CLS-08159; No of Pages 10 Cellular Signalling xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig

Microtubule dynamics regulates Akt signaling via dynactin p150

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Hakryul Jo a,⁎, Fabien Loison b,c, Hongbo R. Luo b,⁎⁎

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Article history: Received 14 March 2014 Accepted 6 April 2014 Available online xxxx

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Keywords: Akt Microtubules Signal transduction Protein phosphorylation Cell death

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Following activation at the plasma membrane, Akt is subsequently deactivated in the cytoplasm. Although activation and deactivation of Akt must sometimes be separated in order to elicit and control cellular responses, the exact details of the spatiotemporal organization of Akt signaling are incompletely understood. Here we show that microtubule dynamics specifically modulate the deactivation phase of Akt signaling. Localization of Akt to microtubules sustains its activity, while disruption of microtubules attenuates Akt signaling independent of its initial activation. Conversely, stabilization of microtubules elevates Akt signaling both in vitro and in muscle tissues in vivo. Localization of Akt to microtubules is mediated by the microtubule binding protein dynactin p150, which is shown to be a direct target of Akt. Finally, microtubule disruption-induced Akt deactivation contributes to delayed cell cycle progression and accelerated cell death. Taken together, we revealed that, after initiation, the overall intensity and duration of oncogenic Akt signaling are determined by microtubule dynamics, a mechanism that could be exploited for therapeutic purposes. © 2014 Elsevier Inc. All rights reserved.

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Environmental Health Sciences, Yale University School of Public Health, New Haven, CT, USA Department of Pathology and Laboratory medicine, Harvard Medical School and Children's Hospital Boston, Boston, MA 02115 USA Department of Microbiology, Faculty of Science, Mahidol University, Phayathai Campus, 272 Rama VI Road, Ratchathewi, Bangkok, 10400, Thailand

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1. Introduction

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Protein phosphorylation is a fundamental signaling process that relays and translates extracellular cues into cellular behaviors. External stimuli activate receptors and recruit kinases to the membrane, which subsequently phosphorylate a cascade of intracellular kinases that amplify and propagate the signal. When considering even a simple model of intracellular signal transduction, it is important to remember that opposing phosphatases regulate signal propagation in the cytoplasm. Thus, dynamic compartmentalization of protein kinases and their deactivating phosphatases is crucially important to avoid futile activation of signaling pathways and fine tune cellular outcomes. The oncogenic Akt kinase is a major mediator of a wide range of cellular responses to external cues and is activated by phosphatidylinositol 3,4,5-triphosphate (PIP3). After activation by various types of receptor, phosphotidylinositol-3′ kinase (PI3K) converts phosphatidylinositol 4,5-biphosphate (PIP2) to PIP3, leading to elevated PIP3 concentrations at the plasma membrane. Akt is then recruited to the membrane via direct binding to PIP3 via its N-terminal pleckstrin homology (PH)

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⁎ Correspondence to: H. Jo, 7th Floor, 1 Church Street, New Haven, CT 06510. Tel.: +1 203 785 5485; fax: +1 203 785 7401. ⁎⁎ Correspondence to: H.R. Luo, Enders Building 811, 320 Longwood Ave, Boston, MA 02115. Tel.: +1 617 919 2303; fax: +1 617 730 0885. E-mail addresses: [email protected] (H. Jo), [email protected] (H.R. Luo).

domain, where it is subsequently phosphorylated and activated by upstream kinases, such as PDK1 and mTOR Complex 2 (mTORC2) [1,2]. PIP3-mediated phosphorylation of Akt is a well-established activating mechanism and is broadly applicable to different cell types in a range of contexts. While the initiation of Akt signaling by phosphorylation at the membrane resembles that of other signaling pathways, the propagation of downstream signaling is rather unique. Activated Akt detaches from the membrane and distributes to different cellular compartments to directly phosphorylate target proteins [3,4]. Live cell imaging studies with fluorescent probes show that phosphorylation of target proteins and deactivation of Akt signaling is variable in both time and space [5–7]. Cellular mechanisms that coordinate the spatial distribution of Akt and opposing phosphatase activity must operate, but the underlying molecular mechanisms that control the spatiotemporal restriction of Akt signaling are ill-defined. In this study, we reveal that microtubule dynamics specifically modulate the deactivation phase of Akt signaling. Activated Akt localizes to microtubules stabilized by a microtubule binding protein dynactin p150. Although microtubule localization is dispensable for the initial activation of Akt, it is required in order to sustain Akt signaling. Finally, we demonstrate that microtubule disruption-induced Akt deactivation contributes to delayed cell cycle progression and accelerated cell death. These results establish that the overall extent of Akt signaling is not only regulated by its initial activation, but also by intrinsic microtubule dynamics mediated by dynactin p150.

http://dx.doi.org/10.1016/j.cellsig.2014.04.007 0898-6568/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: H. Jo, et al., Microtubule dynamics regulates Akt signaling via dynactin p150, Cellular Signalling (2014), http://dx.doi. org/10.1016/j.cellsig.2014.04.007

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2. Materials and methods

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2.1. Cell culture and stable cell lines

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For routine maintenance, both HEK293 and HeLa cells were maintained in DMEM supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin under 5% CO2. All other cells of hematopoietic origin were cultured in RPMI medium with 10% fetal bovine serum and 1% penicillin and streptomycin under 5% CO2. The stable EK293 cell lines expressing the wild-type, Akt1 mutants, or dynactinp150 were generated by transfecting the corresponding expression plasmids. The generation and propagation of stable cell lines were previously described.

