text:
Das Iringlied (auch Iringsage) ist ein Heldenlied, dessen Ursprung im Frühmittelalter, womöglich auch in der ausgehenden Spätantike vermutet wird. Von Mediävisten wird es als "verlorene Heldensage" eingeordnet, da der Originaltext bis heute nicht gefunden wurde. Die Vermutung, das Werk müsse einst in Form heroisch-dichterischer Verse vorgelegen haben, ist selbst fast 200 Jahre alt und geht auf den Literaturwissenschaftler Jacob Grimm zurück.

Überlieferungen

Die wichtigste Quelle sind die über 400 Jahre nach den beschriebenen Ereignissen verfassten Res gestae Saxonicae, eine Chronologie der sächsischen Geschichte des Mönchs Widukind von Corvey. Dieser "kunstvoll komponierte Text" führte die Überlegungen der Historiker zu einer noch kunstvolleren Vorlage, die sich in mündlicher Überlieferung oder gar als schriftlich festgehaltenes Verslied bis in jene Zeit erhalten habe.

In den Quedlinburger Annalen findet die Iringsage ein weiteres historisches Fundament. Allerdings ist umstritten, ob der einschlägige Abschnitt 'unabhängigen' Quellen entstammt oder aus den 10 Jahre zuvor entstandenen Res gestae Saxonicae übernommen wurde. Die konkrete Handlung ist bereits hier stark zusammengefasst, und die im entsprechenden Abschnitt beschriebenen Geschehnisse enthalten keine über die sächsische Quelle hinausgehenden Angaben.

Weitere Erwähnungen findet der Stoff in der Weltchronik des Mönches Frutolf von Michelsberg und der Herkunftserzählung 'De Origine Gentis Swevorum', der Geschichte vom Ursprung der Schwaben.

Handlung
Als der Frankenkönig Chlodwig nach vielen Eroberungskriegen starb, hinterließ er vier Söhne, von denen Dietrich der Älteste war und bis zur Erbaufteilung das Reich zusammenhielt. Dieser schickte Boten an Irmenfried, König von Thüringen, um sich dessen Freundschaft zu versichern und ihn zum Erbmahl des neuen fränkischen Königs einzuladen. Hiervon erfuhr Amalaberga, die Schwester Dietrichs und Gemahlin des Thüringers, und rief den königlichen Gefolgsmann und Ratgeber Iring, "ein Mann ebenso schlau und wortgewaltig wie löwenkühn" (Übersetzung von Gustav Neckel), zu sich. Sie erinnerte ihn an ihr Verwandtschaftsverhältnis mit dem fränkischen Königshaus – als Tochter Chlodwigs hatte auch sie Anspruch auf einen Anteil am Erbe – und schickte Iring zu König Irmenfried. Vom Rat seines Gefolgsmanns bestärkt, sprach Irmenfried zu den fränkischen Boten, Dietrich solle selbst erscheinen und seiner Gemahlin das Erbrecht abkaufen. Der oberste Gesandte gab zur Antwort: "Mein Herr wird kommen, wie du wünschest, König, und wenn sein Gold nicht schwer genug ist, so legt er dir noch einen Berg von Thüringerköpfen auf die Waage!". Als der Frankenkönig die Worte Irmenfrieds vernahm, rüstete er zum Krieg und zog mit seinem Heer nach Thüringen.

Irmenfried eilte den Franken mit seinen Truppen entgegen, und nach ihrer Begegnung entbrannte eine Schlacht. Nach zwei Tagen des Kampfes griffen die Sachsen als Bundesgenossen Dietrichs in die Schlacht ein, und die Thüringer zogen sich zur Veste Scheidungen an der Unstrut zurück. Iring wurde als Unterhändler zu Dietrich geschickt, um die Bedingungen der Kapitulation auszuhandeln. Dieser blieb jedoch unnachgiebig, und erst als Iring ausschließlich um Schonung von Dietrichs Schwester Amalaberga bat, war der König zum Friedensschluss bereit. Der Ratgeber machte auf den Franken einen so großen Eindruck, dass er ihn in einer geheimen Unterredung fragte, ob er sein Diener werden wolle. Als Dienst für seine Gnade, sein Gold sowie die zahlreichen Ehren und Güter verlangte er, Irmenfried aus seiner Burg hervorzulocken und zu enthaupten, sobald er als Zeichen der Niederlage vor Dietrich knien würde. Iring, der seinen König bereits als gefallenen Mann betrachtete, stimmte schweren Herzens zu. Das Vertrauen des Thüringerkönigs nutzend, ließ er Irmenfried im Glauben seiner Sicherheit zu Dietrichs Heerlager kommen. Dort schlug er seinem früheren Herrn den Kopf ab, während dieser dem Frankenkönig auf Knien seine Demut bezeugte. Sofort verurteilte Dietrich die Tat vor seinen Mannen und sagte, niemand werde Iring dafür belohnen. Wort- und waffengewaltig setzte sich Iring zur Wehr. Er tötete Dietrich, legte den Leichnam Irmenfrieds darüber und erklärte seinen König für gerächt. "Und mit dem Schwerte sich eine Gasse bahnend, ging er davon."

Bezug zur gesicherten Geschichte

Der Untergang des Thüringerreiches wird auf das Jahr 531 datiert. Seine unmittelbare Ursache wird in einem verlorenen Krieg gegen Franken gesehen. Besonders ausschlaggebend scheint hierfür eine verlorene Schlacht an den Ufern der Unstrut gewesen zu sein, wobei allerdings umstritten ist, ob die Sachsen überhaupt in den Konflikt verwickelt waren. Ebenso wird die Veste Scheidungen als Austragungsort der Kämpfe angezweifelt.

Nach Angaben Gregors von Tours kam Irings König nicht durch die Hand des eigenen Dieners um, sondern wurde drei Jahre später unter einem Vorwand zu Verhandlungen mit den fränkischen Merowingern unter Theuderich I. und Chlothar I. nach Zülpich, einem Sitz von Theuderich, gelockt und dort von den Festungsmauern gestürzt. Der Geschichtsschreiber merkt hierzu an: A nesquio quo (lateinisch für 'Man weiß nicht von wem'). Die Unklarheit über den Mörder und die nach damaligem Verständnis unehrenhaften Umstände von Herminafrieds Tod haben die Legendenbildung möglicherweise begünstigt.

Theuderich I. sicherte unmittelbar nach dem Tod Chlodwigs den Bestand des gesamten Frankenreiches. Nach der Erbteilung erhielt er den an das Königreich Thüringen grenzenden Ostteil des damaligen Frankenreiches (später 'Austrasien' genannt) und benötigte die Unterstützung seines Halbbruders Chlothar I., um einen Feldzug führen zu können. Auch der Anlass des Krieges ist durch die Herkunft Theuderichs I. inspiriert: Iring nennt ihn in seiner Rede an Irmfried einen "Mägdesohn" und "leibeigenen Knecht". Er spielt damit auf Theuderichs Mutter an, die im Gegensatz zu Chrodechild, der Mutter seiner drei Halbbrüder, keine Adlige war.

Amalaberga war nicht die Schwester von Theuderich I., sondern die Nichte von Theoderich dem Großen. Herminafried hatte durch sie also keine Ansprüche auf die fränkische Krone, sondern auf den Beistand der Italien beherrschenden Ostgoten, die von ihrer Cousine Amalasuntha regiert wurden.

Parallelen zum Nibelungenlied
Sowohl im hochmittelalterlichen Nibelungenlied wie auch in der Bearbeitung von Friedrich Hebbel spielen die Krieger Iring und Irnfrit bzw. Iring und Thüring eine Rolle in der Schlacht am Hof des Hunnenkönigs, in der mittelalterlichen Originalversion als Verbündete von Etzel und damit auch eines Dietrichs, nämlich König Dietrichs von Bern, der in vielen Interpretationen mit Theoderich dem Großen gleichgesetzt wird.

Varia
Iring ist einer der Namen des Asen Heimdall, dem Gott des Schutzes und des Lichts in der Germanischen Mythologie. Aus dieser Benennung leitet sich auch der Iringsweg ab, ein alter Name der Milchstraße.

Ein fränkischer Adeliger mit dem Namen Iring vermachte dem Kloster Fulda im Jahre 822 – rund 150 Jahre vor dem Erscheinen der Res gestae Saxonicae – die Iringsburg. Diese liegt im ehemaligen Grenzbereich der Königreiche Thüringen und Franken, je nach historischer Auffassung könnte sie Teil des Thüringerreiches gewesen sein und der fränkische Edelmann somit der Namenspatron des Irings der Sage.

