-phase exit provided the framework for a molecular understanding of CSF. These pathways have now been linked by the discovery and characterization of the protein Emi2, a meiotic APC/C inhibitor, the activity and stability of which are controlled by the Mos-MAPK pathway. Continued investigation into the mechanism of action and mode of regulation of Emi2 promises to shed light not only on CSF function, but also on the general principles of APC/C regulation and the control of protein function by MAPK pathways.

A gel‐free MS‐based quantitative proteomic approach accurately measures cytochrome P450 protein concentrations in human liver microsomes

Abstract: The human cytochrome P450 (P450) superfamily consists of membrane-bound proteins that metabolize a myriad of xenobiotics and endogenous compounds. Quantification of P450 expression in various tissues under normal and induced conditions has an important role in drug safety and efficacy. Conventional immunoquantification methods have poor dynamic range, low throughput, and a limited number of specific antibodies. Recent advances in MS-based quantitative proteomics enable absolute protein quantification in a complex biological mixture. We have developed a gel-free MS-based protein quantification strategy to quantify CYP3A enzymes in human liver microsomes (HLM). Recombinant protein-derived proteotypic peptides and synthetic stable isotope-labeled proteotypic peptides were used as calibration standards and internal standards, respectively. The lower limit of quantification was approximately 20 fmol P450. In two separate panels of HLM examined (n = 11 and n = 22), CYP3A, CYP3A4 and CYP3A5 concentrations were determined reproducibly (CV or=0.87) and marker activities (r(2)>or=0.88), including testosterone 6beta-hydroxylation (CYP3A), midazolam 1'-hydroxylation (CYP3A), itraconazole 6-hydroxylation (CYP3A4) and CYP3A5-mediated vincristine M1 formation (CYP3A5). Taken together, our MS-based method provides a specific, sensitive and reliable means of P450 protein quantification and should facilitate P450 characterization during drug development, especially when specific substrates and/or antibodies are unavailable.

Cdc2 and Mos Regulate Emi2 Stability to Promote the Meiosis I–Meiosis II Transition

Abstract: The transition of oocytes from meiosis I (MI) to meiosis II (MII) requires partial cyclin B degradation to allow MI exit without S phase entry. Rapid reaccumulation of cyclin B allows direct progre...

Article Aven-Dependent Activation of ATM Following DNA Damage

Abstract: Department of Human ScienceGeorgetown University Medical CenterGeorgetown UniversityWashington, D.C. 20057SummaryBackground: In response to DNA damage, cells undergoeither cell-cycle arrest or apoptosis, depending on the extentof damage and the cell’s capacity for DNA repair. Cell-cyclearrest induced by double-stranded DNA breaks depends onactivation of the ataxia-telangiectasia (ATM) protein kinase,which phosphorylates cell-cycle effectors such as Chk2 andp53 to inhibit cell-cycle progression. ATM is recruited to dou-ble-stranded DNA breaks by a complex of sensor proteins, in-cluding Mre11/Rad50/Nbs1, resulting in autophosphorylation,monomerization, and activation of ATM kinase.Results: In characterizing Aven protein, a previously reportedapoptoticinhibitor,wehavefoundthatAvencanfunctionasanATM activator to inhibit G2/M progression. Aven bound toATMandAvenoverexpressedincyclingXenopuseggextractsprevented mitotic entry and induced phosphorylation of ATMand its substrates. Immunodepletion of endogenous Avenallowed mitotic entry even in the presence of damaged DNA,and RNAi-mediated knockdown of Aven in human cells pre-vented autophosphorylation of ATM at an activating site(S1981) in response to DNA damage. Interestingly, Aven isalsoasubstrateoftheATMkinase.MutationofATM-mediatedphosphorylation sites on Aven reduced its ability to activateATM, suggesting that Aven activation of ATM after DNA dam-age is enhanced by ATM-mediated Aven phosphorylation.Conclusions: These results identify Aven as a new ATM acti-vator and describe a positive feedback loop operating be-tween Aven and ATM. In aggregate, these findings placeAven, a known apoptotic inhibitor, as a critical transducer ofthe DNA-damage signal.IntroductionEntry into mitosis, driven by Cdc2/Cyclin B, can be inhibitedafter DNA damage by DNA-responsive checkpoints [1, 2].These signaling pathways employ DNA-damage sensors andtransducerstoinhibitcell-cycleeffectors[3].Topreventmitoticentry,checkpointpathwaystargetthecriticalCdc2regulators,Wee1 and Cdc25 [4–7], which inhibit and activate Cdc2/CyclinB,respectively[1,2,8].Whencheckpointsareoperative,Chk1/Chk2 kinases phosphorylate Wee1 and Cdc25, inducing acti-vation and inhibition, respectively, of the downstream mole-cules [6, 9]. Cdc25 is phosphorylated and inhibited by Chk1/Chk2-mediated phosphorylation at S287 (Xenopus number-ing), which promotes docking of 14-3-3 [10–14]. Cdc25 activa-tion depends upon active 14-3-3 removal and subsequentPP1-mediated S287 dephosphorylation [15–17].Activation of Chk1/Chk2 requires DNA-damage-mediatedactivation of upstream members of the phosphoinositide-3-ki-nase-related kinases (PIKKs), ATM and ATR [11, 18–21]. In thecase of double-stranded DNA breaks, a complex of proteins,including Mre11, Rad50, and Nbs1 (the MRN complex), accu-mulates at DNA-damage sites to form foci. ATM is then re-cruited to the MRN complex, where it autophosphorylates atS1981 and is converted from an inactive dimer into an activemonomer [22–26]. It is likely that there are other, as-yet-un-identified factors that participate in ATM regulation.A variety of cellular processes have been reconstituted inextracts prepared from Xenopus eggs [27–29]. Cycling eggextracts [30], which oscillate between S and M phases of thecell cycle, can recapitulate DNA-damage-responsive check-points in vitro; addition of annealed oligonucleotides mimick-ing damaged DNA prevents M phase entry, arresting extractswith inactive Cyclin B/Cdc2 complexes [18, 31, 32]. Incubationof egg extracts on the bench for 4–6 hr results in cytochrome crelease from mitochondria and caspase activation [33–35].These cycling and apoptotic properties of the Xenopus eggextracts make them well-suited for the analysis of factorsimplicated in cell-cycle progression or apoptosis.We wished to use egg extracts to analyze the function ofa previously reported apoptotic inhibitor, Aven. Human Avenwas originally identified as an interactor of the anti-apoptoticprotein Bcl-x