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2.2. Reagents and antibodies

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The plasmids encoding human Akt1 was initially obtained from Dana-Farber/Harvard Cancer Center DNA Resource Core. The human dynactin p150 was obtained from Openbiosystems. These plasmids were subcloned into the pcDNA3.1/V5-His-TOPO vector (Invitrogen) by PCR. EGFP-Foxo1 was obtained from Addegene. The siRNAs were obtained from Dharmacon and were mixture of 4 individual siRNAs targeting different regions of each gene. All phosphorylation-specific antibodies were purchased from Cell Signaling Technology; monoclonal V5 antibody was from Invitrogen. Mouse monoclonal antibodies for gamma-Tubulin (T3320), alpha-Tubulin (T6199), and beta-Tubulin (T4026) were from Sigma Aldrich; rabbit polyclonal antibodies for alpha Tubulin (ab18251) and beta-Tubulin (ab6046) were from Abcam. The HRP-conjugated anti-rabbit and anti-mouse secondary antibodies were from GE Health Sciences. All other chemicals unless specified were from Sigma Aldrich and Tocris Bioscience.

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2.3. Immunoblot, immnoprecipitation, and immnunostaining

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Preparation of cell lysates for SDS-PAGE and Western blot and other standard molecular biological technique employed were essentially the same as described previously [8]. For the analysis of microtubule associated phosphorylated Akt, HEK293T cells transfected with the control or dynactin p150 siRNAs were briefly washed in PBS. The cell pellet was frozen on dry ice for 15 minutes and lysed in general tubulin buffer (80 mM PIPES pH 6.9, 2 mM MgCl2 and 0.5 mM EGTA) supplemented with 1 mM GTP, 2 μg Taxol, 0.3% CHAPS, and cocktails of proteases and phophatase inhibitors. The lysate was kept on dry ice for 10 minutes and thawed at room temperature. Once thawed, the cell lysate was immediately centrifuged at 10,000 rpm for 10 minutes at 4 degree and the supernatant and pellet fractions were analyzed separately. For immunoprecipitation, HEK293 cells were lysed in the ice-cold lysis buffer (10 mM TrisCl [pH 7.6], 1 mM EDTA, 150 mM NaCl, 0.3% CHAPS) containing the cocktails of protease inhibitors (Roche) and protein phosphotatase inhibitors (Sigma). The lysates were cleared by centrifugation and incubated with 1–2 μg of V5 antibody for overnight at 4 °C. Thirty microliters of protein G/A-agarose slurry was added and incubated for additional 3 hours. After washing three times with the lysis buffer, the immune complex was resolved on NuPAGE 4–12% BisTris gels and analyzed for the interacting proteins. For immunostaining of Akt1 (1:2000 for V5 antibody) and phospho-Akt (1:200 for pS473), cells were cultured in a 35 mm-glass bottom dish (MatTek Corp.) and fixed in 3% PFA as previously described. For immunostaining of microtubules, cells were fixed for 5 minutes in pre-chilled (−20 °C) methanol. After washing three times in PBS-Triton X-100 (0.05%), the fixed cells were permeabilized for 30 minutes in 5% normal goat serum containing 0.3% Triton X-100. The diluted antibody (1:2000 for primary and 1:1000 for secondary antibody) in the same solution was added and incubated for 3 hours at room temperature. After washing three times in PBSTriton X-100, the Alexa fluor dye-conjugated secondary antibody was added and incubated for 1 hour at room temperature. The staining

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2.4. Time-lapse live cell imaging

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For the analysis of mitotic entrance, HeLa cells growing exponentially (or around 65–70% confluence) in 35 mm dish were tapped several times to detach loosely attached cells (i.e. late G2 and mitotic stages). The resulting culture dishes were washed in 2 mL of Leibovitz's L15 medium supplemented with 10% FBS to remove the detached cells. The plate were replenished with 2 mL of L15 medium and cultured for 10 minutes prior to addition of microtubule agents. The time-lapse movie was taken every 15 minutes for 14 hours. Each movie frame in 14 hour-time period was analyzed for the mitotic entrance. The mitotic cells were identified as they underwent morphological changes from ‘flat to round shape’ and remain mitotic arrested until the end of imaging. For the live cell imaging of PH-EGFP and FOXO-EGFP, HeLa cells expressing each EGFP fusion proteins were plated into a 35-mm glassbottom dish (MatTek) and cultured for 24–48 hours in DMEM medium with serum. Cells were serum-starved for 2 hours in 2 mL Leibovitz's L15 medium and was replaced with 1 mL of fresh serum-free Leibovitz L15 medium containing the microtubule agents and IGF-1. According to experimental requirement, the sequential addition of each drug and IGF-1 in different orders was also performed. The images were taken every 5–10 minutes under a 40× oil objective lens.

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2.5. Measurement of PIP3 level

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The serum-starved HeLa cells (1 × 107) were pre-treated with nocodazole (2 μM) or Taxol (1 μM) for 30 minutes, then stimulated with IGF-1 (20 ng/mL) for 20 minutes. Cells were washed in PBS prior to extraction for membrane lipids. The extraction of PIP3 was done by a sequential centrifugation in Methanol:Chloroform:HCl buffer. The measurement of PIP3 was done using the PIP3 Mass ELISA Kit (echelon; K-2500s), according to the provided instructions. The relative amounts of triplicates of each sample were presented after normalization of the values with the PIP3 standard.

