Cellular Signalling 26 (2014) 748–756

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Dephosphorylation of CaMKII at T253 controls the metaphase–anaphase transition Alexander Hoffman 1, Helen Carpenter 1, Richard Kahl, Lauren F. Watt, Phillip W. Dickson, John A.P. Rostas, Nicole M. Verrills, Kathryn A. Skelding ⁎ School of Biomedical Sciences and Pharmacy, Faculty of Health, The University of Newcastle, Callaghan, New South Wales, Australia The Hunter Medical Research Institute, Faculty of Health, The University of Newcastle, Callaghan, New South Wales, Australia

a r t i c l e

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Article history: Received 10 December 2013 Accepted 29 December 2013 Available online 7 January 2014 Keywords: CaMKII Calcium/calmodulin stimulated protein kinase II Protein phosphorylation Protein phosphatase 2A Metaphase Cell cycle

a b s t r a c t Calcium/calmodulin-stimulated protein kinase II (CaMKII) is a multi-functional serine/threonine protein kinase that controls a range of cellular functions, including proliferation. The biological properties of CaMKII are regulated by multi-site phosphorylation and targeting via interactions with specific proteins. To investigate the role specific CaMKII phosphorylation sites play in controlling cell proliferation and cell cycle progression, we examined phosphorylation of CaMKII at two sites (T253 and T286) at various stages of the cell cycle, and also examined the effects of overexpression of wild-type (WT), T286D phosphomimic, T253D phosphomimic and T253V phosphonull forms of CaMKIIα in MDA-MB-231 breast cancer and SHSY5Y neuroblastoma cells on cellular proliferation and cell cycle progression. We demonstrate herein that whilst there is no change in total CaMKII expression or T286 phosphorylation throughout the cell cycle, a marked dephosphorylation of CaMKII at T253 occurs during the G2 and/or M phases. Additionally, we show by molecular inhibition, as well as pharmacological activation, that protein phosphatase 2A (PP2A) is the phosphatase responsible for this dephosphorylation. Furthermore, we show that inducible overexpression of WT, T286D and T253V forms of CaMKIIα in MDA-MB-231 and SHSY5Y cells increases cellular proliferation, with no alteration in cell cycle profiles. By contrast, overexpression of a T253D phosphomimic form of CaMKIIα significantly decreases proliferation, and cells accumulate in mitosis, specifically in metaphase. Taken together, these results strongly suggest that the dephosphorylation of CaMKII at T253 is involved in controlling the cell cycle, specifically the metaphase–anaphase transition. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Cells divide via a process known as the cell cycle, which consists of two distinct phases in mammalian cells — interphase (itself comprising of Growth/Gap phase 1 (G1), synthesis phase (S), and Growth/Gap phase 2 (G2)) and M phase (consisting of mitosis [prophase, prometaphase, metaphase, anaphase and telophase] and cytokinesis). Changes in calcium concentration are known to control progression through the G1/S, G2/M, and

Abbreviations: ANOVA, one-way analysis of variance; CaMKII, calcium/calmodulin stimulated protein kinase II; D, aspartic acid; DAPI, 4′,6-diamidine-2-phenylindole; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol tetraacetic acid; EV, empty vector; FCS, foetal calf serum; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; PP2A-C, catalytic subunit of protein phosphatase 2A; PP4, protein phosphatase 4; PP6, protein phosphatase 6; PVDF, polyvinylidene fluoride; SDS, sodium dodecyl sulphate; SEM, standard error of the mean; shRNA, short hairpin RNA; siRNA, small interfering RNA; T, threonine; WT, wild-type; V, valine. ⁎ Corresponding author at: School of Biomedical Sciences and Pharmacy, The University of Newcastle, University Drive, Callaghan, New South Wales 2308, Australia. Tel.: +61 2 4921 5982; fax: +61 2 4921 6903. E-mail address: [email protected] (K.A. Skelding). 1 These authors contributed equally to this work. 0898-6568/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellsig.2013.12.015

metaphase–anaphase transitions [1,2], however the downstream pathways involved in this have not been elucidated. Calcium/calmodulin-stimulated protein kinase II (CaMKII) is one of the major calcium sensors in cells, and is normally activated by binding calmodulin, triggered by a rise in intracellular calcium. CaMKII is a multifunctional serine/threonine (T) protein kinase, whose biological properties are controlled by multi-site phosphorylation and via targeting to specific cellular locations through interactions with binding proteins [3,4]. Phosphorylation at the well-characterised phosphorylation site, T286, causes CaMKII to become autonomously active [5] allowing CaMKII to retain its activity even after calcium/calmodulin has dissociated from it. Phosphorylation at another site, T253, has no direct effect on CaMKII activity in vitro, but markedly alters CaMKII targeting [4,6]. Previous studies have implicated CaMKII as an important player in cell cycle regulation; however the specific role is controversial. Pharmacological inhibition of CaMKII can inhibit the cell cycle at G1 [7–9] or G2/M [10,11] depending on the system utilised and the time of drug treatment. It must be noted that the pharmacological inhibitors used in these studies are not specific for CaMKII, so other closely related kinases would also have been inhibited [12]. Specific targeting of CaMKII (by siRNA downregulation or overexpression of a kinase dead mutant form of CaMKII [13]) implicates CaMKII in the control of the G2 and/or M phases, however,