Der Skandinavist Felix Genzmer hat den Versuch einer epischen (Re-)Konstruktion unternommen und mittels seiner Kenntnisse des Althochdeutschen eine 'moderne' Versform des Liedes verfasst.

Quellen
 Widukind von Corvey: Res gestae Saxonicae. Reclam, Stuttgart 1992 (Universal-Bibliothek Nr. 7699).

Literatur

 Hilkert Weddige: Heldensage und Stammessage. Iring und der Untergang des Thüringerreiches in Historiographie und heroischer Dichtung. (= Hermaea. Neue Folge 61). Max Niemeyer Verlag, Tübingen 1989, ISBN 3-484-15061-0,  (kostenpflichtig bei de Gruyter Online).
 Felix Genzmer: Vier altdeutsche Heldenlieder. Wissenschaftliche Buchgesellschaft, Darmstadt 1953,(Libelli Bd. 9).
Belletristik
 Ingmar Werneburg: Irings Falke. Radegundes Klage um Thüringen. Ein Trauerspiel in fünf Aufzugen. Scidinge Hall Verlag Zürich, 2013

Weblinks
 Musikalische Verarbeitung der Iringsage

Anmerkungen

Heldenepik
Thüringer Reich
Literatur (Völkerwanderungszeit)

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text:
Home > Vol 7, No 48 >	Chu
Published in Oncotarget V7N48, Nov 29, 2016
Mitotic arrest-induced phosphorylation of Mcl-1 revisited using two-dimensional gel electrophoresis and phosphoproteomics: nine phosphorylation sites identified
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Rong Chu, Sarah E. Alford, Katherine Hart, Anisha Kothari, Samuel G. Mackintosh, Matthew R. Kovak and Timothy C. Chambers _
Rong Chu1,*, Sarah E. Alford1,*, Katherine Hart1, Anisha Kothari1, Samuel G. Mackintosh1, Matthew R. Kovak1, Timothy C. Chambers1
1Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
*These authors have contributed equally to the work
Timothy Chambers, email: chamberstimothyc@uams.edu
Keywords: Mcl-1, phosphorylation sites, mitotic arrest
Abbreviations: MTA, microtubule targeting agent; Cdk, cyclin-dependent kinase; 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; MS, mass spectrometry
Received: April 04, 2016 Accepted: September 26, 2016 Published: October 12, 2016
Microtubule targeting agents (MTAs) characteristically promote phosphorylation and degradation of Mcl-1, and this represents a critical pro-apoptotic signal in mitotic death. While several phosphorylation sites and kinases have been implicated in mitotic arrest-induced Mcl-1 phosphorylation, a comprehensive biochemical analysis has been lacking. Contrary to previous reports suggesting that T92 phosphorylation by Cdk1 regulates Mcl-1 degradation, a T92A Mcl-1 mutant expressed in HeLa cells was phosphorylated and degraded with the same kinetics as wild-type Mcl-1 following vinblastine treatment. Similarly, when Mcl-1 with alanine replacements of all five putative Cdk sites (S64, T92, S121, S159, T163) was expressed, it was also phosphorylated and degraded in response to vinblastine. To analyze Mcl-1 phosphorylation in more detail, two-dimensional gel electrophoresis (2D-PAGE) was performed. While untreated cells expressed mainly unphosphorylated Mcl-1 with two minor phosphorylated species, Mcl-1 from vinblastine treated cells migrated during 2D-PAGE as a train of acidic spots representing nine or more phosphorylated species. Immunopurification and mass spectrometry of phosphorylated Mcl-1 derived from mitotically arrested HeLa cells revealed nine distinct sites, including several previously unreported. Mcl-1 bearing substitutions of all nine sites had a longer half-life than wild-type Mcl-1 under basal conditions, but still underwent phosphorylation and degradation in response to vinblastine treatment, and, like wild-type Mcl-1, was unable to protect cells from MTA treatment. These results reveal an unexpected complexity in Mcl-1 phosphorylation in response to MTAs and indicate that previous work has severely underestimated the number of sites, and thus encourage major revisions to the current model.
Mitotic arrest-induced phosphorylation of Mcl-1 revisited using two-dimensional gel electrophoresis and phosphoproteomics: nine phosphorylation sites identified | Chu | Oncotarget
Mcl-1 is a pro-survival member of the Bcl-2 family of apoptotic regulatory proteins that plays a key role in the survival and development of diverse cell types [1]. Mcl-1 overexpression occurs in many tumor types and is a principal factor in chemoresistance, especially toward inhibitors of the other major pro-survival proteins, Bcl-2 and Bcl-xL [1–6]. Mcl-1 differs from Bcl-2 and Bcl-xL in that it lacks a BH4 domain and contains an extended N-terminal region [7]. This region includes several PEST sequences and sites for ubiquitination, phosphorylation, and caspase cleavage, all of which are important in regulating Mcl-1 stability [7]. In particular, site-specific phosphorylation has been shown to be a key mechanism controlling Mcl-1 expression level. For example, phosphorylation by ERK at T92 and T163 stabilizes Mcl-1 by prolonging its half-life [8], whereas phosphorylation by GSK3β at S159 acts as a signal for ubiquitination and subsequent proteosomal degradation [9]. Phosphorylation of other sites has also been reported and this alters the binding properties of Mcl-1 for its pro-apoptotic partners. For example, Mcl-1 is phosphorylated on S64 by CDK or JNK and this enhances its binding to Bak, Noxa, and Bim [10]. Data showing that stress-induced Mcl-1 degradation requires the activities of pro-apoptotic JNK and of GSK3, the latter inhibited by pro-survival AKT, emphasize the key role that Mcl-1 plays in coordinating death and survival signals [11].
We initially reported that microtubule targeting agents3 (MTAs), including vinblastine, paclitaxel, and vincristine, induced Mcl-1 phosphorylation and degradation in KB3 cells [12, 13]. Additional work from our laboratory [13–16] and independent studies from several other laboratories [17–19] have shown that this is a general response to mitotic arrest in a wide variety of cell lines. In KB3 cells, a key role for Mcl-1 is to sequester Bak, and MTA-induced phosphorylation and degradation results in release and activation of Bak [13]. The relevant sites of phosphorylation and corresponding protein kinases responsible have been the subject of several studies. It was reported that the major sites of phosphorylation in nocodazole-treated HeLa cells were T92 and S64, and that Mcl-1 destruction during mitotic arrest was initiated by phosphorylation of T92 catalyzed by Cdk1/cyclin B1 and subsequent ubiquitination by APC/CCdc20 [17]. In mitotically arrested HCT116 cells expressing FLAG-Mcl-1, phosphorylation sites were identified as T92, S121, S159 and T163, with Cdk1, p38, CKII and JNK implicated as key protein kinases [19]. Based on these results, a model was proposed whereby Cdk1 phosphorylates T92, displacing bound protein phosphatase PP2A, allowing net phosphorylation at S121, S159, and T163, mediated by p38, CKII, and JNK, respectively, which in turn allowed recruitment of SCFFBW7, resulting in Mcl-1 degradation [19]. Thus, while Cdk1 is hypothesized to play a key priming role to initiate Mcl-1 phosphorylation during mitotic arrest, the nature of the ubiquitin ligase system responsible for subsequent degradation is uncertain.
In this study, two-dimensional gel electrophoresis (2D-PAGE) and phosphoproteomics were employed to analyze phosphorylation of wild-type and phospho-mutant forms of Mcl-1 expressed in HeLa cells. Our results argue against a priming role for Cdk1, show that mitotic arrest-induced Mcl-1 phosphorylation is far more complex than previously suspected, and encourage revisions to current models.
Mcl-1 is highly phosphorylated during mitotic arrest
As we reported previously [13], Mcl-1 undergoes a phosphorylation-induced mobility shift and degradation after vinblastine treatment of HeLa cells (Figure 1A, upper panel, lanes 1 and 2). In order to facilitate analysis of Mcl-1 phosphorylation we sought to identify an antibody which recognized phosphorylated Mcl-1 from mitotically arrested cells. Previously we had utilized a commercially available antibody against S62-phosphorylated Bcl-xL, which recognized phosphorylated Bcl-xL (31 kDa) in extracts from vinblastine–treated HeLa cells [15]. We discovered that this antibody, in addition to recognizing phosphorylated Bcl-xL at 31 kDa, also strongly reacted with a protein of 39 kDa in extracts from vinblastine-treated HeLa cells but not in control extracts (Figure 1A, middle panel, lanes 1 and 2). The 39 kDa protein migrated at a similar position to phosphorylated Mcl-1 observed with the phosphorylation-independent antibody (Figure 1A, upper panel, lane 2). Immunoprecipitation of Mcl-1 from the same samples was performed and Mcl-1 was readily precipitated from both the control and vinblastine-treated cell extracts (Figure 1A, upper panel, lanes 3 and 4). Phosphorylated Mcl-1 immunoprecipitated from vinblastine-treated cells was recognized by the phospho-specific antibody (Figure 1A, middle panel, lane 4), confirming its identity as Mcl-1, whereas phosphorylated Bcl-xL was not detected in the Mcl-1 immunoprecipitate, as expected. When the samples were probed for Bcl-xL (Figure 1A, lower panel), Bcl-xL was observed in the whole cell extracts as expected but not in the Mcl-1 immunoprecipitates, further demonstrating Mcl-1 antibody specificity. Note that with the phosphorylation-independent antibody, phosphorylated (shifted) Bcl-xL reacts more strongly than unphosphorylated Bcl-xL, as we have observed previously [20]. When mock immunoprecipitations were conducted in the absence of antibody, no immunoreactive bands were observed (data not shown). The phospho-specific antibody was next used to assess the status of Mcl-1 phosphorylation in synchronized HeLa cells traversing mitosis without or with vinblastine treatment. As shown in Figure 1B, a band of 39 kDa reactive with the phospho-specific antibody was observed at 9-10 h after release in the absence of vinblastine treatment. Bands corresponding to phospho-H3 histone and reactive with MPM2 antibody, which recognizes mitotic phosphoproteins, were observed with a similar time-course. These results indicate that Mcl-1 is transiently phosphorylated during mitosis, and are consistent with previous results that HeLa cells enter and traverse M phase approximately 9-10 h after release from double thymidine block [21]. Cells were then released in the presence of vinblastine to induce mitotic arrest, confirmed by strong and persistent phospho-H3 and MPM2 staining (Figure 1B). Under these conditions, Mcl-1 underwent a mobility shift and degradation, and corresponding bands reactive with the phospho-specific antibody were observed. The fact that strong immunoreactivity with the phospho-specific antibody was observed concomitant with relatively low levels of total Mcl-1 suggests a very high level of phosphorylation occurs under these conditions, a conclusion supported by subsequent experiments described below. To exclude the possibility that loss of total Mcl-1 expression was an artifact due to reduced affinity of the antibody for highly phosphorylated Mcl-1, we treated cells with vinblastine in the absence or presence of the proteosome inhibitor, MG132. As shown in Figure 1C, MG132 blocked vinblastine-induced loss of Mcl-1 expression, and this was observed with two independent antibodies recognizing different Mcl-1 motifs. This confirms that the loss of Mcl-1 expression was due to protein degradation and was not an artifact resulting from reduced antibody affinity.