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2.6. In vitro phosphorylation of dynactin p150 by Akt

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The HEK293 cells expressing the C-terminal V5/His tagged dynactin p150 were used for partial purification of dynactin p150. HEK293dynactin p150 cells were treated with either DMSO or a PI3K inhibitor LY294002 for 30 minutes and were lysed in the ice-cold lysis buffer (10 mM TrisCl [pH 7.6], 150 mM NaCl, 20 mM Imidazole, and 0.3% CHAPS with cocktails of proteases and phosphotases inhibitors). After clearing the lysates, the Ni-NTA agarose beads (Qiagen) were added (50 μl/1 mL lysate of 2 × 106 cells), and incubated 3–4 hours in the cold room with gentle rotation. The beads were then sequentially washed in the lysis buffer containing imidazole (20 mM, 50 mM, and 75 mM). After the final wash in the lysis buffer, the bead bound fractions were boiled in 1× LDS buffer and analyzed by using the phospho-Akt substrate antibody. The phosho-PKC substrate antibody was used as a negative control. In parallel, HEK293-dynactin p150 cells were treated with LY294002 and the dynactin p150 immune complex was prepared by using the V5 antibody. The immune complex was subjected to in vitro kinase reaction with the active Akt (Cell Signaling Technology) in the presence or absence of Akt inhibitor. The resulting dynactin p150 immune complex was analyzed for phosphorylation using the phospho-Akt substrate antibody.

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was visualized under the fluorescent microscope (Olympus IX71) and the image was taken using the 100 × objective lens. For temperature dependent depolymerization and repolymerization of microtubules, the plasmid expressing EGFP-α-Tubulin was transfected into HeLa cells using the lipofectamine. Two days after transfection, cells were subjected to experimental manipulations and were directly fixed in prechilled methanol followed by washing in PBS prior to imaging.

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Analysis of statistical significance for the indicated data sets was performed using the student's t test capability on Microsoft Excel.

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3. Results

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3.1. Microtubule depolymerization specifically attenuates Akt signaling

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In order to elucidate the regulatory mechanisms that control Akt dephosphorylation, we first used a cell free system in which the rate of Akt dephosphorylation was dependent on cellular phosphatase activity. Cytosolic lysates from IGF-1-treated HeLa cells were incubated at two different temperatures (20 or 37 °C) and the kinetics of Akt dephosphorylation measured. A higher level of Akt phosphorylated at serine 473 (S473) was maintained at 20 °C than at 37 °C (Fig. S1A), potentially reflecting reduced phosphatase activity at the lower temperature. To recapitulate this in vitro result in live cells, HeLa cells were stimulated with IGF-1 and left at two different temperatures in serum-free medium. Surprisingly, in contrast to the cell free assay, the duration and extent of Akt S473 dephosphorylation were enhanced at 20 °C (Fig. S1B). One potential explanation for this discrepancy might be that phosphorylated Akt is spatially separated from cytosolic phosphatases in live cells. Since the cytoskeletal networks that maintain cellular architecture can serve as a barrier to free diffusion of cellular proteins, we therefore examined if perturbation of cytoskeletal networks could affect phosphorylation of Akt. Serum-starved HeLa cells were treated with two microtubule-disrupting pharmacological agents, nocodazole and vinblastine, and the level of Akt S473 phosphorylation (hereafter referred to as p-Akt, or phosphorylation of Akt, unless otherwise specified) following stimulation with IGF-1 examined. Compared to control, these agents decreased or abolished phosphorylation of Akt (Fig. 1A). Since perturbation of microtubules can affect cell adhesion and indirectly affect Akt phosphorylation, HeLa cells and NB4 leukemic cells were analyzed in suspension cultures; microtubule disruption consistently led to a decrease in p-Akt in these cells (Fig. 1B). To determine whether microtubule disruption can also affect Akt phosphorylation stimulated by other signaling ligands, HL-60 human promyelocytic leukemia cells, which can be differentiated into neutrophil-like cells and respond to the chemoattractant fMLP fMLP [9], were studied. In the presence of nocodazole, IGF-1-elicited Akt phosphorylation was decreased in both parental and differentiated HL60 cells and in fMLPstimulated differentiated HL60 cells, in which Akt phosphorylation is mediated by a G protein-coupled receptor (GPCR) (Fig. 1C). Furthermore, consistent decreases in p-Akt were observed upon microtubule disruption when tested in REH pre-B cells stimulated with stromal cell-derived factor-1 (SDF-1) or leukotriene B4 (Fig. 1D). These observations suggest that the microtubule disruption-induced reduction in Akt phosphorylation is a general mechanism that occurs in multiple cell types and in response to both receptor tyrosine kinase- and GPCR-mediated Akt activation. Microtubule disruption may affect multiple intracellular signaling events, and therefore the observed decreases in p-Akt might be a

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HeLa cells transfected with the control or dynactin p150 siRNAs were treated for 30 minutes with BITC (benzyl isothiocyanate). After briefly washing in PBS, cells were incubated for 6 hours in drug-free medium with 1% FBS. For the microscopic analysis, cells were fixed in 3% PFA for 5 minutes and stained with DAPI. The apoptotic cells were identified based on the simultaneous comparison of fragmented nuclei (DAPI image) and collapsed membrane (bright field image). In addition, the total cell lysates were also analyzed for the cleavage of PARP and procaspase-3 as a biochemical indication of cell death.