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the precise role has not been identified. Furthermore, due to the importance of CaMKII autophosphorylation in regulating the cellular functions controlled by CaMKII [14], it is likely that autophosphorylation of CaMKII will be involved in cell cycle regulation. We have recently shown that CaMKII that is phosphorylated at different sites (T286 or T253) has different effects on cell proliferation [4]. We demonstrated that overexpression of a wild-type (WT) or T286 phosphomimic (T286D) form of CaMKII increases proliferation rates of neuroblastoma and breast cancer cells, whereas overexpression of a T253 phosphomimic form (T253D) significantly reduces proliferation rates in these cells [4]. To examine this in more detail, and to determine the role that CaMKII autophosphorylation plays in controlling the cell cycle, we have examined phosphorylation of endogenous CaMKII at T286 and T253 during various phases of the cell cycle. We have shown that endogenous CaMKII becomes dephosphorylated at T253, but not T286, in the G2 and/or M phases of the cell cycle, and that protein phosphatase 2A (PP2A) is the phosphatase responsible for this. Furthermore, we generated MDA-MB-231 breast cancer and SHSY5Y neuroblastoma cells that inducibly overexpress phosphomimic and phosphonull forms of CaMKII. Following induction of the T253D (phosphomimic), but not the T286D (phosphomimic) or T253V (phosphonull), forms of CaMKIIα, cells accumulate in mitosis, specifically in metaphase. Taken together, our data indicates that T253 phosphorylation is essential for progression through mitosis, specifically the metaphase–anaphase transition.

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2.3. Cell cycle analysis At various times post-cell cycle resumption or following induction of CaMKII expression with doxycycline, cells were trypsinised and counted, and 1 × 106 cells were washed twice with phosphate buffered saline (PBS). Cells were fixed overnight in 300 μl 0.1% glucose/PBS and 3 ml ice-cold 70% ethanol mixture. Following this, the cells were washed once in PBS, and resuspended in 50 μg/ml propidium iodide solution (Sigma-Aldrich) containing 200 μg/ml RNase (Sigma-Aldrich) for 45 min at room temperature. The cell cycle was analysed using a FACSCalibur flow cytometer (Becton Dickinson, Sydney, NSW, Australia), by examining the pulse width versus pulse area to exclude cell doublets, and once the single cell population was identified, data was analysed using the CellQuest Software (BD Biosciences). Four independent experiments were performed per assay.

2.4. Cell proliferation MDA-MB-231 and SHSY5Y cells inducibly expressing various phosphomimic and phosphonull forms of CaMKII were seeded in a 96well plate (1 × 104/well), with 2 μg/ml doxycycline. At various times post-CaMKII induction, proliferation was assessed using the CellTiterBlue Cell Viability Assay (Promega, Alexandria, NSW, Australia), as per the manufacturer's instructions. Assays were plated in triplicate, and four independent experiments were performed.

2. Materials and methods 2.1. Cell lines and generation of inducibly expressing cells MDA-MB-231 (ATCC HTB-26; human breast cancer cells) and SHSY5Y (ATCC CRL-2266; human neuroblastoma cells) were purchased from the ATCC and were maintained in DMEM (Life Technologies, Mulgrave, VIC, Australia), supplemented with 15 mM HEPES (Life Technologies), 2 mM glutamine (Life Technologies), and 10% heatinactivated foetal calf serum (FCS; Sigma-Aldrich, Castle Hill, NSW, Australia). MDA-MB-231 and SHSY5Y cells inducibly expressing CaMKIIα mutants were generated using the T-REx Mammalian Expression Vector System (Life Technologies). Inducible expression vectors were created by inserting CaMKIIα cDNA from rat brain (a gift from H Schulman) into the pcDNA5/TO expression vector (Life Technologies), using standard techniques. The point mutations of CaMKII were created with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The following oligonucleotides were used: 5′-CAATAAGATG CTGGACATCAACCCGTCC-3′ for T253D, 5′-CAATAAGATGCTGGTCATCA ACCCGTCC-3′ for T253V and 5′-GCATGCACAGACAGGAGGACGTGGACT GCCTGAAG-3′ for T286D. The presence of the mutation and the absence of other changes were confirmed by sequencing. To generate cells stably expressing the Tet Repressor, MDA-MB-231 and SHSY5Y cells were initially transfected with the pcDNA6/TR vector (Life Technologies) using Lipofectamine 2000 (Life Technologies). Following this, the MDA-MB-231 and SHSY5Y-pcDNA6/TR cells were transfected with pcDNA5/TO vectors containing wild-type (WT), T286D, T253D, or T253V (or empty vector [EV]), using the Lipofectamine 2000 transfection reagent. CaMKII expression was induced by the addition of 2 μg/ml doxycycline (Sigma-Aldrich). 2.2. Cell cycle synchronisation Parental MDA-MB-231 and SHSY5Y cells were synchronised in the G0/G1 stage of the cell cycle via serum starvation for 24 h. The cell cycle was resumed via the addition of 20% FCS/DMEM, supplemented with 15 mM HEPES and 2 mM glutamine, and cells were harvested at various times post-cell cycle resumption.

