DYRK1A phosphorylates caspase at an inhibitory site and is potently inhibited in human cells by harmine Anne Seifert, Lindsey A Allan and Paul R Clarke Biomedical Research Institute, College of Medicine, Dentistry and Nursing, University of Dundee, UK Keywords apoptosis; caspase; DYRK; harmine; protein kinase Correspondence P R Clarke, Biomedical Research Institute, University of Dundee, Level 5, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK Fax: +44 1382 669993 Tel: +44 1382 425580 E-mail: p.r.clarke@dundee.ac.uk (Received 28 August 2008, revised October 2008, accepted 21 October 2008) doi:10.1111/j.1742-4658.2008.06751.x DYRK1A is a member of the dual-specificity tyrosine-phosphorylation-regulated protein kinase family and is implicated in Down’s syndrome Here, we identify the cysteine aspartyl protease caspase 9, a critical component of the intrinsic apoptotic pathway, as a substrate of DYRK1A Depletion of DYRK1A from human cells by short interfering RNA inhibits the basal phosphorylation of caspase at an inhibitory site, Thr125 DYRK1Adependent phosphorylation of Thr125 is also blocked by harmine, confirming the use of this b-carboline alkaloid as a potent inhibitor of DYRK1A in cells We show that harmine not only inhibits the protein–serine ⁄ threonine kinase activity of mature DYRK1A, but also its autophosphorylation on tyrosine during translation, indicating that harmine prevents formation of the active enzyme When co-expressed in cells, DYRK1A interacts with caspase 9, strongly induces Thr125 phosphorylation and inhibits caspase auto-processing Phosphorylation of caspase by DYRK1A involves co-localization to the nucleus These results indicate that DYRK1A sets a threshold for the activation of caspase through basal inhibitory phosphorylation of this protease Regulation of apoptosis through inhibitory phosphorylation of caspase may play a role in the function of DYRK1A during development and in pathogenesis DYRK1A is the most extensively characterized member of the evolutionarily conserved dual-specificity tyrosine-phosphorylation-regulated protein kinase (DYRK) family, which is distantly related to mitogenactivated protein kinases (MAPKs), cyclin-dependent protein kinases (CDKs), CDK-like kinases (CLKs) and glycogen synthase kinase [1] The DYRK family comprises several members in mammals, of which DYRK1A and DYRK1B are predominantly localized to the nucleus, whereas DYRK2 is cytoplasmic [2,3] Functional studies in mammals and Drosophila suggest a conserved regulatory role for DYRK1A in neurogenesis Mutant flies with reduced expression of minibrain kinase, the Drosophila orthologue of DYRK1A, display specific reductions in the size of the optic lobes and central brain hemispheres as well as distinctive behavioural abnormalities [4] The human DYRK1A gene has been implicated as having a role in pathogenesis due to its location in the ‘Down’s syndrome critical region’ (DSCR) on chromosome 21 [5,6], which is present in three copies in Down’s syndrome individuals The molecular mechanisms underlying Down’s syndrome and its associated pathologies are likely to be complex An important role has been proposed for the transcription factor nuclear factor of activated T cells (NFAT), which is dysregulated by increased gene dosage of DYRK1A and DSCR1, another DSCR gene that encodes an inhibitor of the protein phosphatase calcineurin ⁄ PP-2B [7] Phosphorylation of NFAT by DYRKs counteracts its dephosphorylation by calcineurin, thereby retaining NFAT in the cytoplasm and Abbreviations CDK, cyclin-dependent kinase; CLK, CDK-like kinase; DYRK, dual-specificity tyrosine phosphorylation-regulated kinase; ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; MAPK, mitogen-activated protein kinase; NES, nuclear export signal; NFAT, nuclear factor of activated T cells; NLS, nuclear localization signal; siRNA, short interfering RNA; TPA, 12-O-tetradecanoylphorbol-13-acetate 6268 FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS A Seifert et al inhibiting its transcriptional activity [8] However, the substrates and cellular functions of DYRK1A during normal development and in pathological conditions remain to be fully identified Here, we identify a novel substrate for DYRK1A, the cysteine aspartyl protease caspase 9, which is a critical component of the intrinsic or mitochondrial apoptotic pathway Caspase is fully activated by a variety of apoptotic stimuli that trigger the release of cytochrome c from mitochondria Once in the cytosol, cytochrome c induces the oligomerization of Apaf-1 and subsequent recruitment of procaspase into a high-molecular-mass multimeric complex, termed the apoptosome [9] Apaf-1-induced dimerization of procaspase leads to its activation and autocatalytic processing [10] Active caspase initiates a proteolytic cascade by processing and activating downstream effector caspases such as caspase and caspase 7, leading to the organized disassembly of the cell [11] Regulation of apoptosome formation is controlled at the level of cytochrome c release from mitochondria by pro- and anti-apoptotic proteins of the Bcl-2 family [12] In addition, the pathway is controlled downstream of cytochrome c release