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consequence of such pleiotropism. To examine this possibility, we investigated the effect of microtubule disruption on other signaling pathways known to be activated by IGF-1, such as MAPK. At all the time points examined, Akt phosphorylation at both S473 and T308 was reduced, while Erk phosphorylation was unaffected or even slightly increased at the corresponding time points (Fig. 1E). In addition, total tyrosine phosphorylation, a surrogate of global signaling events, appeared unaffected by nocodazole treatment (Fig. S2). Consistent with this observation, IGF- or EGF-induced phosphorylation of growth factor receptors and Erk phosphorylation were unaffected by nocodazole (Fig. 1F). These results indicate that microtubule disruption preferentially and specifically attenuates Akt signaling. To further demonstrate that the effects of nocodazole and vinblastine are indeed mediated by microtubule disruption, we examined whether microtubule stabilization could rescue these effects. HeLa cells were treated with nocodazole and vinblastine alone or with Taxol (paclitaxel), a microtubule stabilizer. Compared to nocodazole or vinblastine alone, p-Akt levels were significantly greater when cotreated with Taxol at each time point examined (Fig. 1G, left panel). Taxol also rescued p-Akt levels when cells were pre-treated with nocodazole (Fig. 1G, right panel). Furthermore, when tested with a variety of structurally diverse antimicrotubule agents, a similar rescue effect was observed (Fig. S3A). To further test this rescue effect at the cellular level, the Akt signaling-dependent cytosolic retention or inhibition of nuclear translocation of FoxO, a direct downstream target of Akt, was examined using live cell imaging [10,11]. Consistent with reductions in p-Akt, EGFP-FoxO was translocated to the nucleus within 30 minutes of nocodazole treatment, which was reversed by subsequent treatment with Taxol (Fig. 1H). The structure of Taxol-rescued microtubules was similar to Taxol treatment alone (Fig. S3B), suggesting that Taxolstabilized microtubules, without the ‘normal’ architecture of microtubules, are sufficient to overcome microtubule depolymerizationinduced deactivation of Akt. To corroborate this finding in vivo, we examined Akt phosphorylation in skeletal muscles from fed or starved mice following intraperitoneal (Fig. 1I) or intramuscular (Fig. S4) injection of Taxol. In fed animals, vehicle and Taxol-treated p-Akt levels were comparable. However, in starved animals, a higher level of p-Akt was retained in the Taxoltreated group compared to vehicle controls (Fig. 1I). Together, these results confirm that stabilization of microtubules also delays Akt deactivation in vivo.

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3.2. Microtubule integrity is required for sustaining Akt signaling, but not 298 for initial activation 299 To determine the causes of microtubule disruption-induced reductions in Akt signaling, we first examined whether overstimulation with growth factors could overcome the effects of microtubule disruption. Excess IGF-1 failed to recover Akt phosphorylation by nocodazole (Fig. 2A). We next examined if the membrane production of PIP3 was affected by nocodazole using live cell imaging to monitor membrane localization of the PIP3-binding probe PH-EGFP (the PH domain from Akt1 fused to EGFP). Unlike with the PI3K inhibitor wortmannin, neither nocodazole nor Taxol affected IGF-induced membrane localization of PHEGFP (Fig. 2B). A similar result was observed with another PIP3binding probe, PHItk-EGFP (the PH domain from IL2-inducible T-cell kinase (Itk) fused to EGFP) (Fig. S5). To further confirm this result, we directly measured and compared PIP3 levels following IGF-1 stimulation in the presence of nocodazole or Taxol. In line with the observed membrane localization of PIP3-binding probes, the levels of PIP3 did not differ between the control, nocodazole, or Taxol-treated cells (Fig 2C). These results demonstrate that PIP3 production was unaffected by changes in microtubule structure. Microtubules are intrinsically sensitive to low temperatures, which allows for temperature dependent manipulation of microtubule depolymerization and repolymerization in live cells. Using this paradigm, we

Please cite this article as: H. Jo, et al., Microtubule dynamics regulates Akt signaling via dynactin p150, Cellular Signalling (2014), http://dx.doi. org/10.1016/j.cellsig.2014.04.007

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Please cite this article as: H. Jo, et al., Microtubule dynamics regulates Akt signaling via dynactin p150, Cellular Signalling (2014), http://dx.doi. org/10.1016/j.cellsig.2014.04.007

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whether microtubule stabilization by TSA also results in sustained phosphorylation of Akt, a similar deactivation experiment was performed. Compared with control, TSA-treated HeLa cells retained a higher level of p-Akt when subjected to deactivation in serum-free medium (Fig. 2G). Both Taxol and TSA-stabilized microtubules were resistant to cold temperature-induced depolymerization (Fig. 2H, top). In line with this observation, HeLa cells with stabilized microtubules also retained a higher level of p-Akt during deactivation on ice (Fig. 2H, bottom). Together, these results suggest that microtubule stabilization sustains Akt signaling under deactivating conditions. In support of our hypothesis, calyculin A, a protein phosphatase 1 (PP1) and 2A (PP2A) inhibitor, effectively prevented reduction of Akt phosphorylation by nocodazole. However, in the presence of the PI3K inhibitor wortmannin (which prevents the initial phosphorylation of Akt), calyculin A failed to elevate p-Akt levels (Fig. S7). Together, these results suggest that microtubule disruption affects the deactivation of Akt signaling.