2.5. Apoptosis Apoptosis was measured using the Annexin V FITC apoptosis detection kit (BD Biosciences). MDA-MB-231 and SHSY5Y cells inducibly expressing various phosphomimic and phosphonull forms of CaMKII were seeded in a 6-well plate (5 × 105/well). Following an overnight incubation, CaMKII expression was induced by the addition of 2 μg/ml doxycycline. Control cells were treated with apoptosis inducing agents as a positive control (MDA-MB-231: 1 μM staurosporine, 6 h treatment, Sigma-Aldrich; SHSY5Y: 10 μM Lovastatin, 24 h treatment, SigmaAldrich). At various times post-CaMKII expression, cells were trypsinised, washed twice with PBS, and cells were stained with FITC Annexin V and/or 50 μg/ml propidium iodide, as per the manufacturer's instructions. Following incubation for 15 min at room temperature, samples were run on a FACSCalibur flow cytometer (BD Biosciences), and data analysed using the CellQuest Software (BD Biosciences).

2.6. Immunofluorescent staining MDA-MB-231 and SHSY5Y cells inducibly expressing CaMKII were grown on glass coverslips (5 × 104/well), and CaMKII expression was induced for 48 h using 2 μg/ml doxycycline. Coverslips were washed once with PBS, fixed in methanol for 10 min at room temperature, and then washed once in PBS. The coverslips were blocked in 10% FCS/ PBS for 20 min at room temperature, and incubated with 1:500 antiα-tubulin (DM1A; Sigma-Aldrich) or 1:100 anti-Ki67-FITC (SP6; Abcam, Cambridge, MA, USA) for 1 h at room temperature. Coverslips were washed three times (5 min/wash) with PBS, and anti-α-tubulin coverslips were incubated with 1:1000 α-mouse Alexa Fluor-594conjugated secondary antibody (Life Technologies). Coverslips were washed another three times (5 min/wash) with PBS, and counterstained using a 4′,6-diamidino-2-phenylindole (DAPI) and mounting solution (Life Technologies). Images were obtained using a FluoView FV1000 confocal microscope (Olympus, North Ryde, NSW, Australia) and the FV10-ASW v2.0 software (Olympus). Five fields of views were counted per coverslip, and each assay was performed four times.

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Fig. 1. Phosphorylation of CaMKIIγ and δ at T286 and T253 throughout the cell cycle. MDA-MB-231 and SHSY5Y parental cells were synchronised in G0/G1 via serum starvation. At various times post-cell cycle resumption, cells were harvested, and the level of phosphorylation of CaMKII at T286 (white triangle) and T253 (black square) was determined by western blot. Phosphorylation levels are normalised to total CaMKII expression (i.e. phosphorylation/total CaMKII expression). (A) Phosphorylation of CaMKIIδ in MDA-MB-231 cells, (B) phosphorylation of CaMKIIγ in MDA-MB-231 cells, (C) phosphorylation of CaMKIIδ in SHSY5Y cells throughout the cell cycle. At the same times, the proportion of cells (expressed as a percentage) in G1/S (white bars) and G2/M (grey bars) was determined by flow cytometry. * denotes statistical significant difference from 0 h, as determined by one-way ANOVA (p b 0.05), n = 4.