at the level of the apoptosome [13] Caspase activation is subject to modulation by protein kinases activated in signal transduction pathways initiated by extracellular signals or cellular stresses [14–16] We have shown previously that extracellular signal-regulated kinase (ERK)1 and ERK2 MAPKs, which are activated in response to survival signals, restrain caspase activation by direct phosphorylation on a critical inhibitory site, Thr125 [14,17] Furthermore, CDK1–cyclin B1 protects mitotic cells from apoptosis induced by microtubule poisons by phosphorylating caspase on the same residue [18] During these studies, we obtained evidence that Thr125 may be subject to phosphorylation by additional protein kinase activities, because a basal level of Thr125 phosphorylation persists when ERK1 ⁄ and CDK1–cyclin B1 are inhibited [14,18] Here, we identify DYRK1A as an additional kinase that targets Thr125 of caspase in cells These results suggest a function of DYRK1A in the regulation of apoptosis that may be relevant to its roles during development and in pathogenesis We also present evidence that harmine, a potent and specific inhibitor of DYRKs in vitro [19], efficiently inhibits DYRK1A activity towards caspase in cells and also blocks the co-translational activating tyrosine autophosphorylation of DYRK1A, showing that this b-carboline alkaloid can be used to test proposed cellular targets of DYRK1A and potentially could be used to reverse the effects of DYRK1A overexpression Phosphorylation of caspase by DYRK1A Results Identification of DYRK1A as a Thr125 kinase in cells Previous studies have shown that caspase is phosphorylated on a single major site, Thr125, catalysed by the proline-directed kinases ERK1 ⁄ MAPKs and CDK1–cyclin B1 in response to growth factors and during mitosis, respectively However, residual phosphorylation when ERK1 ⁄ and CDK1 are inhibited suggests that an additional kinase also targets this site [14,18] In serum-starved U2.C9–C287A cells, a U2OSderived cell line stably expressing catalytically inactive caspase [18], the MEK1 inhibitors PD0325901 and U0126, which block ERK1 ⁄ activation, did not reduce basal Thr125 phosphorylation (Fig 1A) By contrast, both inhibitors blocked ERK1 ⁄ 2-dependent phosphorylation of Thr125 induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (Fig 1B) Furthermore, although the CDK1–cyclin B1-dependent phosphorylation of Thr125 induced by the microtubule poison nocodazole, which arrests cells in mitosis, was inhibited by the CDK inhibitors alsterpaullone, purvalanol A or roscovitine (Fig 1C), only roscovitine and purvalanol A, but not alsterpaullone, caused a reduction in basal phospho-Thr125 levels (Fig 1A) These results suggest that the basal Thr125 phosphorylation in unstimulated cells involves a novel kinase that is not dependent on ERK1 ⁄ or CDK activity, but is sensitive to roscovitine and purvalanol A DYRK1 has been reported to be inhibited by roscovitine and purvalanol A, but not by alsterpaullone [20,21] Consistent with the idea that DYRK1A might be a physiologically relevant Thr125 kinase, the amino acid context of Thr125 fulfils the reported primary sequence requirements of DYRK kinases (Fig 1D) DYRK1A and related DYRKs are proline-directed kinases which require a proline immediately C-terminal to the phosphorylated serine or threonine residue They also favour the presence of an arginine residue at the )2 or )3 position N-terminal to the phosphorylated serine or threonine residue [22,23], although exceptions exist [24,25] DYRK1A in particular favours an additional proline at the )2 position [22,23], as found in caspase (Fig 1D) Our initial results therefore lead us to investigate further the potential role of DYRK1A as a Thr125 kinase To test the role of DYRK1A independently of chemical inhibitors, we depleted DYRK1A from U2.C9–C287A cells using RNA interference Transfection of two distinct synthetic short interfering RNA (siRNA) duplexes targeting DYRK1A mRNA resulted FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS 6269 Phosphorylation of caspase by DYRK1A A Seifert et al – TPA B – – p-Casp9 (T125) PD U0 PD U0 Alst PA Rosc A p-Casp9 (T125) Casp9 Casp9 p-ERK1/2 p-ERK1/2 ERK1/2 ERK1/2 Actin Actin C – – D Alst PA Rosc Nocodazole p-Casp9 (T125) H.sapiens M.musculus R.norvegicus Casp9 Actin Fig Small-molecule inhibitors suggest DYRK1A as a potential kinase that phosphorylates caspase on Thr125 (A) U2.C9–C287A cells stably expressing caspase 9(C287A) were serum-starved and treated with protein kinase inhibitors for 30 as indicated (B) U2.C9–C287A cells were serum-starved and preincubated with inhibitors 30 prior to addition of TPA for 15 or (C) incubated with nocodazole for 16 h before addition of inhibitors for 15 Inhibitors used were PD0325901 (PD; 0.1 lM), U0126 (U0; 10 lM), alsterpaullone (Alst; 10 lM), purvalanol A (PA; 10 lM) or roscovitine (Rosc; 20 lM) Cell lysates were probed on blots with antibodies against the specified proteins (D) Comparison of the amino acid sequence surrounding Thr125 of human, mouse and rat caspase with the reported DYRK and DYRK1A consensus phosphorylation sequences [22,23] Phosphorylated residues are highlighted