Figure 1: Validation of phospho-specific Mcl-1 antibody, kinetics of Mcl-1 phosphorylation in response to vinblastine, and effect of MG132. A. HeLa cells were untreated or treated with vinblastine (VBL) as indicated and whole cell extracts (WCE, lanes 1 and 2) or immunoprecipitated (IP) Mcl-1 (lanes 3 and 4) subjected to immunoblotting for Mcl-1 (upper panel) or with a phospho-specific antibody (p-Ab) originally developed to detect phospho-Bcl-xL (middle panel) or with a phosphorylation-independent Bcl-xL antibody (lower panel). B. HeLa cells were synchronized at the G1/S boundary by double thymidine block and were either untreated or treated with 30 nM vinblastine (VBL) 1 h after release, then harvested at the indicated times. In the untreated group, time points corresponding to late G2-M and G1 phases are indicated. Extracts were subjected to immunoblotting for the proteins indicated, with GAPDH used as a loading control. Phosphorylated Mcl-1 (P-Mcl-1) was detected with an antibody developed to detect phosphorylated Bcl-xL as in Figure 1A. C. HeLa cells were treated with 30 nM vinblastine (VBL) for 24 h, in the presence or absence of 20 μM MG132 added 4 h prior to harvest, and extracts prepared and subjected to immunoblotting for Mcl-1. In the left panel (Mcl-1 Ab-1), Mcl-1 antibody sc-12756 was used, and in the right panel (Mcl-1 Ab-2), Mcl-1 antibody sc-20679 was used.
Phosphorylation of Mcl-1 by Cdk1 is dispensable for vinblastine-induced degradation
Several reports have indicated that Cdk1/cyclin B1 phosphorylates Mcl-1 at T92 during mitotic arrest thus priming it for additional phosphorylation events [17, 19]. Incubation of recombinant Mcl-1 with purified active Cdk1 under phosphorylation conditions confirmed Mcl-1 as an in vitro substrate (Figure 2A). To identify site(s) of Cdk1 phosphorylation, excised gel bands were processed and subject to mass spectrometric analysis, as described in Materials and Methods. As shown in Figure 2B, the MS/MS spectrum of phosphorylated Mcl-1 peptide (VARPPPIGAEVPDVTApTPAR) unambiguously indicate phosphorylation of T92. The peak at 998.88 m/z is consistent with neutral loss of 79.97 Da characteristic of phosphorylation. The b16, b17-98, and b17 ions shown in the detail from 1550-1800 m/z localize phosphorylation of the peptide to T92. Supplementary Figure S1 shows the MS/MS spectrum of the unphosphorylated peptide (VARPPPIGAEVPDVTATPAR), derived from the reaction conducted in the absence of Cdk1 (Figure 2A), for comparison. The b and y ion series are indicative of the same peptide sequence without the mass shift and neutral loss signatures characteristic of phosphorylation.
Figure 2: Phosphorylation of Mcl-1 on T92 by Cdk1 and analysis of Mcl-1 harboring mutations of Cdk consensus sites. A. His-tagged recombinant Mcl-1 was incubated under phosphorylation conditions with [γ-32P]ATP either in the absence or presence of purified active Cdk1/cyclin A2, as indicated, as described in Materials and Methods. Samples were resolved by SDS-PAGE and stained (Coomassie) and subjected to phosphor-image analysis (32P), as indicated. B. Identification of major phosphorylation site as T92. MS/MS spectrum of phosphorylated Mcl-1 peptide VARPPPIGAEVPDVTApTPAR (2093.07 Da monoisotopic molecular weight) with prominent b and y ions and neutral loss product indicated. The peak at 998.88 m/z is consistent with neutral loss of 79.97 Da characteristic of phosphorylation. The b16, b17-98, and b17 ions shown in the detail from 1550-1800 m/z indicate phosphorylation of Thr92. C, D. HeLa cells stably overexpressing wild-type (WT) or T92A (C) or 5A (D) Mcl-1 were untreated or treated with 30 nM vinblastine (VBL) for 24 h, and extracts immunoblotted for the proteins indicated. Parental HeLa cells are shown in the left lanes of each panel; endogenous Mcl-1 is only weakly detected under these conditions. GAPDH was used as a loading control.
HeLa cells overexpressing wild-type and T92A mutant Mcl-1 were generated and treated with vinblastine. As show in Figure 2C, T92A mutant Mcl-1 underwent phosphorylation and degradation similar to wild-type. Furthermore, when Mcl-1 with alanine mutations of all five putative Cdk1 (proline-directed) phosphorylation sites (S64, T70, T92, S121, T163) (5A) was expressed, it too was phosphorylated and degraded (Figure 2D). These results suggest that Cdk1-mediated phosphorylation is dispensable for mitotic arrest-induced Mcl-1 phosphorylation/degradation, and prompted more detailed biochemical analysis.
Analysis by two-dimensional gel electrophoresis
To examine mitotic arrest-induced Mcl-1 phosphorylation in more detail, two-dimensional gel electrophoresis (2D-PAGE)/immunoblotting was employed. In preliminary experiments using standard conditions of sample preparation where proteins such as Bcl-2 or Bcl-xL were readily detected [22], we failed to detect Mcl-1 on the gels after immunoblotting (data not shown). Several different procedures were tested and it was found that the technique of Herbert et al. [23], which improves protein solubility though the use of tributyl phosphine, provided conditions for the detection of Mcl-1. Thus, asynchronous control cells showed one major and two minor, more acidic, spots, the latter sensitive to acid phosphatase treatment (Figure 3A, 3B). Extracts from mitotically arrested cells showed a train of acidic spots that varied in independent experiments from eight to nine distinct species, all but one of which was eliminated by acid phosphatase (Figure 3C, 3D). Whether the non-eliminated spot represents an acidic modification distinct from phosphorylation, or a phospho-species resistant to dephosphorylation, is unclear. The pI of the most acidic form of Mcl-1 was 4.9 (Figure 3C), corresponding closely to the calculated pI of 4.84 for Mcl-1 containing nine phosphates (www.phosphosite.org). Importantly, mitotic cells showed only three phosphorylated Mcl-1 species (data not shown), indicating that mitotic arrest promotes the phosphorylation of many additional sites. After vinblastine treatment of cells expressing the 5A mutant, several phosphorylated species were observed (Figure 3E), but the pattern was much simpler than that of the wild-type protein, consistent with the absence of five sites present in the wild-type, and consistent too with the presence of several non-proline directed sites. These results indicate that Mcl-1 undergoes phosphorylation on a large number of sites during mitotic arrest, including many that are not normally phosphorylated during mitosis and including several that are not proline-directed.
Figure 3: Analysis of Mcl-1 phosphorylation by 2D-PAGE. Samples were prepared for analysis as described in Materials and Methods with isoelectric focusing from right to left and SDS-PAGE from top to bottom. Approximate pH values are shown at the bottom, and arrows indicate unphosphorylated Mcl-1. A. Asynchronous (control) HeLa cells. B. As in A, except that extract was treated with lambda phosphatase (PTASE) before analysis. C. Vinblastine (VBL) treated: HeLa cells were synchronized at the G1/S boundary by double thymidine block, treated with 30 nM VBL 1 h after release, and harvested during mitotic arrest 12 h post-release, with the final 2 h in the presence of 20 μM MG132. D. As in C, except that the extract was treated with PTASE. E. Asynchronous HeLa cells expressing the 5A Mcl-1 mutant. F. HeLa cells expressing the 5A mutant and synchronized and treated with VBL, as in C.
Identification of mitotic arrest-associated Mcl-1 phosphorylation sites
In order to identify the sites of Mcl-1 phosphorylation associated with mitotic arrest, Mcl-1 was purified by preparative immunoprecipitation from synchronized, vinblastine treated HeLa cells, as described in Materials and Methods. Because this type of phosphorylation leads to ubiquitination and proteosome-mediated degradation [17, 19] (Figure 1C), the proteosome inhibitor MG132 was added 2 h before harvest to protect Mcl-1. Analysis of the preparative Mcl-1 immunoprecipitate by SDS-PAGE and Coomassie Blue staining is shown in Supplementary Figure S2A. Several prominently staining bands were evident which were presumed to be protein A/G and light and heavy chains from IgG. A stained band of the expected molecular mass of ~40 kDa was observed (marked by an asterisk in Supplementary Figure S2A), excised, and subjected to tandem MS (MS/MS) using an LTQ Orbitrap Velos mass spectrometer, as described in Materials and Methods. To identify peptides, a UniProt database search using Mascot was run, and Scaffold was then used to validate MS/MS-based identifications. Peptide identifications were accepted if they were scored at ≥ 95% probability by the Scaffold local false-discovery rate (FDR) algorithm. Peptides corresponding to Mcl-1 were observed, with overall sequence coverage of 63% (Figure 4A). MS/MS spectra corresponding to phosphopeptides were also manually validated for unambiguous site localization. Nine phosphorylation sites meeting the FDR algorithm threshold were identified; these were S64, T68, T70, T92, S121, T156, S159, S162, T163 (Figure 4A, 4B). Five of these sites were manually validated (Figure 4B); the corresponding MS/MS spectra are shown in Supplementary Figure S2, panels B-H. Importantly, the total number of sites identified through MS analysis was consistent with data derived from 2D-PAGE analysis. Furthermore, the same nine sites were predicted when the human Mcl-1 sequence was scanned for phospho-motifs (http://scansite3.mit.edu). The kinase(s) whose recognition motifs best match each site is shown in Figure 4B, and similar information was obtained by searching other kinase/phospho-motif databases. Three of the sites are novel and, to our knowledge, have not been reported previously (T68, T70, T156), and the others are established with various kinases ascribed to them [7], with the exception of S162 which was more recently reported [24].
Figure 4: Mcl-1 phosphorylation sites associated with mitotic arrest. A. Immuno-purified Mcl-1 was digested with trypsin and peptides were analyzed by MS/MS, as described in Methods and Materials and Supplementary Figure S2, which identified the peptide sequences shown in bold representing 63% coverage. Sites of phosphorylation are underlined. B. Sites of phosphorylation, sequence context, and predicted kinases based on matches to established recognition motifs. Sites marked with an asterisk were manually validated. See text for details.
Characterization of 9A phospho-mutant Mcl-1
A plasmid encoding Mcl-1 harboring alanine mutations of the nine sites shown in Figure 4 was created, and mutations were verified by sequence analysis. Several clones of HeLa cells stably overexpressing 9A mutant Mcl-1 (Mcl-1-9A) were prepared, and chosen for further study based on exhibiting comparable levels of overexpression versus wild-type Mcl-1. Results using a representative clone are presented in Figure 5A–5C; similar results were obtained with two other clones. Mcl-1 expression in cells stably overexpressing wild-type or Mcl-1-9A is shown in Figure 5A. A time-course of vinblastine treatment confirmed loss of Mcl-1 expression with a concomitant increase in phospho-Mcl-1 level in cells overexpressing wild-type Mcl-1 (Figure 5B), similar to findings with endogenous Mcl-1 (Figure 1). Essentially the same results were found for cells overexpressing Mcl-1-9A; the overall kinetics, and degree of reduction in expression of Mcl-1 and increase in phospho-Mcl-1 levels, were highly similar (Figure 5B). Collectively, these results strongly suggested that Mcl-1-9A, despite mutation of multiple identified phosphorylation sites, still undergoes vinblastine-induced phosphorylation and degradation. To further test this possibility, cells expressing Mcl-1-9A were untreated or treated with vinblastine, and extracts subjected to 2D-PAGE. As shown in Figure 5C, Mcl-1-9A from control cells resolved as a single spot which underwent a discrete shift to a more acidic form after vinblastine treatment, consistent with phosphorylation of the mutated protein possibly at a single additional site.