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2.7. Activation and inhibition of protein phosphatase 1 (PP1) and 2A (PP2A) Parental MDA-MB-231 and SHSY5Y cells were transiently transfected with small interfering RNA (siRNA)-PP1 or short hairpin RNA (shRNA)-PP2A-Cα (or relevant scrambled control sequences) by Lipofectamine 2000 (Life Technologies), or were treated with the PP2A activator, FTY720 (Cayman Chemical, Ann Arbor, MI, USA), or phosphatase inhibitor, okadaic acid (Sigma-Aldrich). Pools of three to five target-specific 19–25 nucleotide siRNAs designed to knockdown PP1 expression (and control siRNA sequences) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). shRNA-EV and PP2A-Cα constructs in pSUPER were a gift from S Strack (University of Iowa, Iowa City, IA, USA). Briefly, cells (1 × 106) were mixed with DNA (40 μM pan PP1 or control siRNA; 5 μg shRNA-EV or shRNA-PP2A-Cα) and Lipofectamine 2000 transfection reagent (40 μl) in serum free DMEM. FTY720 and okadaic acid were dissolved in dimethyl sulfoxide (DMSO) at 50 mM at 1 mM, respectively. Parental MDA-MB-231 and SHSY5Y cells were treated with 3 μM FTY720 for 24 h, or 1 μM okadaic acid for 45 min. Cells were then lysed as described below (Section 2.8), and effects on CaMKII expression and phosphorylation at T286 and T253 determined by western blotting (Section 2.8). 2.8. Western blotting At various times post-cell cycle resumption or post-transfection, cells were pelleted and then resuspended in lysis buffer (1% NP-40/ Tris-buffered saline, 2 mM ethylenediaminetetraacetic acid [EDTA], 20 μM sodium orthovanadate, 50 mM sodium fluoride, complete protease inhibitor cocktail) for 20 min at 4 °C. Lysates were homogenised using a glass-Teflon Dounce Homogeniser (20 strokes; 700 rpm). Sodium dodecyl sulphate (SDS) sample buffer (final concentration: 67 mM Tris, 2% SDS, 25 mM β-mercaptoethanol, 2 mM ethylene glycol tetraacetic acid [EGTA], 10% glycerol, 0.03% bromophenol blue, pH 6.8) was added, and the lysate boiled for 10 min. Cell lysates (10–20 μg) were separated using 10% SDS-polyacrylamide gel electrophoresis (PAGE), and then transferred to polyvinylidene fluoride (PVDF) membranes, using standard techniques. The primary antibodies used were as follows: phospho-T253-CaMKII (1:20,000 [6]), total pan CaMKII (1:5000; Millipore, North Ryde, NSW, Australia), total CaMKIIα antibody (6G9; 1:5000; Abcam), phospho-T286-CaMKII (1:1000; Abcam), GAPDH (1:2000, BioVision, Milpitas, CA, USA), PP1 (1:1000, Santa Cruz Biotechnology), PP2A-Cα/β (1D6; 1:5000, Santa Cruz Biotechnology), and actin (AC-15; 1:50,000, Sigma-Aldrich). Binding of the primary antibody was detected by incubation with sheep anti-mouse or donkey anti-rabbit horseradish peroxidase-linked secondary antibody, and the ECL Plus Immunoblotting Detection System (GE Healthcare, Castle Hill,

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NSW, Australia). Blots were scanned with a Fujifilm LAS-3000 Imaging System and analysed with MultiGauge Software (Fujifilm, Brookvale, NSW, Australia). 2.9. Data analysis All statistical analyses were conducted using GraphPad Prism software V6.0 (GraphPad, San Diego, CA, USA). Comparisons between lines were made using one-way analysis of variance (ANOVA), with a Bonferonni post-test. All data is presented as the mean ± standard error of the mean (SEM) for the number of replicates (n). 3. Results 3.1. CaMKII becomes dephosphorylated at T253, but not T286, during the G2 and/or M phases of the cell cycle To investigate the role that CaMKII autophosphorylation plays in regulating cell cycle progression, we examined the phosphorylation of endogenous CaMKIIγ and δ at various stages throughout the cell cycle. MDA-MB-231 and SHSY5Y cells were synchronised in G0/G1 via serum starvation, cells were harvested at various times post-cell cycle resumption, and phosphorylation of endogenous CaMKII at T253 and T286 was examined. Even though these two cell lines express different isoforms of CaMKII (MDA-MB-231 cells express equivalent amounts of CaMKIIγ and CaMKIIδ, whereas the predominant isoform expressed in SHSY5Y cells is CaMKIIδ), we found that total CaMKIIγ and δ expression did not change throughout the cell cycle (Supplementary Fig. 1). Additionally, whilst phosphorylation of both CaMKIIγ and δ at T286 remained relatively constant throughout the cell cycle in both cell lines examined, phosphorylation of both CaMKIIγ and δ at T253 was significantly reduced to ~ 30% of that observed in the G1 phase (p b 0.05 for both lines) during the G2 and/or M phases of the cell cycle (Fig. 1A–C). 3.2. PP2A Is the phosphatase responsible for dephosphorylating CaMKII at T253 To determine the phosphatase responsible for this selective dephosphorylation of CaMKII at T253 during the G2 and/or M phases of the cell cycle, we used molecular inhibitors to inhibit the major phosphatases within cells, and measured the effect on phosphorylation of CaMKII at T286 and T253. Both PP1 and PP2A can dephosphorylate CaMKII at T286 in vitro [15] depending on the subcellular location of CaMKII within the cell. However, the phosphatase responsible for dephosphorylating CaMKII at T253 has not been examined. We transiently transfected MDA-MB-231 and SHSY5Y cells with siRNA directed against the

Fig. 2. Effect of pharmacological inhibition or activation of protein phosphatases on CaMKIIγ and δ phosphorylation at T286 and T253. MDA-MB-231 and SHSY5Y parental cells were transiently transfected with (A) siRNA against protein phosphatase 1 (PP1; +) or a control siRNA sequence (−), (B) shRNA against protein phosphatase 2A (PP2A; +), or a shRNA-EV (−) or (C) were treated with 3 μM FTY270. 24 h post-transfection or treatment, cells were harvested, and total endogenous CaMKIIγ and δ, pT286-CaMKII, pT253-CaMKII or actin expression was measured by western blot. Blots are representative of four independent experiments.