in bold in a strong decrease of endogenous DYRK1A protein levels (Fig 2A) These siRNA duplexes had no effect on the expression of the most closely related family A member DYRK1B (85% identical amino acids in the catalytic domain) [3] when this kinase was expressed as a green fluorescent protein (GFP) fusion protein, indisiRNA B Luc 1A #1 1A #2 Luc 1A #1 1A #2 1B siRNA DYRK1A GFP-DYRK1B-p69 Actin Actin D p-Casp9 (T125) p-ERK1/2 ERK1/2 ERK1/2 Actin 1B Casp9 p-ERK1/2 1A #2 p-Casp9 (T125) Casp9 1A #1 Luc siRNA 1B 1A #2 Luc 1A #1 siRNA C Actin Fig DYRK1A depletion inhibits phosphorylation of caspase on Thr125 in cells (A) Transfection with two DYRK1A-targeting siRNA duplexes ablates endogenous DYRK1A protein U2.C9–C287A cells were transfected with siRNA duplexes targeting DYRK1A (1A#1 and 1A#2) or luciferase (Luc) as control, serum-starved for 48 h after transfection and lysed 24 h later Endogenous DYRK1A was precipitated from cell lysates using Ni2+-NTA agarose (B) Transfection of U2.C9–C287A cells with DYRK1A targeting siRNA duplexes (1A#1 and 1A#2) has no effect on protein levels of overexpressed GFP–DYRK1B–p69, whereas transfection with DYRK1B targeting siRNA (1B) decreases GFP–DYRK1B–p69 protein levels siRNA transfections were carried out 24 h prior to DNA transfections Cells were lysed 72 h after siRNA transfections (C) Depletion of DYRK1A decreases Thr125 phosphorylation in serum-starved U2.C9–C287A cells Transfections were carried out as in (A) (D) Depletion of DYRK1A decreases Thr125 phosphorylation in U2.C9–C287A cells in the presence of serum Transfections were carried out as in (A) In all cases, proteins were detected on immunoblots probed with the indicated antibodies 6270 FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS A Seifert et al Phosphorylation of caspase by DYRK1A cating their specificity for DYRK1A (Fig 2B) Depletion of DYRK1A by siRNA significantly inhibited basal Thr125 phosphorylation in cells both in the absence (Fig 2C) and in the presence (Fig 2D) of serum, demonstrating a role for DYRK1A in the phosphorylation of caspase in cells DYRK1A depletion did not impinge on ERK1 ⁄ phosphorylation (Fig 2C,D) nor did it prevent the phosphorylation of caspase at Thr125 induced by the phorbol ester TPA (data not shown), showing that DYRK1A is not required for the ERK1 ⁄ 2-dependent phosphorylation of caspase In contrast to the effect of depleting DYRK1A, a DYRK1B-specific siRNA (see Fig 2B for validation of knockdown efficiency) had no effect on Thr125 phosphorylation (Fig 2C,D) Therefore, we B FLAG-DYRK1A WT K188R Y321F To establish whether DYRK1A can phosphorylate caspase directly, we carried out an in vitro kinase assay using [32P]ATP[cP] and catalytically inactive His6– caspase 9(C287A) as a substrate Active DYRK1A produced in bacteria catalysed the incorporation of radiolabelled phosphate into caspase 9, whereas a caspase mutant in which Thr125 was mutated to alanine (T125A) was not phosphorylated (Fig 3A) Thus, DYRK1A phosphorylates caspase directly in vitro His-C9 (T125A) His-C9 His-C9 +DYRK1A Direct phosphorylation of caspase by DYRK1A EV A conclude that DYRK1A is required for basal phosphorylation of caspase at Thr125 and we can exclude DYRK1B as a significant Thr125 kinase in U2OS cells p-Casp9 (T125) Casp9 p-ERK1/2 ERK1/2 Autorad FLAG Coomassie p-Casp9 (T125) C9 T125A + DYRK WT GFP-DYRK1B D EV GFP-DYRK1A C DYRK WT DYRK K188R C9 C9 + DYRK WT C9 + DYRK K188R C9 T125A Actin Casp9 IP: -FLAG FLAG Casp9 p-ERK1/2 Casp9 ERK1/2 GFP Actin Input FLAG Actin Fig DYRK1A interacts with caspase and induces its phosphorylation on Thr125 in cells (A) DYRK1A directly phosphorylates caspase at Thr125 Recombinant His6–caspase (His–C9) or His6–caspase 9(T125A) (both containing the catalytically inactivating C287A mutation) was incubated with recombinant DYRK1A in the presence of [32P]ATP[cP] as indicated Samples were analysed by SDS ⁄ PAGE, followed by autoradiography (B) Expression of DYRK1A causes Thr125 phosphorylation in cells U2.C9–C287A cells were transiently transfected with empty vector (EV) or wild-type (WT), K188R or Y321F mutant DYRK1A in pcDNA3–FLAG (C) Expression of DYRK1B also induces Thr125 phosphorylation U2.C9–C287A cells were transiently transfected with empty vector (EV), pEGFP–DYRK1A or pEGFP–DYRK1B-p69 (D) DYRK1A interacts with caspase in cells U2OS cells were co-transfected with empty vector, wild-type (WT) or K188R DYRK1A in pcDNA3–FLAG and caspase 9(C287A) (C9) or caspase 9(T125A ⁄ C287A) in pcDNA3 A portion of each cell lysate was retained as an input sample and FLAG-immunoprecipitations were performed on the remainder *Indicates bands resulting from IgG; arrows indicate bands of interest In (B–D), cell lysates were prepared 24 h after transfection and proteins were detected on immunoblots probed with the indicated antibodies FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS 6271 Phosphorylation of caspase by DYRK1A A Seifert et al and that Thr125 is the sole phosphorylation site on caspase targeted by DYRK1A Expression of exogenous FLAG-tagged DYRK1A in U2.C9–C287A cells caused a strong increase in the phosphorylation of caspase on Thr125 (Fig 3B), confirming the