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We next examined if Akt is associated with microtubules. Indeed, Akt was localized to the mitotic spindles as revealed by immunostaining (Fig. 3A, top). The gain-of-function mutant Akt 1 (e17k), whose activity was downregulated by microtubule disruption, was also localized to the mitotic spindles (Fig. 3A, bottom). The localization of phosphorylated

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microtubule repolymerization was abolished with nocodazole, p-Akt levels were comparable at the initial phase of recovery, with differences in Akt phosphorylation only becoming obvious at later time points (Fig. 2E). This suggests that microtubule integrity is not needed for initial Akt activation. To further explore the role of microtubules in the initial activation of Akt, we used a gain-of-function mutant Akt1 (e17k; identified in human cancers), which can be constitutively localized to the membrane independent of PIP3 levels [12]. Microtubule disruption still reduced Akt1 (e17k) mutant phosphorylation (Fig. S6) and, as revealed by the immunostaining, nocodazole did not affect the membrane localization of Akt 1 (e17k) (Fig. S6). These results again suggest that microtubule integrity is not required for the initial activation of Akt. Since the initial activation of Akt is unaffected by microtubule disruption, the rescuing effects of Taxol might be due to prevention of Akt deactivation; if this is the case, then microtubule stabilization should sustain Akt signaling. To test this possibility, HeLa cells grown in serum-rich medium were left in serum-free medium in the presence of nocodazole or Taxol, and the levels of p-Akt were examined over time. Compared to control and nocodazole treatment, Taxol treatment led to the sustained phosphorylation of Akt and its target proteins as expected (Fig. 2F). Inhibitors of histone deacetylases (HDACs), such as trichostatin A (TSA), have also been shown to stabilize microtubules by increasing levels of acetylated alpha tubulin [13]—a different mechanism to Taxol, which directly binds to microtubules. To determine

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Fig. 2. Microtubule integrity is critical for sustaining Akt signaling but is not required for initial Akt activation. (A) Initial activation of Akt was unaffected by microtubule disruption. Excess IGF-1 failed to overcome the inhibitory effects of nocodazole on p-Akt levels. Whole cell lysates were analyzed after 30 minutes of IGF-1 stimulation. (B) Representative live cell images of membrane localization of PIP3-binding probe PH (Akt)-EGFP in the presence of nocodazole (2 μM), Taxol (1 μM), or wortmannin (100 nM). (C) The effect of nocodazole or Taxol on PIP3 production by IGF-1 stimulation. The relative amounts of triplicates of each sample were presented after normalization of the values with the PIP3 standard. (D) Temperature dependent depolymerization and repolymerization of microtubules as revealed by EGFP-α-Tub. (E) Changes in p-Akt during microtubule repolymerization in the presence or absence of nocodazole. (F) Microtubule stabilization sustained Akt signaling. Phosphorylation of Akt and its target proteins in DMSO, nocodazole, or Taxol-treated HeLa cells upon removal of serum. (G) The level of IGF-stimulated p-Akt in control or TSA-treated HeLa cells upon removal of IGF. Akt phosphorylation is expressed as the ratio of phospho-Akt to total Akt and normalized to time "0" of untreated cells. The results are the means (±SD) of three independent experiments. (H) The effect of microtubule stabilization on p-Akt levels during cold temperature-induced deactivation. Following pre-stimulation with IGF-1, cells were left on ice in the presence of IGF 1 for the indicated periods of time.

Fig. 1. Microtubule depolymerization specifically attenuates Akt signaling. (A) Inhibition f IGF-1-elicited Akt phosphorylation by nocodazole (2 μM) and vinblastine (1 μM). Serum-starved HeLa cells were pre-treated with the indicated chemicals for 5 minutes followed by IGF-1 (20 ng/mL) for an additional 30 minutes. Phosphorylated and total Akt were detected by western blotting using anti-phospho-Akt (Ser473) and anti-Akt antibodies, respectively. (B) The effect of nocodazole on IGF-1-elicited Akt phosphorylation in NB4 cells in suspension culture. Serum-starved NB4 cells were treated as in (A). (C) Inhibition of IGF-1 or fMLP-elicited Akt phosphorylation by nocodazole in HL60 and differentiated HL60 cells. (D) Inhibition of SDF-1 or LTB4-elicited Akt phosphorylation by nocodazole in REH cells. (E) The phosphorylation levels of Akt and Erk at different time points following IGF-1 stimulation in the presence or absence of nocodazole. (F) The effect of nocodazole on the phosphorylation of Akt, Erk, IGF, and EGF receptors upon stimulation with IGF-1 or EGF (20 ng/mL of each growth factor for 15 minutes). (G) Stabilization of microtubules rescued nocodazole and vinblastine-induced reductions in Akt phosphorylation. The levels of IGF-1-elicited Akt phosphorylation in the presence of nocodazole (2 μM), vinblastine (1 μM), or co-treatment with Taxol (1 μM) at different time points. Serum-starved HeLa cells were pre-stimulated with IGF-1 for 15 minutes. Whole cell lysates were analyzed after 15, 30, or 45 minutes after drug treatment (left panel). The sequential treatment of Taxol-rescued nocodazole-inhibited p-Akt levels (right panel). HeLa cells were treated with nocodazole for 30 minutes as described above and subsequently treated with Taxol for an additional 30 minutes prior to analysis. (H) Live cell imaging of subcellular translocation of FoxO-EGFP. Nocodazole-induced nuclear translocation of FoxO-EGFP was rescued by subsequent treatment with Taxol. (I) The effect of Taxol on p-Akt levels in skeletal muscle. Animals were injected intraperitoneally twice (8-hour interval) with the vehicle or Taxol (2.5 mg/kg body weight) and left with (n = 4 mice) or without (n = 6 mice) food overnight. The level of p-Akt in the skeletal muscles was compared. The results are the means (±SD). *, P b 0.05 compared to vehicle control.