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Fig. 3. CaMKIIα controls cellular proliferation in MDA-MB-231 and SHSY5Y cells. Expression of CaMKIIα in MDA-MB-231 and SHSY5Y cells at various times post-doxycycline treatment. (A) MDA-MB-231 and (B) SHSY5Y cells inducibly expressing empty vector (EV), wild-type (WT), T286D, T253D, or T253V CaMKIIα were generated. At various times post-treatment with 2 μg/ml doxycycline (0, 24, and 48 h), cells were lysed, and expression of CaMKIIα was determined by western blot. Actin expression was used as a loading control. Blots are representative of three independent experiments. CaMKIIα expression was induced by 2 μg/ml doxycycline in (C) MDA-MB-231 and (D) SHSY5Y cells, and 48 h post-CaMKII expression, cells were fixed, and nuclei were stained for the proliferation marker, Ki67 (green), and counterstained with DAPI (blue). Arrows indicate Ki67 positive cells. Five fields of view were counted per coverslip, and four independent experiments were performed. Ki67 results are presented as the percentage of positive Ki67 cells/100 cells for (E) MDA-MB-231 and (F) SHSY5Y cells. Cell viability was measured at 0, 24, 48, and 72 h post-CaMKII expression via CellTiter Blue Assay in (G) MDA-MB-231 and (H) SHSY5Y cells. * denotes statistical significant difference from EV control cells, as determined by one-way ANOVA (p b 0.05). n = 4.

catalytic subunit of PP1 or shRNA directed against the catalytic subunit of PP2A (PP2A-C). Knockdown of PP1 increased phosphorylation of endogenous CaMKIIγ and δ at T286, with no change in total CaMKII expression or T253 phosphorylation (Fig. 2A). By contrast, knockdown of PP2A-C increased phosphorylation of CaMKIIγ and δ at T253, with no change in total CaMKII expression or T286 phosphorylation (Fig. 2B). To further confirm a role for PP2A in T253 dephosphorylation, MDAMB-231 and SHSY5Y cells were treated with a pharmacological activator of PP2A, FTY720 [16] and phosphorylation of endogenous CaMKIIγ and δ at T286 and T253 was examined. FTY720 treatment of MDAMB-231 and SHSY5Y cells for 24 h resulted in dephosphorylation of CaMKIIγ and δ at T253, with no change in total CaMKII expression or T286 phosphorylation (Fig. 2C). Taken together with the knockdown data, this indicates that PP2A is the phosphatase responsible for the selective dephosphorylation of CaMKII at T253 during mitosis.

3.3. Phosphorylation of CaMKII at T253 decreases cell proliferation rates Studies in a range of cell types using various approaches [7,17–20] have established that CaMKII is an important regulator of the cell cycle, however the precise role and mechanism are not known. As we have shown that CaMKII that is phosphorylated at different sites affects cell proliferation rates differently [4], we investigated the role that CaMKII phosphorylation plays in controlling the cell cycle. As cells stably expressing a T253D phosphomimic form of CaMKIIα stop dividing [4], precluding detailed analysis, we generated stable tet-inducible cell lines (MDA-MB-231-6/TR and SHSY5Y-6/TR) that inducibly overexpress a WT, T286 phosphomimic (T286D), T253 phosphomimic (T253D), T253 phosphonull (T253V) form of CaMKIIα, or an empty vector (EV). CaMKII expression was induced by doxycycline, and effects on cell proliferation were determined. All cell lines transfected with inducible CaMKII

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Fig. 4. CaMKIIα controls progression through the G2 and/or M phases in MDA-MB-231 and SHSY5Y cells. (A) MDA-MB-231 and (B) SHSY5Y cells inducibly expressing empty vector (EV), wild-type (WT), T286D, T253D, or T253V CaMKIIα. CaMKIIα expression was induced by 2 μg/ml doxycycline, and the proportion of cells in each phase of the cell cycle was determined 48 h later by flow cytometry. Histograms are representative of four independent experiments. Results are presented as the proportion of cells (expressed as a percentage) in G1 (white bars), S (grey bars) and G2/M (black bars) as determined by flow cytometry. * denotes statistical significant difference from EV cells, as determined by one-way ANOVA (p b 0.05).