ability of DYRK1A to target caspase in cells The FLAG–DYRK1A mutants K188R or Y321F, the catalytic activity of which is reduced due to a mutation in the ATP-binding site or the activation-loop, respectively [26,27], did not result in strong Thr125 phosphorylation (Fig 3B), confirming that the protein kinase activity of DYRK1A is required Expression of DYRK1B as a GFP fusion protein caused strong Thr125 phosphorylation like DYRK1A (Fig 3C), showing that DYRK1B is capable of catalysing Thr125 phosphorylation even though it is not responsible for basal Thr125 phosphorylation in U2OS cells Many protein kinases interact with their substrates in complexes that can be co-precipitated For example, caspase associates with CDK1–cyclin B1 in cells during G2 and mitosis [18] To test for the interaction between caspase and DYRK1A, U2OS cells were transiently co-transfected with expression vectors encoding caspase and FLAG–DYRK1A or FLAG– DYRK1A(K188R) Both wild-type and K188R mutant FLAG–DYRK1A were able to precipitate caspase 9, indicating caspase and DYRK1A indeed associate in cells, but DYRK1A kinase activity is not required Furthermore, caspase 9(C287A ⁄ T125A) lacking Thr125 still precipitated with FLAG–DYRK1A (Fig 3D), showing that Thr125 and its phosphorylation are dispensable for the interaction Taken together with the ability of DYRK1A to catalyse the phosphorylation of caspase at Thr125 in vitro, these results strongly indicate that DYRK1A targets caspase directly in cells Harmine is a potent inhibitor of DYRK1A in cells The b-carboline alkaloid harmine has recently been reported as a specific DYRK inhibitor in vitro by Bain et al [19] Harmine inhibits purified DYRK1A in the nanomolar range, with DYRK2 and DYRK3 inhibited  10-fold less potently, and little or no inhibition by lm harmine of a panel of 67 other protein kinases [19] We found that the basal phosphorylation of Thr125 in U2.C9–C287A cells that is due to DYRK1A was potently inhibited by harmine, with a partial inhibition even at a concentration of 0.01 lm and almost complete inhibition at lm (Fig 4A) We did not observe any impairment of the basal phosphorylation of ERK1 ⁄ by harmine, excluding the possibility that 6272 the reduction of Thr125 phosphorylation is due to defective ERK1 ⁄ activation (Fig 4A) In agreement, we also found no effect of lm harmine on TPAinduced ERK1 ⁄ activation and the subsequent phosphorylation of Thr125 (Fig 4B) A previous study identified harmine as an inhibitor of CDKs at micromolar concentrations, with an IC50 = 17 lm for CDK1–cyclin B [28] Bain et al [19], however, demonstrated no significant inhibitory activity against another cyclin-dependent kinase, CDK2–cyclin A, at lm harmine Consistent with the latter study, we observed that lm harmine had no effect on CDK1–cyclin B1-dependent Thr125 phosphorylation induced by nocodazole (Fig 4C) These results confirm that harmine selectively inhibits the basal phosphorylation of caspase at Thr125 that is due to DYRK1A, but not the ERK1 ⁄ 2-dependent phosphorylation induced by mitogens or the CDK1– cyclin B1-dependent phosphorylation induced by mitotic arrest When the phosphorylation of Thr125 in caspase was strongly induced by the expression of FLAG–DYRK1A, this activity was also completely inhibited by harmine, although higher concentrations of the inhibitor were required, presumably because of the elevated levels of DYRK1A in the cells (Fig 4D) We also analysed the effect of lm harmine on the phosphorylation of Thr125 on endogenous caspase immunoprecipitated from HeLa cells Immunoblots showed a reduction of Thr125 phosphorylation in response to harmine, confirming that endogenous caspase is also phosphorylated in a DYRK1A-dependent manner (Fig 4E) DYRKs are unusual dual-specificity kinases that require autophosphorylation of an essential Tyr–Xaa– Tyr motif in the activation loop to form a mature kinase that has specificity towards serine and threonine residues in substrate proteins [21,26,29] Therefore, we were interested in studying whether harmine, like purvalanol A [21], inhibits the autophosphorylation of DYRK1A on tyrosine in addition to the phosphorylation of exogenous substrates Using an assay developed by Lochhead et al [21], we translated FLAG– DYRK1A in rabbit reticulocyte lysate in the absence or presence of harmine and found that harmine also blocks the tyrosine autophosphorylation of DYRK1A, with almost complete inhibition at lm (Fig 4F) Inhibition of caspase auto-processing by DYRK1A Previous work has shown an inhibitory effect of Thr125 phosphorylation on caspase activation, thereby blocking downstream caspase activation and FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS Phosphorylation of caspase by DYRK1A M) – TPA B Harmine( p-Casp9(T125) M) p-Casp9(T125) Casp9 Casp9 p-ERK1/2 ERK1/2 p-ERK1/2 ERK1/2 Actin Actin M) – – D M) 0.1 50 Flag Actin – -caspase-9 – – F EV p-Casp9(T125) Casp9 FLAG-DYRK1A Harmine Inhibitor ( M) Casp9 – – PA p-Tyr Actin Input – – Casp9 Mock Harmine( M) 10 Harmine( p-Casp9(T125) p-Casp9(T125) Casp9 Actin E EV FLAG-DYRK1A 0.1 10 100 Harmine( 0.1 0.01 0.001 Nocodazole 100 C IP – – 0.1 0.01 0.001 Harmine( 0.1 A 0.01 0.001 A Seifert et al