Please cite this article as: H. Jo, et al., Microtubule dynamics regulates Akt signaling via dynactin p150, Cellular Signalling (2014), http://dx.doi. org/10.1016/j.cellsig.2014.04.007

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To identify potential cytosolic factors that mediate and/or strengthen Akt localization to microtubules, we first sought those MAPs known to be localized to the minus-end of mitotic spindles and that possess a consensus Akt phosphorylation site (RxRxxS/T) that might indicate direct interaction with Akt. A consensus Akt phosphorylation site, well-conserved from Drosophila to humans (Fig. S8A), was identified within the first coiled-coil domain of dynactin p150. Dynactin p150 is a multifunctional protein that directly binds to the dynein intermediate chain and enables dynein motor-mediated transport of vesicles along microtubules. In metaphase cells, dynactin p150 localization was evident at the centrosome and toward the minus-end of microtubule spindles, which was different to that of dynein intermediate chain localization (Fig. S8B). Consistent with the presence of a consensus Akt phosphorylation site in dynactin p150, we revealed that dynactin p150 was a direct target of Akt and was associated with p-Akt in intact cells (Fig. S9). To examine p-Akt and dynactin p150 co-localization, HeLa cells were transfected with dynactin p150 and treated with TSA to stabilize microtubules. In control cells, dynactin p150 staining was disorganized, reflecting the dynamic changes in microtubule structures, with p-Akt staining throughout the cytoplasm and the nucleus (Fig. 4A, upper panel). However, in TSA-treated cells, staining of dynactin p150 was much better organized and, importantly, a fraction of p-Akt was colocalized with dynactin p150 along stabilized microtubules (Fig. 4A, lower panel). To further substantiate this finding, HEK293T cells containing a higher level of p-Akt were investigated and, similar to control HEK293T cells, p-Akt staining was found predominantly at the plasma membrane (Fig. S10A). However, in dynactin p150 expressing

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cells p-Akt was localized on the microtubules as well as the plasma membrane (Fig. S10B). Overexpression of dynactin p150 is known to induce microtubule bundle formation [16,17] and therefore p-Akt could simply be associated with these structures rather than being actively recruited by dynactin p150. To address this, endogenous dynactin p150 was depleted and co-localization of p-Akt with the acetylated alpha-tubulin (a marker for the stabilized microtubules) examined following IGF-1 stimulation and TSA treatment. Reduced levels of dynactin p150 did not affect induction of acetylated α-tubulin by TSA treatment (Fig 4B). In DMSO-treated cells, acetylated α-tubulin staining appeared disorganized and p-Akt staining was indistinguishable from control- or dynactin p150 siRNA-transfected cells (Fig 4C), while in TSA-treated cells, well-organized acetylated α-tubulin and prominent p-Akt staining on microtubule bundles were observed. However, microtubule bundle p-Akt staining was significantly reduced when dynactin p150 was knocked down (Fig. 4C), demonstrating that dynactin p150 is required for localization of Akt to the stabilized microtubules. We next examined if dynactin p150 is required for sustained Akt signaling mediated by microtubules. Knockdown of dynactin p150 did not affect initial activation of Akt stimulated by IGF-1 (Fig. 4B) or in HEK293T cells grown in serum-rich conditions. However, in low serum conditions, the p-Akt levels were much lower in HEK293T cells lacking dynactin p150 compared to control HEK293T cells (Fig. 4D), indicating that dynactin p150-mediated Akt localization on microtubules is essential for sustained Akt signaling. To further examine the contribution of dynactin p150 to microtubuledependent Akt signaling, we explored other cellular contexts that affect microtubule structures, including cell adhesion (during which microtubules undergo dynamic reorganization) and pharmacological intervention. Compared to control cells, knockdown of dynactin p150 resulted in reduced levels of p-Akt during cell adhesion (Fig 4E). Having shown earlier that Taxol prevents or rescues the reduction in p-Akt caused by microtubule disruption (Fig. 1), when dynactin p150 was knocked down the Taxol-mediated rescue of p-Akt was abolished (Fig 4F). This effect appeared to be specific to dynactin p150 since knockdown of EB1, a microtubule plus-end binding protein, had no effect (Fig 4G).

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Akt was assessed by immunostaining with an anti-p-Akt antibody, and when serum-starved HeLa cells were stimulated with IGF-1 a transient accumulation of p-Akt at the mitotic spindles was detected (Fig. 3B). Together, these results show that at least some Akt is associated with microtubules. To further determine the relationship between Akt and microtubules, we examined if Akt can associate with α-tubulin, a structural component of microtubules. HEK293-Akt1 cells were transfected with EGFP fused to full-length α-tubulin (EGFP-α-tubulin) or the Cterminal deletion mutants EGFP-α-tubulin (Δ15) and EGFP-α-tubulin (Δ50). As well as differing between isotypes and defining the isotypes, the 15 amino acids at the C-terminus of α-tubulin are subjected to various posttranslational modifications but are dispensable for formation of the α-/β- tubulin heterodimer, the building block for microtubules [14,15]. Thus, as expected, both EGFP-α-tubulin and EGFP-α-tubulin (Δ15) were incorporated into mitotic spindles. However, EGFP-αtubulin (Δ50) failed to be incorporated into the spindles (Fig. 3C, top). When these cell lysates were co-immunoprecipitated with Akt1, both EGFP-α-tubulin and EGFP-α-tubulin (Δ15), but not EGFP-α-tubulin (Δ50), were pulled down (Fig. 3C, bottom). Some microtubule-associated proteins (MAPs) display polarity with respect to their location at mitotic spindles (i.e., proximal or distal from the centrosome). To see whether Akt is uniformly localized or polarized, Akt staining was examined along transverse sections of mitotic spindles. Intriguingly, while Akt1 was co-localized with tubulin toward the minus end (or proximal to the centrosome), co-localization was lacking toward the plus end (or closer to the metaphase plane) (Fig. 3D), indicating that localization of Akt at microtubules might be mediated factors other than tubulins.