constructs were shown to express CaMKIIα by 24 h post-induction with doxycycline (Fig. 3A and B), whereas the control EV expressing MDAMB-231 (Fig. 3A) and SHSY5Y (Fig. 3B) cells did not express detectable levels of CaMKIIα. To determine the effect of CaMKII phosphorylation on cell proliferation, doxycycline-induced cells were stained for Ki67, a marker of cell proliferation [21], and proliferation was also measured using a CellTiter Blue Assay. Using these inducible cells, we have confirmed and extended our previous findings [4]. Overexpression of a WT, T286D phosphomimic, or T253V phosphonull form of CaMKIIα significantly increased proliferation rates over 72 h when compared to control cells expressing the EV, as determined by Ki67 staining (Fig. 3C–F) and CellTiter Blue Assay (Fig. 3G, H). These findings suggest that overexpression of a WT or constitutively active (T286D phosphomimic mutant form) of CaMKIIα positively increases cell proliferation. By contrast, overexpression of a T253D phosphomimic form of CaMKIIα significantly reduces proliferation in

both MDA-MB-231 and SHSY5Y cells (Fig. 3), suggesting that CaMKII that is phosphorylated at T253 can negatively regulate cell proliferation. 3.4. Overexpression of a T253D phosphomimic form of CaMKII causes cells to accumulate in mitosis, specifically in metaphase, and induces apoptosis As CaMKII becomes dephosphorylated at T253 during the G2 and/or M phases, we next investigated the effects on cell cycle progression following overexpression of phosphomimic and phosphonull forms of CaMKIIα. As determined by flow cytometry, the majority of EV MDAMB-231 (57% ± 0.4; Fig. 4A) and SHSY5Y (73% ± 1.4; Fig. 4B) cells were found to be in G1; the next most abundant phase was G2/M (MDA-MB-231: 26% ± 1.7; SHSY5Y: 22% ± 1.1), and the smallest proportion was noted to be in S phase (MDA-MB-231: 17% ± 1.3; SHSY5Y: 5% ± 0.3). Overexpression of WT, T286D, and T253V CaMKIIα did not significantly alter these proportions for either MDA-MB-231 (Fig. 4A)

Fig. 5. Proportion of cells expressing phosphomimic and phosphonull forms of CaMKIIα in each phase of mitosis. (A) MDA-MB-231 and (B) SHSY5Y cells inducibly expressing empty vector (EV), wild-type (WT), T286D, T253D, or T253V CaMKIIα. CaMKIIα expression was induced by 2 μg/ml doxycycline, and 48 h post-CaMKII expression, cells were fixed, stained with anti-αtubulin (red), and counterstained with DAPI (blue). Representative fields for each cell line are shown. Five fields of view were counted per coverslip, and four independent experiments were performed. Results are presented as the percentage of cells in each phase. * denotes statistical significant difference from EV control cells, as determined by one-way ANOVA (p b 0.05).

754 A. Hoffman et al. / Cellular Signalling 26 (2014) 748–756 Fig. 6. Cell survival following expression of phosphomimic and phosphonull forms of CaMKIIα in each phase of mitosis. (A) MDA-MB-231 and (B) SHSY5Y cells inducibly expressing empty vector (EV), wild-type (WT), T286D, T253D, or T253V CaMKIIα. CaMKIIα expression was induced by 2 μg/ml doxycycline, and apoptosis was measured at 0 or 72 h later (Annexin V staining, flow cytometry). Histograms are representative of four independent experiments. The proportion of cells in each quadrant was calculated, and the % of Annexin V positive cells following expression of each construct in (C) MDA-MB-231 and (D) SHSY5Y cells from 3 independent experiments were calculated. * denotes statistical significance from EV control cells, as determined by one-way ANOVA (p b 0.05).