FLAG-DYRK1A Fig Harmine inhibits DYRK1A-dependent phosphorylation of caspase on Thr125 in cells (A) Harmine inhibits the basal phosphorylation of Th125 Serum-starved U2.C9–C287A cells were treated with indicated concentrations of harmine for 30 (B) Harmine does not inhibit ERK1 ⁄ activation or TPA-induced Thr125 phosphorylation Serum-starved U2.C9–C287A cells were incubated with harmine for 15 prior to addition of TPA for further 15 (C) Harmine does not inhibit mitotic phosphorylation of Thr125 U2.C9–C287A cells were incubated in the presence of nocodazole for 16 h prior to addition of harmine for 30 (D) Harmine inhibits Thr125 phosphorylation caused by DYRK1A overexpression in cells U2OS cells were transfected with pcDNA3–caspase 9(C287A) and pcDNA3–FLAG–DYRK1A or empty vector (EV) Twenty hours after transfection, cells were incubated with indicated concentrations of harmine for 30 (E) HeLa cells were incubated in the presence of lM harmine where indicated for 30 min, followed by immunoprecipitation of endogenous caspase using a caspase 9-specific antibody Mock immunoprecipitations were carried out using an anti-Myc IgG (F) FLAG–DYRK1A was in vitro translated in rabbit reticulocyte lysate in the absence or presence of indicated concentrations of harmine or purvalanol A (PA), followed by immunoprecipitation using anti-(FLAG agarose) EV indicates empty vector In all cases, proteins were detected on immunoblots probed with the indicated antibodies apoptosis [14,18] Although proteolytic processing of caspase is not required for its activation, generation of a processed p35 form is dependent on the catalytic activity of the enzyme [30] When we expressed catalytically active caspase in U2OS cells, generation of the p35 auto-processed form was significantly reduced by co-expression of DYRK1A (Fig 5) Auto-processing of caspase was not antagonized by DYRK1A if the Thr125 residue of caspase was converted to alanine (Fig 5A) Furthermore, inhibition of caspase autoprocessing required the kinase activity of DYRK1A, because co-expression of the DYRK1A–K188R mutant did not block caspase auto-processing (Fig 5B) Thus, DYRK1A inhibits the auto-processing of caspase in cells through the phosphorylation of Thr125 on caspase This result indicates that phosphorylation of Thr125 by DYRK1A inhibits caspase activation, consistent with previous studies on the effects of phosphorylation of this site by other kinases [14,18] DYRK1A phosphorylates caspase in the nucleus DYRK1A has been reported as a predominantly nuclear kinase [2] We therefore wished to determine whether DYRK1A and caspase co-localize As anticipated, DYRK1A expressed as a fusion protein with GFP was found to be predominantly nuclear by confocal microscopy (Fig 6A) When caspase was expressed as the catalytically inactive mutant C287A in FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS 6273 Phosphorylation of caspase by DYRK1A EV DYRK1A WT To test if caspase phosphorylation takes place in the nucleus where DYRK1A is localized, we engineered GFP–caspase fusion constructs tagged with either a nuclear localization signal (NLS) or a nuclear export signal (NES) When expressed in cells, the NLS–GFP–caspase and NES–GFP–caspase proteins localized to the nucleus and cytoplasm, respectively (Fig 6B) When co-expressed with DYRK1A, GFP–caspase and NLS–GFP–caspase exhibited a higher level of Thr125 phosphorylation than NES– GFP–caspase (Fig 6C) In agreement with this result, we also found that the endogenous basal Thr125 kinase had a stronger preference for nuclear caspase than for cytoplasmic caspase (Fig 6D) This nuclear kinase activity towards caspase was sensitive to harmine and thus due to DYRK1A (Fig S2) Together, these results demonstrate that DYRK1A and caspase co-localize to the nucleus and that DYRK1A phosphorylates caspase in the nuclear compartment T125A EV DYRK1A A A Seifert et al Casp9 Procaspase9 Processed Casp9 p-Casp9(T125) Procaspase9 Processed Casp9 FLAG EV DYRK1A WT B Casp9 p-Casp9(T125) DYRK1A K188R Actin Procaspase9 Processed Casp9 Procaspase9 Processed Casp9 DYRK1A Discussion Actin Fig Phosphorylation of Thr125 by DYRK1A inhibits caspase activation (A) U2OS cells were transfected with pcDNA3 vectors encoding catalytically active caspase (wild-type; WT) or catalytically active caspase 9(T125A) and FLAG–DYRK1A or empty vector (EV) (B) U2OS cells were transfected with pcDNA3 vectors encoding catalytically active caspase (wild-type) and wild-type (WT) or K188R FLAG–DYRK1A or empty vector (EV) Cell lysates were prepared h after transfection and blotted with the indicated antibodies U2OS cells, it localized to both the cytoplasm and the nucleus Nuclear speckle-like foci were detected by the pThr125 antibody in the absence of transfected caspase (Fig 6A) These speckles were not removed by siRNA-mediated depletion of endogenous caspase (Fig S1); therefore the speckles are not likely to correspond to phosphorylated caspase and probably originate from another epitope However, in cells in which caspase was co-expressed with DYRK1A, the signal detected by the pThr125 antibody was strongly increased in the nucleus, and pThr125 epitopes also appeared in the cytoplasm