Figure 5: Vinblastine-induced phosphorylation and degradation, protein stability, and Bak binding properties, of Mcl-1 9A mutants. A. HeLa cells were untransfected, or transfected with vector or with plasmids encoding wild-type (WT) or Mcl-1-9A (with replacements of all nine phosphorylation sites, see Figure 4), and extracts subjected to immunoblotting for Mcl-1. B. Time-course of VBL treatment in HeLa cells expressing WT or Mcl-1-9A. Cells were treated with 30 nM VBL for the times indicated and extracts subjected to immunoblotting for the indicated proteins. C. HeLa cells expressing Mcl-1-9A were untreated (control) or treated with 30 nM VBL for 16 h. Samples were prepared for 2D-PAGE analysis as described in Materials and Methods with isoelectric focusing from right to left and SDS-PAGE from top to bottom. Approximate pH values are shown at the bottom, and arrows indicate unphosphorylated Mcl-1. D. Parental HeLa cells, or cells overexpressing wild-type (WT) or 9A mutant Mcl-1 (two independent clones) were treated with 1 μg/mL cycloheximide for the times indicated, and extracts subjected to immunoblotting for Mcl-1 or GAPDH as indicated. E. Both wild-type and 9A mutant Mcl-1 interact with Bak. Whole cell extracts (WCE) from HeLa cells overexpressing wild-type (WT) or 9A mutant Mcl-1 were immunoprecipitated with Mcl-1 antibody, and subjected to immunoblotting for either Mcl-1 or Bak, as indicated. Mock immunoprecipitations conducted in the absence of Mcl-1 antibody are also shown.
Other studies have indicated that certain phosphorylation sites, including T92 and T163 which are among the sites mutated in Mcl-1-9A, regulate Mcl-1 stability and prolong its half-life [7, 8]. To determine whether mutation of the identified phosphorylation sites altered Mcl-1 stability, parental HeLa cells, cells overexpressing wild-type Mcl-1, or two independent clones overexpressing Mcl-1-9A, were treated with cycloheximide to block protein synthesis, and at intervals up to 4 h, extracts were prepared for the analysis of Mcl-1 by immunoblotting. The results from a representative experiment are shown in Figure 5D. It is evident that the 9A mutants exhibit more persistent expression over this time-course. In order to quantitate the results, the experiment was repeated twice, and band intensity determined, as described in Methods and Materials, which was plotted over time to determine the half-life of Mcl-1 in each case. The following values, given as mean ± S.D., were obtained: endogenous Mcl-1, 26 ± 4 min; wild-type Mcl-1, 50 ± 10 min; 9A-1, 113 ± 9 min; and 9A-2, 118 ± 4 min. Thus mutation of the major phosphorylation sites significantly increased Mcl-1 half-life (p ≤ 0.002, WT vs. 9A-1 or 9A-2).
We have previously shown that an important role for Mcl-1 in HeLa cells is to bind and sequester the pro-apoptotic effector protein Bak [13]. To determine whether mutation of the nine phosphorylation sites altered this property, Mcl-1 was immunoprecipitated from cells overexpressing wild-type or 9A mutant Mcl-1, and immunoblotting performed for Mcl-1 and Bak. As shown in Figure 5E, Bak co-immunoprecipitated with both wild-type and 9A mutant Mcl-1. Quantitation of the results indicated that the ratio of Mcl-1 to Bak was essentially identical in each case (data not shown). Because vinblastine treatment causes Mcl-1 degradation, quantitative assessment of the presence of Bak in Mcl-1 immunoprecipitates from vinblastine treated cells was not possible.
To determine whether expression of Mcl-1-9A affected vinblastine sensitivity, several clones of HeLa cells stably overexpressing the protein (Supplementary Figure S3), were subjected to cell viability assays after vinblastine treatment, as described in Materials and Methods. While there was some variation in sensitivity, the IC50 values obtained were similar in general to that of cells expressing wild-type Mcl-1 and similar to parental HeLa cells (Table 1). Importantly, we have previously shown that wild-type Mcl-1 when transduced into leukemia cells confers resistance to the BH3 mimetics ABT-263 and ABT-199 [25]. Thus in the appropriate context, when the protein remains stable after drug challenge, it confers resistance as expected, and the lack of effect in cells treated with vinblastine can be attributed to the accompanying loss of expression. Furthermore, caspase-3 assays were conducted, and the kinetics and degree of activation of caspase-3 were similar for cells expressing either wild-type or Mcl-1-9A (Figure 6). These results indicate that expression of 9A Mcl-1 does not alter vinblastine sensitivity of HeLa cells.
Table 1: Vinblastine sensitivity of parental HeLa cells and HeLa cells expressing wild-type or 9A mutant Mcl-1
IC50(nM)
0.40 ± 0.05
Mcl-1 WT
Mcl-1 9A #1
IC50 values were determined as described in Materials and Methods. Data shown are mean ± S.D. (n = 6).
Figure 6: Caspase-3 activation in HeLa cells expressing Mcl-1-9A. HeLa cells expressing wild-type (WT) or Mcl-1-9A were treated with 30 nM vinblastine (VBL) for the indicated times, and extracts subjected to caspase-3 assay, as described in Materials and Methods. Results are shown as fold-activation relative to untreated cells, and represent mean ± S.D. (n = 6).
Mcl-1 is subject to several different types of phosphorylation during the cell cycle and in response to various cellular stimuli which alter the stability and properties of this key pro-survival Bcl-2 family member. During normal mitosis two Cdk1-regulated sites have been reported; S64 [10, 17], the phosphorylation of which appears to enhance Mcl-1 pro-survival function [10], and T92, the phosphorylation of which may trigger Mcl-1 degradation [17]. Because mitotic T92 phosphorylation is transient and incomplete, cells can enter and exit mitosis keeping Mcl-1 largely intact and can thus divide successfully without approaching a death threshold [26]. The most dramatic changes in Mcl-1 phosphorylation occur during mitotic arrest, the consequence of which is widespread Mcl-1 degradation [27]. Indeed, Mcl-1 phosphorylation/degradation has been observed in a many cell lines during prolonged mitotic arrest and thus appears to be a universal and conserved response [16]. Existing models of mitotic arrest-induced Mcl-1 phosphorylation [27] suggest that T92 phosphorylation is required, either alone or in concert with phosphorylation of S121, S159, and T163, to trigger Mcl-1 degradation.
The results presented here indicate that current models are incomplete and underestimate the number of sites and in turn the complexity of mitotic arrest-induced Mcl-1 phosphorylation. In addition, contrary to the current paradigm, our results indicate that T92 phosphorylation is not required during mitotic arrest to trigger Mcl-1 phosphorylation and degradation. We identified nine sites of phosphorylation in this context, derived from several independent lines of investigation, including 2D-PAGE, tandem MS/MS, and a database search for phospho-motifs. Each of these approaches gave consistent results with respect to the high number and identity of the phosphorylation sites. Analysis by 2D-PAGE of wild-type Mcl-1 from vinblastine treated cells revealed a complex pattern of phospho-species, the most acidic of which exhibited a pI corresponding to Mcl-1 with nine phosphates (Figure 3C), further reinforcing this conclusion. Analysis of a 5A mutant (Mcl-1 lacking all proline-directed S/T sites), revealed a much simpler but still fairly complex pattern after vinblastine treatment (Figure 3F), confirming the presence of additional, non-proline directed sites. The 9A mutant, lacking all identified phospho-acceptor sites, underwent a discrete incremental acidic shift on 2D-PAGE after vinblastine treatment (Figure 5D) This confirmed that the mutated sites were indeed those phosphorylated endogenously to give rise to the complex train of acidic spots observed with wild-type Mcl-1. That the 9A mutant underwent vinblastine-induced phosphorylation suggests the existence of at least one site in addition to those identified by MS/MS. Several serine and threonine residues are present in the portion of sequence not covered by MS/MS analysis (Figure 4), and thus an additional phosphorylation site may have remained undetected. Alternately, there may be a certain degree of redundancy among the sites of phosphorylation, such that when preferred sites are absent, secondary sites become available for phosphorylation. In the latter case, secondary sites would escape detection upon analysis of wild-type Mcl-1.
Phosphorylation has been widely regarded as a key mechanism triggering Mcl-1 ubiquitination and subsequent degradation during mitotic arrest [26, 27]. In order to rigorously test this hypothesis, a phosphorylation-defective Mcl-1 mutant is needed. The 9A mutant described here is severely under-phosphorylated versus wild-type Mcl-1, but still retains the ability to become phosphorylated and degraded during mitotic arrest. If phosphorylation of the 9A mutant is indeed the trigger for its degradation, this again implies redundancy among the sites. Alternately, phosphorylation may be one pathway leading to Mcl-1 destruction during mitotic arrest, but there may be other pathways. In this regard, the role of Mcl-1 ubiquitination in the regulation of Mcl-1 expression and degradation is instructive. Using a mutant lacking the lysine residues required for ubiquitination, it was observed that murine Mcl-1 degradation by the proteasome can occur in the absence of ubiquitination [28]. Assuming the same holds true for the human homolog, and since the major role of phosphorylation is to promote ubiquitination, it follows that phosphorylation of Mcl-1 may also be dispensable for its degradation during mitotic arrest. Experiments with a completely phospho-defective Mcl-1, which has yet to be described, will be needed to answer this question. Although the lack of many major phosphorylation sites did not alter the ability of Mcl-1 to undergo vinblastine-induced degradation, the absence of these sites did significantly prolong Mcl-1 half-life under basal conditions (Figure 5D). The value of 24 min we obtained for the half-life of endogenous Mcl-1 is similar to a previous study, which reported a value of 40 min [29], emphasizing that Mcl-1 is a short-lived protein.
Many different protein kinases have been reported to phosphorylate Mcl-1; these include GSK3, JNK, ERK, p38, Cdk1/cyclin B1, and CK2 [7]. Recently, evidence was presented that Cdk2/cyclin E also phosphorylates and stabilizes Mcl-1 [30]. In this study, several different protein kinases were predicted to be responsible for the phosphorylation of the sites in Mcl-1 we identified (Figure 4B). For the sites already described in the literature, the identifications in general gave a corresponding match. For example, S64 and T92 were predicted in this study to be phosphorylated by Cdk1, and T163 by GSK3 or ERK, consistent with earlier reports [7]. We identified several sites which had not been previously reported; these included T68, T70, and T156. Interestingly, T156 was predicted to be phosphorylated by AMPK, which is the first preliminary indication that this kinase may play a role in Mcl-1 phosphorylation. These results broaden the number of kinases potentially involved in Mcl-1 phosphorylation.