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or SHSY5Y (Fig. 4B) cells. By contrast, overexpression of T253DCaMKIIα significantly decreased the proportion of cells in G1 phase (MDA-MB-231: 24% ± 3.9, p b 0.0001; SHSY5Y: 15% ± 2.9, p b 0.0001), which was accompanied by a significant increase in the proportion of cells in the G2 and/or M phases (MDA-MB-231: 61% ± 2.8, p b 0.0001; SHSY5Y: 71% ± 2.3, p b 0.0001), when compared to EV expressing cells (Fig. 4A and B). To identify the precise phase of the cell cycle in which this accumulation was occurring, cells expressing phosphomimic and phosphonull forms of CaMKIIα were co-stained with anti-α-tubulin and DAPI, and then examined by fluorescent microscopy. The majority of EV MDAMB-231 (71% ± 5.7; Fig. 5A) and SHSY5Y (67% ± 5.6; Fig. 5B) were in interphase. Overexpression of WT, T286D, and T253V CaMKIIα did not alter these proportions for either cell line (Fig. 5A and B). Overexpression of T253D-CaMKIIα significantly decreased the proportion of cells in interphase (MDA-MB-231: 42% ± 2.2, p b 0.001; SHSY5Y: 31% ± 1.4, p b 0.0001), and significantly increased the proportion of cells that were in metaphase (MDA-MB-231: 25% ± 2.5, p b 0.001; SHSY5Y: 34% ± 4.2, p b 0.0001), when compared to EV expressing cells (Fig. 5A and B). Inhibition of cell cycle progression can result in either a delay or in a complete block of progression. If a delay is induced, cells are able to overcome the block and continue to divide, whereas if a complete block occurs, cells will be unable to continue proliferating and, in most cases, will commit to apoptosis. To determine whether the accumulation of cells expressing T253D-CaMKIIα resulted from a delay or a complete block in cell cycle progression, we examined induction of cell death by Annexin V and propidium iodide staining. A significant increase in cell death was observed following expression of T253DCaMKII for 72 h in both MDA-MB-231 and SHSY5Y cells (Fig. 6), indicating that the accumulation of cells in metaphase following overexpression of T253D-CaMKIIα results from a complete block. Taken together with the phosphorylation of endogenous CaMKII data, this suggests that it is the dephosphorylation of CaMKII at T253 that is involved in controlling progression through metaphase into anaphase. 4. Discussion Research in a variety of cell types has established that CaMKII is an important regulator of the cell cycle. Studies utilising pharmacological inhibition [10,11,22,23], siRNA downregulation [13], or expression of a kinase dead mutant form of CaMKII [13] implicate CaMKII as a regulator of progression through the G2 and/or M phases of the cell cycle, but the precise role and mechanism is not known. Our data presented herein shows that CaMKII functions in mitosis, specifically at the metaphase– anaphase transition, and that progression through mitosis is regulated by dephosphorylation of CaMKII at T253. Furthermore, we have shown that PP2A is responsible for this selective dephosphorylation of CaMKII at T253. Thus we have identified a new mechanism for controlling progression through mitosis. CaMKII is a multifunctional kinase that is encoded by four closely related genes (α, β, γ, and δ) that produce highly homologous proteins expressed in virtually every tissue. The autophosphorylation sites, T253 and T286, are conserved across all isoforms. Whilst we used CaMKIIα in this study, as overexpression of WT-CaMKIIα has previously been shown to control osteosarcoma [24], neuroblastoma and breast cancer cell [4] proliferation, CaMKIIγ and CaMKIIδ have also been shown to be involved in controlling leukaemia [25] and vascular smooth muscle [13,26,27] cell proliferation, respectively. Furthermore, overexpression or knockdown of CaMKII was shown to produce the same proliferative effects in each of these studies, irrespective of cell type or isoform examined. This indicates that cell proliferation is a cellular function that is controlled by all isoforms of CaMKII. Evidence for a role of CaMKII in controlling progression through mitosis, and specifically the metaphase–anaphase transition, has come from studies examining oocyte activation. Mammalian oocytes are