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3.5. Microtubule disruption-induced Akt deactivation contributes to de- 472 layed cell cycle progression and accelerated cell death 473 We next asked whether a reduction in Akt signaling caused by microtubule disruption has functional consequences. Akt signaling is required for cell cycle progression during interphase [18–22]. We therefore used time-lapse live cell imaging to count cells entering mitosis under different experimental conditions [23]. First, to validate the experimental strategy, cells in interphase were treated with a low concentration of nocodazole (50 nM), which is sufficient to inhibit mitotic spindle function and causing mitotic arrest but insufficient to affect p-Akt levels; as expected, the percentage of cells entering mitosis was similar to DMSO-treated controls. However, at a higher concentration of nocodazole (2 μM), significant inhibition of entrance into mitosis was observed (45% versus less than 10%), with similar results also observed with another microtubule depolymerizer vinblastine (1 μM) (Fig. 5A). A dynamic and constant depolymerization and repolymerization of microtubules are essential for various cellular functions [24], and stabilization of microtubules is also known to impair a multitude of cellular functions. As previously reported [23–25], stabilization of microtubules by Taxol leads to cell cycle arrest; however, both low (50 nM) and high

Fig. 3. Some fractions of Akt are associated with microtubules. (A) Localization of wild-type Akt1 or Akt1 (e17k) mutant on the mitotic spindles of metaphase cells. Black arrow heads indicate a mitotic spindle localization of wild-type Akt and Akt1 (e17k). White arrow heads indicate membrane localization of Akt1 (e17k). (B) Localization of endogenous p-Akt on the mitotic spindles. Serum-starved HeLa cells with or without IGF stimulation were stained with anti-p-Akt and anti-α-tubulin antibodies. (C) Akt is associated with α-tubulin. Representative images of EGFP-α-tubulin and EGFP-α-tubulin (Δ15), but not EGFP-α-tubulin (Δ50), incorporated into the mitotic spindles (left). Both EGFP-α-tubulin and EGFP-α-tubulin (Δ15), but not EGFP-α-tubulin (Δ50), were pulled down by Akt1 (right). (D) Differential localization of Akt1 along the mitotic spindles during metaphase. Four different focal planes of transverse sections of mitotic spindles co-stained with Akt1 and α-tubulin are shown. An absence of co-localization between Akt1 and α-tubulin is obvious toward the plus end (plane #4).

Please cite this article as: H. Jo, et al., Microtubule dynamics regulates Akt signaling via dynactin p150, Cellular Signalling (2014), http://dx.doi. org/10.1016/j.cellsig.2014.04.007

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Please cite this article as: H. Jo, et al., Microtubule dynamics regulates Akt signaling via dynactin p150, Cellular Signalling (2014), http://dx.doi. org/10.1016/j.cellsig.2014.04.007

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Fig. 5. Microtubule disruption-induced Akt deactivation leads to delayed cell cycle progression and accelerated cell death. (A) Microtubule disruption delayed cell cycle progression. Representative still frames of time-lapse movies are shown. Interphase cells entering mitosis in the presence of different concentrations of nocodazole, vinblastine or Taxol were taken every 15 minutes and at least three independent experiments for each treatment were quantified (right panel). (B) Dose-dependent effect of nocodazole or Taxol on p-Akt levels. (C) Phosphorylation of myr-ΔPH-Akt (active form) or ΔPH-Akt (inactive form) in the presence of nocodazole or Taxol. (D) Ectopic expression of myr-_PH-Akt rescued the cell cycle delay caused by microtubule disruption. HeLa cells transfected with myr-ΔPH-Akt or ΔPH-Akt were treated with nocodazole (2 μM) and time-lapse images taken every 15 minutes. Interphase cells entering mitosis were quantified (right panel). (E) BITC led to a dose-dependent reduction in p-Akt levels that was rescued by co-treatment with Taxol. p-Akt levels in the presence of BITC alone or co-treated with Taxol were assessed by western blotting. (F) Dynactin p150 was required for Taxol-mediated rescue of cell death induced by BITC. HeLa cells transfected with control or dynactin p150 siRNAs were incubated with BITC alone (10 μM) or with Taxol (1 μM) for 30 minutes followed by incubation in drug-free medium (1% FBS) for 6–8 hours. Dying cells were quantified based on membrane blebbing and fragmented nuclei (indicated by black arrows). Mitotically arrested but healthy cells (indicated by white arrows) on cotreatment with Taxol were excluded. The results are the means (±SD) of 10 transfected cells in three independent experiments.

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concentrations (1 μM) of Taxol failed to affect the mitotic entry of interphase cells (Fig. 5A), again suggesting that impaired entry into mitosis induced by vinblastine and nocodazole is due to reduced Akt signaling (Fig. 5B). To further demonstrate the role of Akt signaling in microtubule disruption-induced delay of cell cycle progression, we attempted to rescue these defects by constitutively expressing activated Akt. Ectopic expression of a constitutively active form of Akt, myr-ΔPH-Akt, effectively

rescued microtubule disruption-induced inhibition of mitosis, while ΔPH-Akt, an inactive form, was essentially ineffective (Fig. 5C, D and Supplementary Movies 1, 2). Collectively, these results suggest that microtubule disruption attenuates Akt signaling and leads at impairing cell cycle progression. Finally, we explored whether dynactin p150 also contributes to those cellular functions mediated by microtubule-dependent regulation of Akt. Several anti-cancer agents exert their apoptotic effects by