In previous studies we have found that HeLa cells are critically dependent on Mcl-1 for survival, and if Mcl-1 levels are depleted using siRNA knockdown, the cells die via apoptosis [31]. However, overexpression of wild-type Mcl-1 in HeLa cells does not increase resistance to vinblastine and other inducers of mitotic arrest, whereas overexpression of Bcl-2 or Bcl-xL does impart resistance [15]. The basis for this observation appears to be that vinblastine causes loss of Mcl-1 expression via degradation, whereas Bcl-2 and Bcl-xL remain stably overexpressed after treatment. In this report we have confirmed this result, showing that parental HeLa cells and cells overexpressing Mcl-1 have similar degrees of sensitivity to vinblastine in cell viability assays (Table 1). Similarly, cells overexpressing Mcl-1-9A exhibit sensitivity to vinblastine in the same concentration range (Table 1). That Mcl-1-9A is subject to degradation like wild-type Mcl-1 is likely the reason that cells overexpressing this mutant are not more resistant to vinblastine. The generation and expression of a degradation resistant Mcl-1 will be needed to test the effect of Mcl-1 overexpression on cellular sensitivity to inducers of mitotic arrest. Given that redundant pathways likely exist for Mcl-1 degradation during mitotic arrest, this may be challenging to accomplish.
Antibodies to Mcl-1 (sc-12756 and sc-20679) and Protein A/G PLUS-Agarose Beads (sc-2003) were from Santa Cruz Biotechnology (Dallas, TX); antibody to phospho-Bcl-xL (ab30655) and full length His-tagged human Mcl-1 protein (ab131682) were from Abcam (Cambridge, MA); antibodies to Bcl-xL (2762S), (phospho-histone H3 (9701S) and GAPDH (2118S) were from Cell Signaling Technology (Danvers, MA); purified active Cdk1/cyclin A2 was obtained from SignalChem (Richmond, BC, Canada); [γ-32P]ATP (BLU002A250UC) was from PerkinElmer (Waltham, MA); lambda protein phosphatase (P0753S) was from New England BioLabs (Ipswich, MA); OmniCleave™ endonuclease was from Epicentre (Madison, WI); tributylphosphine (T7567-10VL) and cycloheximide (C104450) were from Sigma-Aldrich (St. Louis, MO); Bio-Lyte 5/7 Ampholytes (163-1112) were from Bio-Rad (Hercules, CA); and porcine sequencing grade modified trypsin was from Promega (Madison, WI).
Cell culture and synchronization
The HeLa human carcinoma cell line was maintained in monolayer culture at 37°C in 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 U/mL penicillin, and 50 g/mL streptomycin. Cell line clones overexpressing Mcl-1 or phospho-mutants were prepared by transfecting HeLa cells with untagged human Mcl-1 in pcDNA3.1 vector followed by selection and maintenance in media containing 0.5 mg/mL G418, as described [22]. Cells were synchronized by double thymidine block method as described previously [32]. Briefly, 106 cells in a 100-mm dish were incubated in medium containing 2 mM thymidine for 16 h, released into normal medium for 9 h, and then incubated for 16 h in medium containing 2 mM thymidine. To assess Mcl-1 protein stability, cells were treated with 1 μg/mL cycloheximide for intervals up to 4 h, and extracts prepared for immunoblotting.
Preparation of cell extracts and immunoblotting
Whole cell extracts were prepared by suspending HeLa cells in 0.25 mL of lysis buffer (40 mM HEPES [pH 7.4], 120 mM NaCl, 1% Triton X-100, 50 mM NaF, 1 mM EDTA, EDTA-free complete protease inhibitor tablets, 1% aprotinin, 50 μg/ml leupeptin, 10 μM pepstatin, 1mM phenylmethylsulfonyl fluoride, 20 mM β-glycerophosphate, 1 mM Na3VO4, and 1 μM okadaic acid). The suspension was incubated for 45 min on ice with occasional mixing, insoluble material was removed by centrifugation (15 min at 12,000 x g), and protein concentration in the supernatant was determined using the BioRad protein assay. Equal amounts of extract were subjected to SDS-PAGE and immunoblotting, and band intensity was quantified using an ImageQuant LAS4000 digital imaging system.
Cells were lysed in 0.5 mL of lysis buffer (40 mM HEPES, pH 7.4, 120 mM NaCl, 1% Triton X-100, 50 mM NaF, 1 mM EDTA, supplemented with protease and phosphatase inhibitors (EDTA-free complete protease inhibitor tablets, 1% aprotinin, 50 μg/ml leupeptin, 10 μM pepstatin, 1 mM phenylmethylsulfonyl fluoride, 20 mM β-glycerophosphate, 1 mM Na3VO4, and 1 μM okadaic acid]) by incubating on ice for 45 min and centrifugation at 12,000 x g for 15 min. The extract (1 mg) was precleared with 50 μL agarose beads, according to the manufacturer's directions (Santa Cruz), and to the supernatant was added 5 μg of monoclonal antibody to Mcl-1. After mixing for 3 h at 4°C, the lysates were then incubated with 100 μL of protein A/G PLUS-Agarose beads overnight at 4°C. The beads were pelleted by centrifugation at 1,000 x g for 5 min and washed three times with 0.3 mL of lysis buffer. The beads were resuspended in 100 μL of 2 × SDS loading buffer and incubated for 5 min at 95°C. The immunoprecipitated samples were resolved by 12.5% acrylamide SDS-PAGE gels (Bio-Rad) and analyzed by immunoblotting. Immunoprecipitation of Bcl-xL was performed as described previously [22].
Preparative immuno-purification of phosphorylated Mcl-1
HeLa cells growing in 14 x 100-mm dishes were synchronized at the G1/S boundary by double thymidine block, treated with 30 nM vinblastine for 15 h, with the final 2 h in the presence of 20 μM MG132, cells were lysed, and 30-35 mg of protein subject to immunoprecipitation with 0.8 mL Mcl-1 antibody and 3 mL of protein A/G PLUS-Agarose beads, as described above. The following steps were carried out essentially as described previously for immunopurification of phosphorylated Bcl-xL [33]. Briefly, washed beads were incubated in 0.5 M NH4OH, 0.5 mM EDTA to elute bound proteins, samples were dried in a speed vacuum, dissolved in 80 μL SDS-PAGE sample buffer, resolved by SDS-PAGE using a 15-cm separation gel, and stained with Colloidal Coomassie Blue.
In vitro phosphorylation of Mcl-1
Reactions mixtures of 0.1 mL contained 25 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 5 mM DTT, 10 μg His-tagged Mcl-1, 0.1 μg Cdk1/cyclin A2, 1 mM ATP, and 1 μCi [γ-32P]ATP. After 2 h at 30°C, reactions were terminated by the addition of EDTA to 20 mM, and subjected to SDS-PAGE. Gels were stained with Colloidal Coomassie Blue, destained, dried and exposed to a phosphoimager screen. For the analysis of Cdk1-catalyzed phosphorylation sites by mass spectrometry, the same reaction was carried out but with the omission of [γ-32P]ATP.
LC-MS/MS methods
Protein gel bands were excised and subjected to in-gel trypsin digestion as follows. Gel slices were destained in 50% methanol, 100 mM NH4HCO3, followed by reduction in 10 mM Tris[2-carboxyethyl]phosphine and alkylation in 50 mM iodoacetamide. Gel slices were then dehydrated in acetonitrile, followed by addition of 100 ng porcine sequencing grade modified trypsin in 100 mM NH4HCO3 and incubation at 37°C for 12-16 hours. Peptide products were then acidified in 0.1% formic acid. Tryptic peptides were separated by reverse phase Jupiter Proteo resin (Phenomenex) on a 100 x 0.075 mm column using a nano2D HPLC system (Eksigent) or a nanoAcquity UPLC system (Waters). Peptides were eluted using a 30 min gradient from 97:3 to 35:65 buffer A:B ratio (buffer A = 0.1% formic acid, 0.5% acetonitrile; buffer B = 0.1% formic acid, 75% acetonitrile). Eluted peptides were ionized by electrospray (1.9 kV) followed by MS/MS analysis using collision induced dissociation on an LTQ Orbitrap or LTQ Orbitrap Velos mass spectrometer (Thermo). MS data were acquired using the FTMS analyzer in profile mode at a resolution of 60,000 over a range of 375 to 1500 m/z. MS/MS data were acquired using the ion trap analyzer in centroid mode and normal mass range with a normalized collision energy of 35.0. Proteins and phosphopeptides were identified by database search using Mascot (Matrix Science), with phosphopeptide MS/MS spectra manually verified.
Site-directed mutagenesis
A plasmid encoding untagged human Mcl-1 in pcDNA3.1 vector served as a template. Serine or threonine to alanine mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The nucleotide sequences of all cDNA constructs were verified by automated DNA sequencing.
Two-dimensional polyacrylamide gel electrophoresis
This was carried out essentially as described [23]. Briefly, cells were washed in PBS and resuspended in 7 M urea, 2M thiourea, 4% CHAPS, 2 mM tributylphosphine (TBP), and 1% Omni Cleave™ Endonuclease. The suspension was incubated on ice for 30 min, and centrifuged at 4°C at 9,200 x g for 15 min. After determination of the protein concentration, samples were rehydrated with rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 2 mM TBP, 0.2% 5/7 Bio-Lyte Ampholytes, and 0.001% Bromophenol Blue). Isoelectric focusing was carried out in 11-cm pH 5/7 strips for 50,000 Vh. The strips were equilibrated with equilibration buffer (0.375 M Tris-HCl, pH 8.8, 2% SDS, 20% Glycerol, and 5 mM TBP) and SDS-PAGE in the second dimension was performed using 8-16% polyacrylamide gels. After transfer to polyvinylidene difluoride membrane, immunoblotting was performed for Mcl-1.
Phosphatase treatment
Cell extracts were prepared by lysing cells under nondenaturing conditions in 40 mM HEPES (pH 7.5), 120 mM NaCl, 50 mM NaF, 1% Triton X-100, 1 mM EDTA, 1% aprotinin, 50 μg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride. Aliquots of 100 μg were incubated with 400 U of lambda protein phosphatase in 25 μL phosphatase buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 5 mM DTT, 0.01% Brij 35, and 2 mM MnCl2) at 30°C for 30 min. The samples were precipitated by the addition of trichloroacetic acid to 25% v/v, dissolved in rehydration buffer and analyzed by 2D PAGE/immunoblotting as described above.
Cells (2,000 per well) were seeded in 96-well plates, and vinblastine at half-log increments from 0 to 1 μM was added in a fixed final concentration of 0.1% DMSO. After incubating for 48 h, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (50 μg/10 μL/well) was added and incubated overnight at 37°C. The following day, 0.1 mL of 10% SDS in 0.01 M HCl was added, and after overnight incubation, absorbance readings were taken at 540 nm. Each condition was conducted with n = 6, and the data plotted and analyzed using GraphPad Prism software.
Caspase-3 assay
Caspase-3 activity was determined as described previously [31].
We thank Dr. Joshua Eichhorn for instruction on isolation of stable transfectants and for critical review of the manuscript, Magdalena Tillman for assistance with the figures, and Dr. Karl Boehme for providing cycloheximide.
The authors declare that they have no conflicts of interest with the contents of this article.
This work was supported by National Institutes of Health grant CA-109821 from the National Cancer Institute (to TCC).
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redpajama_set_name: RedPajamaCommonCrawl