The increased signal generated by DYRK1A expression was due entirely to the phosphorylation of caspase at Thr125, because no increased signal was detected when cells were co-transfected with a non-phosphorylatable mutant of caspase 9(T125A) (Fig 6A) The increase in Thr125 phosphorylation also required the kinase activity of DYRK1A, because it was not induced by the ATPbinding site mutant K188R (data not shown) 6274 The dual-specificity tyrosine phosphorylation-regulated protein kinase DYRK1A plays important roles during development and in human pathologies However, little is currently known about the substrates through which it exerts these effects Here, we have identified the apoptotic protease caspase as a substrate for phosphorylation by DYRK1A at a critical site, Thr125 Previously we have shown that phosphorylation of this site inhibits the activation of caspase and restrains apoptosis in human cells [14,18] We propose that basal phosphorylation of Thr125 in caspase by DYRK1A sets a threshold in the response to apoptotic stimuli that is augmented in proliferating cells through the activities of ERK1 ⁄ and CDK1–cyclin B1 kinases [14,18] Although DYRK1A appears to be synthesized as a constitutively active enzyme, work on the cytoplasmic Caenorhabditis elegans DYRK orthologue MBK-2 has shown a cell-cycle and developmental stimulus-dependent regulation of DYRK activity [31], and DYRK1A may also be regulated through alterations of its expression during development and the cell cycle in mammalian cells [32,33] Thus, the level of phosphorylation of caspase catalysed by DYRK1A and its significance for cell survival is likely to be modulated by changes in DYRK1A expression in vivo DYRK1A has been mapped to the Down’s syndrome critical region on chromosome 21 that is present as an additional copy in Down’s syndrome individuals [5,6] DYRK1A is overexpressed in Down’s syndrome brains FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS A Seifert et al Phosphorylation of caspase by DYRK1A A Casp9 GFP p-Casp9(T125) DNA Casp9 GFP-DYRK1A p-Casp9(T125) DNA Casp9 GFP p-Casp9(T125) DNA Casp9 GFP-DYRK1A p-Casp9(T125) DNA Casp9/ GFP Casp9/ GFP-DYRK1A Casp9(T125A)/ GFP Casp9(T125A)/ GFP-DYRK1A NLS-Casp9 Casp9 NLS-Casp9 NLS-Casp9 GFP-Casp9 DNA Casp9 GFP NES-Casp9 EV NES-Casp9 FLAGDYRK1A C NES-Casp9 B p-Casp9 (T125) GFP-Casp9 Actin NES-GFP-Casp9 FLAG D Casp9 NLS-GFP-Casp9 p-Casp9(T125) GFP-Casp9 Actin Fig Phosphorylation of caspase on Thr125 by DYRK1A in the nucleus (A) Immunofluorescence staining of U2OS cells transiently transfected with vectors encoding caspase 9(C287A), caspase 9(T125A ⁄ C287A), GFP or GFP–DYRK1A Cells were fixed 20–24 h after transfection and stained with antibodies directed against total caspase and caspase phosphorylated on Thr125 DNA was DAPI-stained and cells were analysed by confocal microscopy Scale bars, 10 lm (B) Localization of GFP–caspase 9(C287A), NES–GFP–caspase 9(C287A) and NLS–GFP–caspase 9(C287A) in U2OS cells Cells were transiently transfected with pEGFP vectors encoding the respective fusion proteins, followed by fixation after 8–9 h Scale bars, 10 lm (C) Overexpressed DYRK1A predominantly phosphorylates nuclear caspase U2OS cells were transfected as in (B), but in combination with empty vector (EV) or DYRK1A in pcDNA3–FLAG and lysed 8–9 h after transfection (D) Endogenous Thr125 kinase(s) preferentially phosphorylate(s) nuclear caspase U2OS cells were transfected as in (B) In (C, D), cell lysates were blotted with antibodies against the specified proteins Note that samples in (C) were harvested 8–9 h after transfection, whereas cells in (D) were lysed 20–24 h after transfection This difference in expression time accounts for the absence of a p-Casp-9(T125) signal in lanes 1–3 in (C) FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS 6275 Phosphorylation of caspase by DYRK1A A Seifert et al [34], suggesting a role in neurogenesis like its Drosophila orthologue, minibrain (mnb) [4] Studies in both mouse and Drosophila have found an important role for DYRK1A ⁄ minibrain kinase in determining the number of neurons during post-embryonic neurogenesis: mutation of minibrain causes reduction of the size of the optic lobes and central brain hemispheres [4], whereas mice lacking one copy of the DYRK1A gene exhibit region-specific reductions in brain size [35] Although the molecular mechanisms underlying this phenotype are not understood, regulation of apoptosis might be involved This idea is particularly appealing because caspase also has an essential function in mouse brain development [36,37] DYRK1A is a predominantly nuclear kinase that is localized to intranuclear splicing speckles, which are sites of mRNA processing [38] Our pThr125 antibody also detects these speckles (Fig 6; data not shown), although the phosphoepitope is probably not due to caspase (Fig S1) This study shows that caspase is partially localized to the nucleus and its phosphorylation by DYRK1A is promoted by nuclear targeting and diminished by cytoplasmic targeting Previously, although caspase was reported to be mainly localized to mitochondria and cytosol when analysed by subcellular fractionation of Jurkat cells [39], GFP–caspase was found partially localized to nuclei in Jurkat and HEK293 cells [40] Nuclear caspase has also been observed in mammary epithelial cells [41] Our results show that caspase is distributed in both the nucleus and cytoplasm in U2OS cells Caspase would encounter DYRK1A in the nucleus and become phosphorylated, before being redistributed to the cytoplasm In this way, a nuclear kinase, DYRK1A, can regulate the cytoplasmic activity of caspase as an initiator of apoptosis It does, however, remain possible that caspase has a distinct function within the nucleus that is controlled by DYRK1A Identification of caspase as a bona fide substrate for DYRK1A in cells has enabled us to confirm the b-carboline alkaloid harmine as an intracellular inhibitor of DYRK1A, as suggested by its identification as a potent and selective inhibitor of DYRKs in vitro [19] b-Carbolines are present in Peganum harmala and other plants which have been used as medicinal preparations as well as hallucinogens in traditional rituals Harmine has a long history of use as a chemotherapeutic drug for a number of diseases, including malarial infection and Parkinson’s disease [42] Harmine and related b-carbolines have cytotoxic activity towards human tumour cell lines in culture [43], suggesting a possible use in anti-cancer therapy Several putative molecular targets for harmine have been identified 6276 [28,44], but inhibition of DYRK1A at low (sub-micromolar) concentrations in cells strongly suggests that inhibition of this kinase is involved in the biological activity of harmine in vivo Interestingly, our results show that harmine not only inhibits the protein–serine ⁄ threonine kinase activity of the mature enzyme, but also the tyrosine autophosphorylation that is required for maturation of the active enzyme [21] This indicates that harmine also inhibits formation of active DYRK1A in cells Identification of harmine as a cellpermeable DYRK1A inhibitor is anticipated to facilitate the identification of further DYRK1A substrates in vivo and also suggests its potential use to reverse the pathological effects of DYRK1A overexpression Experimental procedures Plasmids and recombinant proteins Caspase cDNA in pcDNA3 (Invitrogen, Carlsbad, CA, USA) or pET28a (Novagen, Madison, WI, USA) has been described previously [14] To generate an expression construct for GFP–caspase fusion protein, caspase cDNA was subcloned into pEGFP(C2) Vectors encoding NLS– GFP–caspase and NES–GFP–caspase were constructed by insertion of the SV40T NLS or the NES from the protein kinase A inhibitor between the initiating ATG and the second codon of EGFP using the QuikChangeÔ sitedirected mutagenesis kit Wild-type and K188R mutant pEGFP(C1)–DYRK1A (rat) as well as pEGFP(C1)– DYRK1B-p69 (human) were kind gifts from W Becker (Aachen, Germany) An expression construct for FLAG– DYRK1A was generated by subcloning DYRK1A cDNA into pcDNA3–FLAG (kindly provided by D Meek, University of Dundee, UK) Expression of recombinant His6–caspase 9(C287A) and His6–caspase 9(T125A ⁄ C287A) proteins was carried out as described previously [14] All expression constructs encoding proteins bearing amino acid substitutions were generated by site-directed mutagenesis using the QuikChangeÔ kit (Stratagene, Cedar Creek, TX, USA) according to the manufacturer’s instructions Recombinant DYRK1A, expressed as a fusion protein with glutathione S-transferase (GST–DYRK1A) in Escherichia coli, was purchased from Millipore (Watford, UK) Antibodies and reagents The following antibodies were used for western blotting and immunological staining according to standard protocols: caspase mAb (Chemicon, Temecula, CA, USA), phospho-ERK1 ⁄ 2, phospho-Tyr-100 (both Cell Signalling Technology, Beverly, MA, USA), ERK1 ⁄ (Millipore), GFP, DYRK1A G-19 (both Santa Cruz Biotechnology, Santa Cruz, CA), Actin, FLAG-M2 (both Sigma-Aldrich, FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS A Seifert et al St Louis, MO, USA), myc-9E10 (Cancer Research UK, London, UK) Generation and characterization of rabbit anti-[phospho-caspase 9(T125)] IgG was described previously [14] Reagents used were: nocodazole (Sigma), TPA and protein kinase inhibitors (Calbiochem, San Diego, CA, USA), harmine (Sigma-Aldrich) and PD0325901 (kindly provided by P Cohen, University of Dundee, UK) DYRK1A kinase assay Recombinant His6–caspase (1.5 lg) was added to a total reaction volume of 15 lL kinase assay buffer (50 mm Tris pH 7.5, 10 mm MgCl2, 100 lm ATP, mm dithiothreitol) containing 1.5 lCi [32P]ATP[cP] (from a 10 mCiỈmL)1 stock with specific activity 3000 CiỈmmol)1) The kinase assay was initiated by adding 30 ng of active recombinant GST–DYRK1A Reaction mixtures were incubated at 30 °C for 30 Reactions were stopped by boiling in SDS ⁄ PAGE sample buffer and half the volume of a reaction was analysed by SDS ⁄ PAGE, followed by autoradiography Cell culture, DNA transfections and treatments HeLa and U2OS cells (obtained from Cancer Research UK Cell Services) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 50 mL)1 penicillin, 50 lgỈmL)1 streptomycin and mm l-glutamine (Invitrogen, Carlsbad, CA) For U2.C9– C287A cells [18], which stably express