Fig. 4. Dynactin p150 mediates localization of Akt to stabilized microtubules. (A) Colocalization of p-Akt and dynactin p150 on microtubules in TSA-treated cells. Arrow indicates staining of Akt on stabilized microtubules. (B) The levels of p-Akt and acetylated α-tubulin in control or dynactin p150 knocked-down cells. (C) p-Akt does not localize to stabilized microtubules upon dynactin p150 knockdown. HeLa cells transfected with control or dynactin p150 siRNA were treated with DMSO or TSA. Cells were serum starved and stimulated with IGF-1, followed by immunostaining for p-Akt and acetylated α-tubulin. (D) The levels of p-Akt in control or dynactin p150 depleted cells grown in different serum conditions. (E) Bright field images of control or dynactin p150 depleted cells during cell adhesion (top) and the levels of p-Akt in the corresponding cells (bottom). (F) Taxol-mediated rescue of p-Akt was diminished by knockdown of dynactin p150. (G) Taxol-mediated rescue of p-Akt was not affected by knockdown of EB1.

Please cite this article as: H. Jo, et al., Microtubule dynamics regulates Akt signaling via dynactin p150, Cellular Signalling (2014), http://dx.doi. org/10.1016/j.cellsig.2014.04.007

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Conflict of interest statement

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The spatial separation of the activity of kinases and opposing deactivators remains poorly defined. Once activated at the membrane, Akt is transported to different cellular compartments to phosphorylate target proteins, a process that must occur in the presence of cytoplasmic phosphatases cytoplasm [31]. However, the cellular factors that coordinate the spatiotemporal aspects of Akt signaling are unknown. Here we show that microtubule dynamics is a mechanism that specifically deactivates Akt signaling. Microtubules play crucial roles in various intracellular processes, including cell division, transport of vesicles, maintenance of cell shape, and positioning of subcellular organelles. Perturbation of microtubules might therefore be expected to globally affect intracellular signal transduction pathways. However, we found that Akt signaling was particularly sensitive to microtubule disruption, with total levels of phosphorylated tyrosine residues and the MAPK pathway remaining unaffected. The inhibitory effect was not simply due to changes in cell shape or loss of cell adhesion, since a similar reduction in Akt phosphorylation was seen in different cell types in suspension. Several lines of evidence in this study suggest that microtubule-dependent Akt regulation is specifically restricted to the deactivation phase. First, we showed that the phosphorylation of growth factor receptors, such as IGF-1 and EGF, could occur normally in the presence of microtubule depolymerizers. Second, pharmacological disruption or stabilization of microtubules failed to alter levels of PIP3 at the plasma membrane. Third, the IGF-1 elicited membrane translocation of PIP3-binding probes was unaffected by microtubule agents. Finally, the mutant Akt1 (e17k), which is constitutively active, was still susceptible to deactivation by microtubule disruption. How is microtubule-dependent regulation of Akt signaling achieved? Akt phosphorylation may be spatially controlled by the microtubule cytoskeleton regulating access to protein phosphatases. Cytoskeletonassociated Akt is inaccessible to, and sequestered from, cytoplasmic phosphatases, while the Akt that is “freed” following microtubule disassembly may be susceptible to dephosphorylation. Alternatively, microtubules may directly or indirectly inhibit local phosphatase activity, ensuring sustained phosphorylation of microtubule-associated Akt. Finally, protection from dephosphorylation may also result from the direct interaction of phosphorylated Akt with other microtubuleassociated proteins, such as dynactin p150, with binding protecting the phosphorylated sites from dephosphorylation.

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The putative Akt phosphorylation site in dynactin p150 is evolutionarily well conserved. The fact that dynactin p150 is a direct target of Akt suggests that the functional relationship plays important roles in cell motility and vesicle trafficking. Akt may directly influence intrinsic microtubule dynamics and other microtubule-mediated cellular processes via phosphorylation of dynactin p150. The physiological significance of this regulation needs to be further investigated. These findings have important implications with respect to in vivo regulation of Akt signaling. Unlike cells in culture, most cells in differentiated tissues only have limited access to growth factors but their microtubules tend to be more stable. Under these conditions, the overall intensity and duration of Akt signaling might be more influenced by the rate of deactivation than conditions of initial activation. Intrinsic microtubule dynamics and structural integrity are likely to be a key determinant of cell type-specific and context-dependent Akt signaling, especially when external stimuli are limited and upon exposure to therapeutic agents that target microtubules. Due to the significant role that Akt signaling plays in tumorigenesis, our findings provide novel therapeutic strategies and targets for cancer treatment. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cellsig.2014.04.007.

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The authors thank all members of the Luo laboratory. H.R. Luo was 589 supported by NIH grants HL085100, HL095489, and AI076471 and a 590 Research Scholar Grant from the American Cancer Society. 591

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targeting tubulin [26–30]; for example, it has recently been shown that isothiocyanates (ITCs) (such as benzyl isothiocyanate (BITC)) bind covalently to tubulin, leading to microtubule depolymerization and cell death [26,27]. Interestingly, BITC-induced apoptosis occurs over a relatively short time frame, independent of mitotic arrest. Using this paradigm, we evaluated the role of dynactin p150 in the apoptotic function of Akt signaling. Similar to conventional microtubule agents, BITC led to a dose-dependent reduction in p-Akt that could be rescued by co-treatment with Taxol (Fig. 5E). Transient treatment with BITC was sufficient to induce apoptosis, which was prevented by Taxol (Fig. 5F). Knockdown of dynactin p150 further enhanced cell death caused by BITC. Importantly, the rescue effects of Taxol were diminished by dynactin p150 knockdown (Fig. 5F). These results are consistent with dynactin p150 contributing to a microtubule-dependent function of Akt and establish that dynactin p150 could act as a novel therapeutic target in cancer.

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