text:
Q: Maximum call stack error for geofire ( firebase-real time database ) I am using real time database as firebase and i used geofire javascript package to get near by data from firebase. but some time it automatically gives error like :
RangeError: Maximum call stack size exceeded
at ChildrenNode.isLeafNode (api/node_modules/firebase-admin/node_modules/@firebase/database-compat/dist/index.standalone.js:8956:50)
at ChildrenNode.equals (api/node_modules/geofire/dist/geofire/index.cjs.js:8461:24)

this is my sample query to find near by things from firebase realtime database using geofire
while (some amount) {
 geoDriverId = await(new Promise((resolve, reject) => {
try {
  geoDriverId = []
  geoDriverIds = []
  const geoQuery = config.firebase.table.query({
    center: [parseFloat(pick_up_latitude), parseFloat(pick_up_longitude)],
    radius: i
  })
  let listener = geoQuery.on("key_entered", (key, location, distance) => {
    geoDriverIds.push({ key, distance });
  });
  geoQuery.on("ready", (key, location, distance) => {
    listener.cancel();
    geoQuery.cancel();
    resolve(geoDriverIds);
    return;
  });
} catch (e) {
  console.log("e", e)
}
}))
}

A: If the intention is to poll for the initial results of the query, consider using the following method instead (this is effectively the code you provided in the question, cleaned up and turned into a method):
// Executes a GeoFire query around the given center and returns only the initial results as a Promise.
function pollGeofireLocation(center: Geopoint, radius: number) {
  return new Promise(resolve => {
    const queryResults = [],
          geoQuery = config.firebase.table.query({ center, radius });