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arrested in metaphase of meiosis II, and fertilisation is characterised by a series of calcium spikes, which result in activation of CaMKII and resumption of the metaphase–anaphase transition. Studies using pharmacological inhibitors [28–30], gene knockout [31], or morpholinoinduced downregulation of CaMKII [32] have demonstrated that oocyte activation is prevented by CaMKII inhibition, as oocytes remain arrested at metaphase II. Further evidence indicates that CaMKII is involved in controlling the metaphase–anaphase transition in somatic cells, as pharmacological inhibition of CaMKII in ovarian cancer and endothelial cells results in a transient arrest in metaphase [11]. Our finding that dephosphorylation of CaMKII at T253 is involved in controlling the progression through metaphase extends these previous studies, and provides evidence, for the first time, that CaMKII autophosphorylation at T253 plays a role in controlling the cell cycle. Our findings are consistent with the vast majority of previous studies examining the role of CaMKII in controlling the cell cycle which have used pharmacological inhibitors of CaMKII kinase activity [9,17], and suggest that CaMKII can regulate the cell cycle. CaMKII is a multifunctional kinase that has been shown to phosphorylate several cell cycle regulatory proteins, including cdk2, cyclin E, cyclin D1 [7,27], p53 [27] and pRb [7]. However, the T253D phosphomimic mutation does not inhibit CaMKII activity [6], but it alters the interaction between CaMKII and specific binding partners. Our results suggest that it is not the total activity of CaMKII that is important for regulating the cell cycle, but rather that targeting to specific complexes, regulated by T253 phosphorylation/dephosphorylation that is crucial for the metaphase–anaphase transition, suggesting that dephosphorylation of CaMKII at T253 induces a molecular switch, and targets CaMKII to cell signalling pathways that either allow or prevent progression through metaphase into anaphase. One of the strengths of our study is our use of two different cell lines that have different tissues of origin, different rates of proliferation, and express different endogenous CaMKII isoforms (MDA-MB-231 cells express equivalent amounts of CaMKIIγ and δ, whereas the predominant isoform found in SHSY5Y cells is CaMKIIδ). This shows that it is phosphorylation at the T253 site that is important for controlling progression through mitosis rather than the isoform of CaMKII that is predominantly expressed in the cell. We saw no alteration in cell cycle progression following overexpression of a T286D phosphomimic form of CaMKIIα in breast cancer and neuroblastoma cells, when compared to WT-CaMKIIα and EV expressing cells (Figs. 4 and 5). This is in direct contrast to a study performed by Beauman et al. [33], in which a T286D form of CaMKIIα was inducibly overexpressed in HeLa cells. In that study, cells were synchronised in S phase and CaMKII expression was induced by the addition of doxycycline. When compared to untransfected cells, HeLa cells transfected with T286D-CaMKII possessed a higher proportion of cells in the G2 phase (as determined by flow cytometry), even in the absence of doxycycline (and hence, absence of CaMKIIα expression). This indicates that the transfection procedure may have fundamentally altered these cells, and perhaps accounts for the difference in results. By contrast, in our study, two different cell types overexpressing WT, T286D, and T253V CaMKIIα were examined, and cell cycle profiles were similar to those observed in EV expressing cells, as determined by flow cytometry (Fig. 4) and confocal microscopy (Fig. 5). As cell cycle profiles were compared between CaMKIIα overexpressing and EV expressing cells, as opposed to CaMKIIα expressing cells and untransfected parental cells, our study has accounted for any perturbations in cellular function caused by the transfection procedure. Additionally, our study directly addresses the question of what effect CaMKIIα phosphorylation has on cell cycle progression, rather than simply the presence of CaMKIIα itself. As the biological functions of CaMKII are regulated by autophosphorylation at several sites, including T286 and T253, it is interesting that phosphorylation at these sites affects proliferation differently. It is important to note that these differences in functional outcomes are not due to changes in kinase activity, as T253D phosphomimic mutation

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does not affect activity in vitro [6], and yet overexpression of WT and T253D-CaMKIIα has opposite effects. Furthermore, T286D-CaMKIIα is autonomously active (activity is independent of the presence of calcium and calmodulin); whereas WT-CaMKIIα depends on calcium/calmodulin for activity, and yet the functional effect of overexpression is the same. This suggests that the difference in functional outcomes is produced by T253 phosphorylation-mediated CaMKII targeting, and hence changes in the subcellular location of CaMKII, rather than by alterations in CaMKII activity. As overexpression of a form of CaMKIIα that cannot be dephosphorylated at T253 (T253D phosphomimic mutant) causes an accumulation of cells in metaphase, whereas expression of non-phosphorylated CaMKIIα (WT and T253V phosphonull mutant) has no effect on cell cycle progression (Figs. 4 and 5), our data suggests that it is the dephosphorylation of CaMKII at T253 that is important for progression through metaphase into anaphase (Fig. 1). We have shown that PP2A is the phosphatase responsible for this selective dephosphorylation (Fig. 2). A role for PP2A in cell cycle regulation has long been suspected, with extensive studies in yeast, Drosophila and Xenopus demonstrating its involvement at virtually every stage [34]. PP2A has recently been shown to control both entry into and exit from mitosis [35,36]. PP2A activity is essential for the establishment of chromosome–microtubule attachments during prometaphase [37], and is involved in regulating the spindle checkpoint, by controlling APC/Ccdc20 activity [38] and by interacting with BubR1 to counteract Aurora B kinase activity [39]. Additionally, an endogenous inhibitor of PP2A (namely I2PP2A) colocalises with PP2A at the centromere during meiosis II, and this colocalisation is essential for the separation of sister chromatids in oocytes [40]. Furthermore, PP2A has also been implicated in the maintenance of metaphase II arrest in oocytes [41]. Taken together with our data, these studies suggest that PP2A-mediated dephosphorylation of CaMKII at T253 may be involved in controlling the transition through metaphase into anaphase. 5. Conclusions Taken together, our findings have identified a new targetingmediated mechanism for controlling cell progression through mitosis that involves a specific change in phosphorylation of CaMKII at T253. Furthermore, we have shown that PP2A is the phosphatase responsible for this dephosphorylation. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cellsig.2013.12.015.

supported by a CINSW fellowship. The authors would like to thank Mr Matthew Morten for his technical assistance on various aspects of this project. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33]

Disclosure/conflict of interest The authors declare that they have no conflict of interest regarding any of the work conducted within this manuscript. Acknowledgements

[34] [35] [36] [37] [38] [39]

This work was supported by research funds from the Hunter Medical Research Institute, the Hunter Translational Cancer Research Unit, and the University of Newcastle. LW was supported by an APA, CINSW, and University of Newcastle Vice Chancellor's Scholarships. NV was

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