    geoQuery.on("key_entered", (key, location, distance) => {
      queryResults.push({ key, distance }); // consider storing location
    });

    geoQuery.on("ready", () => {
      geoQuery.cancel(); // unsubscribe all event listeners and destroy query
      // consider sorting queryResults before resolving the promise (see below)
      resolve(queryResults);
    });
  });
}

Usage:
const pick_up_geopoint = [parseFloat(pick_up_latitude), parseFloat(pick_up_longitude)];
const results = await pollGeofireLocation(pick_up_geopoint, 10);
// results is an array of { key: string, distance: number } objects,
// in the order they were discovered by the query - not necessarily by distance

To sort the results by the distance from the center point:
const pick_up_geopoint = [parseFloat(pick_up_latitude), parseFloat(pick_up_longitude)];
const nearbyDriverIDs = await pollGeofireLocation(pick_up_geopoint, 10);
// nearbyDriverIDs is an array of { key: string, distance: number } objects,
// in the order they were discovered by the query - not necessarily by distance

nearbyDriverIDs.sort(({ distance: a_dist }, { distance: b_dist }) => a_dist - b_dist);
// nearbyDriverIDs is now an array of { key: string, distance: number } objects,
// ordered by their distance from the center point, closest to furthest.
// nearbyDriverIDs[0], if present (nearbyDriverIDs may be empty!), is the closest driver

If the intention is to find only the closest result, you can use the following method to progressively search for results:
// Executes a GeoFire query around the given center, performing an expanding search where the first result is returned in a Promise.
function findClosestResultToGeofireLocation(center: Geopoint, maxRadius: number, stepRadius: number = 5, startRadius: number = 0) {
  return new Promise((resolve, reject) => {
    const queryResults = [],
          geoQuery = config.firebase.table.query({
            center,
            radius: Math.min(maxRadius, startRadius || stepRadius) // if startRadius is provided, use its value, otherwise start at stepRadius
          });

    geoQuery.on("key_entered", (key, location, distance) => {
      queryResults.push({ key, distance }); // consider storing location
    });

    geoQuery.on("ready", () => {
      // found at least one result? return the closest one
      if (queryResults.length > 0) {
        geoQuery.cancel(); // unsubscribe all event listeners and destroy query
        const closest = queryResults
          .reduce((closest, result) => result.distance < closest.distance ? result : closest);
        resolve(closest);
        return;
      }

      // no results for search criteria and at max search radius? reject with an error
      if (geoQuery.radius() >= maxRadius) {
        geoQuery.cancel(); // unsubscribe all event listeners and destroy query
        reject(new Error(`No results found within ${maxRadius}km`));
        // could also use resolve(null);
        return;
      }

      // expand search area by increasing the radius by stepRadius kilometers,
      // stopping at maxRadius kilometers
      // note: you may want to add a delay before calling this
      geoQuery.updateCriteria({
        radius: Math.min(maxRadius, geoQuery.radius() + stepRadius)
      });
    });
  });
}

Usage:
const pick_up_geopoint = [parseFloat(pick_up_latitude), parseFloat(pick_up_longitude)];
const closestDriverID = await findClosestResultToGeofireLocation(pick_up_geopoint, 25);
// closestDriverID will be the closest driver to pick_up_geopoint.
// With the above arguments, the search will try for drivers <5km away, then
// <10km away, then <15km away, then <20km away and then finally <25km away.
// If a driver is not in range, the returned promise will reject with an error.

redpajama_set_name: RedPajamaStackExchange


FM Broadcast Antennas - Communications Technologies, Inc.Communications Technologies, Inc.
CTI has extensive work experience with most major North American and European FM broadcast antenna manufacturing firms. This experience, coupled with extensive design efforts on directional, nondirectional, single frequency and master antenna systems has placed CTI in a position to offer expert RF engineering design services to broadcasters, tower owners and management companies. In 2003/2004 CTI subcontracted to Riser Management Services of Burlington, Vermont the RF design specification services which resulted in the implementation of a new FM master antenna for The Durst Organization's 4Times Square facility in New York City. Partnering with Riser Management Systems on 4 Times Square is but one example of the benefits that CTI can bring to clients through effective partnerships.


Friction causes power losses and impacts acceleration.
Formulated to flow fast, Castrol POWER1 products minimise internal losses due to friction to deliver superior acceleration at the touch of the throttle.
Castrol Power 1 delivers superior acceleration at the touch of the throttle. Power Release Technology flows fast and minimises internal losses due to friction.  Specially designed to enable your bike to deliver maximum and sustained power right up to very high operating RPM thus offering a significant advantage over other motorcycle engine oils.


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Carpenter bee activity tends to be a nuisance and a threat to your McAllen & Surrounding Area residence. Too many carpenter bees burrowing at once can cause costly structural and aesthetic damage to the side of your home. Woodpeckers will often peck into house siding seeking carpenter bee larvae for food. While they do not sting, male carpenter bees are territorial. Female carpenter bees can sting if provoked which can be a danger to your family and pets.
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SCIPS RESOURCE – 1Please acknowledge. 3/08 How To Write A Letter Of Application Or Cover Letter The usual way of completing an application for a job is to complete a letter explaining why you... How to written an application letter written essay guidelines pay someone to do your research paper how a business plan can help in starting a business help me write a cover letter for my resume 2.2 - written analysis critical thinking questions how to help your child develop problem solving skills pay someone to do thesis.
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Application letter, commonly referred to as a cover letter, is uploaded with your resume and sent to an employer. It indicates your desire to obtain a chosen position and will help management make a decision.


Following my last post on DNS query port usage, here are some more interesting DNS graphs.
The following graphs are based on a packet capture taken from the network interface of a recursive DNS server. This DNS server is one of the primary recursive DNS servers for a small Internet service provider. The capture includes all UDP DNS traffic to the DNS server as well as UDP DNS traffic from the DNS server to addresses within the local AS.
Would you like to share the scripts that you used to generate these graphs?
Hi Rupam, it's been a long time. I don't think I have these scripts anymore.


text:
Your question: Who led the Vietnamese nationalist movement?
Who led the Vietnamese rebellion?
How did nationalism led to independence in Vietnam?
Who led resistance to French colonization and oppression in Vietnam in the 20th century?
Who led Vietminh forces at the battle of Dien Bien Phu?
Who is the dictator of Vietnam?
Who assumed leadership of North Vietnam after 1954 South Vietnam?
Despite financial assistance from the United States, nationalist uprisings against French colonial rule began to take their toll. On May 7, 1954, the French-held garrison at Dien Bien Phu in Vietnam fell after a four month siege led by Vietnamese nationalist Ho Chi Minh.
Many times feelings of nationalism lead a group to demand certain rights and freedoms. This was the case with India and Vietnam in the mid 1900s when nationalist sentiments drove the people of these countries to gain independence from their colonial rulers in Europe.
A new national movement arose in the early 20th century. Its most prominent spokesman was Phan Boi Chau, with whose rise the old traditionalist opposition gave way to a modern nationalist leadership that rejected French rule but not Western ideas, science, and technology. In 1905 Chau went to Japan.
In northwest Vietnam, Ho Chi Minh's Viet Minh forces decisively defeat the French at Dien Bien Phu, a French stronghold besieged by the Vietnamese communists for 57 days.
IT IS IMPORTANT: What to have made in Hoi An?
President of Vietnam
President of the Socialist Republic of Vietnam
Chủ tịch nước Cộng hòa xã hội chủ nghĩa Việt Nam
Incumbent Nguyễn Xuân Phúc since 5 April 2021
Style Mr President (informal) His Excellency (diplomatic)
Type Head of state
In July, a cease fire agreement was reached by the Geneva Accords dividing Vietnam into two provisional states at the 17th parallel of latitude. Ngô Đình Diệm became Prime Minister of South Vietnam and Ho Chi Minh became President of North Vietnam.


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text:
Home»Forts/Historical Places
Kharpocho Fort
Kharphocho Fort (also known as Skardu Fort) is located in Skardu Valley of Gilgit-Baltistan. It is constructed on the eastern side of the Khardong Hill, about 12 meters above Skardu city. As Khardong Hill is centered in the middle of Skardu city, the view from Kharphocho fort captures the entire valley, the Indus River and the mountains around the city.
Kharpocho fort which means king of forts in Balti is the mesmerizing site for a tourist. Fort stands at a height of 40 m above the city. Monarchs of Baltistan built this seven storey fort. An instinctive wad of stone and rock along with the rough edges make it look like being forged out of the mountain itself. This fort dates back to 8th century.
Shigar Fort
The Shigar Fort is located in Shigar Valley of Skardu District, Gilgit-Baltistan. As it is constructed on top of a huge boulder and partially surrounds a large cone-shaped rock, Shigar Fort is also known as Fong-Khar in the Balti language, meaning Palace on the Rock.
Khaplu Palace
Khaplu Palace (meaning "The fort on the roof"), is an old fort and palace located in Khaplu, in the Baltistan region of Gilgit-Baltistan Province in Northern Pakistan. The palace, considered an architectural heritage and a tourist attraction, was built in the mid-19th century, replacing an earlier fort located nearby. It served as a royal residence for the Raja of Khaplu.
From 2005 to 2011 the palace, having earlier stood in vulnerable conditions, underwent a restoration project carried out by the Aga Khan Trust for Culture under the Aga Khan Historic Cities Programme. The palace now houses a hotel operated by Serena and a museum depicting the history and culture of Baltistan.
Baltit Fort
Baltit Fort is an ancient fort in the Hunza valley in Gilgit-Baltistan, Pakistan. Founded in the 1st century, it has been on the UNESCO World Heritage Tentative list since 2004.
In the past, the survival of the feudal regime of Hunza was ensured by the impressive fort, which overlooks Karimabad. The foundations of the fort dated back to 700 years ago, with rebuilds and alterations over the centuries. In the 16th century the local prince married a princess from Baltistan who brought master Balti craftsmen to renovate the building as part of her dowry.
The Mirs of Hunza abandoned the fort in 1945, and moved to a new palace down the hill. The fort started to decay which caused concern that it might possibly fall into ruin. Following a survey by the Royal Geographical Society of London a restoration programme was initiated and supported by the Aga Khan Trust for Culture Historic Cities Support Programme. The programme was completed in 1996 and the fort is now a museum run by the Baltit Heritage Trust.
Altit Fort
Altit Fort is an ancient fort at Altit town in the Hunza valley in Gilgit Baltistan, Pakistan. It was originally home to the hereditary rulers of the Hunza state who carried the title Mir, although they moved to the somewhat younger Baltit fort nearby three centuries later. Altit Fort and in particular the Shikari tower is around 1100 years old, which makes it the oldest monument in the Gilgit–Baltistan.
Kalam Darchi Fort (KD Fort)
Kalam Darchi Fort (KD Fort). KD fort reminds the colonial architecture of the region. This fort was named after a naked saint and was built by British soldier during 1932-33 to check the invasion and inflow of people from China and USSR through Wahkan corridor. It remained the abode of Gilgit Scouts.
The historical buildings and mousques of Ganish Hunza
GANISH, a beautiful village in Hunza valley in Gilgit-Baltistan, is famous for its rich history, splendid wooden mosques, impressive village guest house, imposing towers that mark the landscape. It is situated on the right bank of Hunza River and is one of the oldest settlements of the valley.


Complete analysis for the best course of action.
In today's era where everything is changing and new technologies evolve with much speed it is difficult to catch up with that speed without proper help and guidance. We win the competition only if an organization has the best employees as their asset such an asset is an onsite consultant. Companies go through the changes in all stages; there could be change in staff, departments and also the technologies of course. To process that changes and grow with the change is where onsite consultants come into place and hold a seat for themselves. The changes in an organization could be positive or could not be positive all the time as there will be fluctuations at time as well and that is when the external members or support staff such as onsite consultants takes their place to be a part of your team and help build the growth to it.
While choosing an onsite consultant who will be an external to your organization the business tends to go through its own share of discussions and operational analysis on whether this online consultant is needed or not or also consider the new direction. The review of the discussion is in the best interest of the company and it also acts as an interview for the consulting team to know if the hired online consultant is required and will be able to assist you in the new direction of the job role given to him/her.
There are many situations where business owners will choose the virtual meetings and conferences when they think of choosing external consulting services. Organization basically tend to ask for a firm to check their systems and diagnose by coming up with a plan, or their scope of work, which the business entities can utilize for their work helping their technologies, and operational process to function smoothly. No one can look at a company from outside and decide on what the operational flow is and the requirements with a clear picture.
However if you have an onsite consultant to use or to make recommendations of the directions of the system, team and also the processing, proper analysis in simple words comes from an onsite consultant. Onsite consultant meets the entire team from owners to executives. When you are settling for an onsite consultant firm the organization needs to have the capability to make sure the onsite consultant fills in the gaps for which he/she is hired. Onsite consultant needs to know that day to day operations, and also need to familiarize themselves with the staff members.
The result of onsite consultant is that they can determine the best course of action and takes the company forward. Once the onsite consultant discovers the portion to be completed for an organization a good consulting firm like Techgene will then reevaluate the finding and deliver the proposed path of service as promised in the course of action. This could range from re-implementations of the complete process of the business graph, to the process corrections that need to happen.
Online Consultant would be formal in the recommended scope of work, their resources and duties provided with a time frame estimate the complete scope of the project. Onsite consultants identify the opportunities to keep the business growing. Onsite consultants strive to decrease the employee absence, increase employee productivity, and help decrease the overall cost of disability. Onsite consultant will help increase employee managements at work place, and this will improve their level of work satisfaction, and engagement. The financial burden of employee absence is massive, with costing millions every year for an organization. Having an on-site consultant in place aids employers in keeping costs down and helps their employees stay working.
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