catalytically inactive caspase 9(C287A), the growth medium was supplemented with G418 sulfate (400 ngỈmL)1, Calbiochem) Where indicated, serum starvation was performed by culturing cells in Dulbecco’s modified Eagle’s medium containing 0% fetal bovine serum for 20–24 h DNA transfections were carried out using CsCl-purified plasmid DNA and Superfect (Qiagen, Valencia, CA, USA) according to manufacturer’s protocol To arrest cells in mitosis, cells were treated with 100 ngỈmL)1 nocodazole for 16 h To activate ERK1 ⁄ MAPK signalling, cells were incubated with lm TPA for 15 The protein kinase inhibitors PD0325901 (0.1 lm), U0126 (10 lm), alsterpaullone (10 lm), purvalanol A (10 lm), roscovitine (20 lm) or harmine (routinely, lm) were added as indicated The specificity of these inhibitors towards a panel of purified protein kinases is reported by Bain et al [19] For analysis by immunoblotting, cells were lysed in SDS ⁄ PAGE sample buffer Phosphorylation of caspase by DYRK1A following siRNA duplexes were used to deplete DYRK1A (sense strands): 5¢-UAAGGAUGCUUGAUUAUGAdTdT-3¢ (DYRK1A#1), 5¢-AAACUCGAAUUCAACCUUAdTdT-3¢ (DYRK1A#2) Other siRNAs used were 5¢-CGUACG CGGAAUACUUCGAdTdT-3¢ (Luciferase), 5¢-CGACCU GACUGCCAAGAAAdTdT-3¢ (Caspase 9) and DYRK1B SmartPool (Dharmacon) comprising four different duplexes: 5¢-GAAAUUGACUCGCUCAUUGrUrU-3¢, 5¢-ACACGG AGAUGAAGUACUArUrU-3¢, 5¢-GCCAGAGGAUCUA CCAGUArUrU-3¢, 5¢-GCACAUCAAUGAGGUAUACr UrU-3¢ Single siRNA duplexes were synthesized by MWG (Martinsried, Germany) Caspase and FLAG immunoprecipitations Cells were lysed in IP buffer (20 mm Tris pH 7.6, 137 mm NaCl, mm EDTA, mm Na3VO4, 50 mm NaF, mm b-glycerophosphate, 1% Triton X-100, lm okadaic acid, mm phenylmenthanesulfonyl fluoride, lgỈmL)1 each aprotinin, leupeptin and pepstatin A) Immunoprecipitation of endogenous caspase from HeLa cells was carried out as described previously [14] For co-immunoprecipitation of FLAG–DYRK1A and caspase from U2OS cells, cell lysate (0.5 mg) was incubated with 15 lL anti-(FLAG agarose) (Sigma) for h at °C Beads were washed three times in IP buffer and boiled in SDS ⁄ PAGE sample buffer Samples were analysed by western blotting Ni2+-pulldown of endogenous DYRK1A from cells DYRK1A contains an internal stretch of 13 consecutive histidine residues, enabling the endogenous DYRK1A protein to bind Ni2+-NTA agarose [45] U2.C9–C287A cells were lysed in buffer A (6 m guanidine–HCl, 10 mm Tris, 0.1 m phosphate buffer, pH 8.0) supplemented with mm imidazole for min, sonicated and incubated with 30 lL Ni-NTA–agarose (Qiagen) for 4–5 h Beads were pelleted and washed once in buffer A, followed by one wash in buffer B (8 m urea, 10 mm Tris, 0.1 m phosphate buffer, pH 8.0), one wash in buffer C (8 m urea, 10 mm Tris, 0.1 m phosphate buffer pH 6.5, 0.2% Triton X-100) and one wash in buffer D (buffer C supplemented with 0.1% Triton X-100) For elution, beads were boiled in SDS ⁄ PAGE sample buffer Supernatant was analysed by western blotting and endogenous DYRK1A was detected with a DYRK1A-specific antibody RNA interference In vitro translation and immunoprecipitation of FLAG–DYRK1A from rabbit reticulocyte lysate For siRNA transfections, cells were transfected with 100 nm siRNA duplex and Lipofectamine 2000 following the manufacturer’s instructions (Invitrogen) Then, cells were trypsinised and cultured for 72 h before analysis The In vitro translation of FLAG–DYRK1A was performed in absence or presence of inhibitors using the TNT Quick coupled transcription ⁄ translation protocol (Promega, Madison, WI, USA) and pcDNA3–FLAG–DYRK1A as template FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS 6277 Phosphorylation of caspase by DYRK1A A Seifert et al Following incubation of the reaction mixture at 30 °C for h, IP buffer containing mm Na3VO4 and washed anti(FLAG–M2) agarose beads (Sigma) were added Immunoprecipitation was carried out as outlined below Protein was eluted in SDS ⁄ PAGE sample buffer containing mm Na3VO4 at 37 °C Immunofluorescence Cells were transfected on coverslips and fixed with 3% paraformaldehyde, permeabilised with 0.2% Triton X-100 in Tris-buffered saline and incubated in blocking buffer (5% fetal bovine serum in Tris-buffered saline) for h at room temperature Samples were incubated overnight at °C with anti-(caspase 9) (1 : 100) and anti-[phospho-caspase 9(T125)] (1 : 50) purified sera, followed by incubation with AlexaFluor647-conjugated anti-mouse (1 : 100; Invitrogen) and TRITC-conjugated anti-rabbit (1 : 20; DAKO) IgG for 45 at room temperature DNA was stained with DAPI Microscopy was carried out using a LSM 510 confocal microscope (Zeiss) with a 63· Plan Apochromat objective Fluorescent signals of EGFP (excitation 488 nm using an argon laser), TRITC (excitation 543 nm using a HeNe1 laser) and AlexaFluor-647 (excitation 633 nm using a HeNe3 laser) were detected using band-pass 505–530 nm, 560–615 nm and long-pass 650 nm emission filters, respectively Acknowledgements We thank P Cohen (Dundee, UK) for reagents and helpful discussion of this work We are 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supplementary material is available: Fig S1 Deletion of caspase by siRNA does not reduce nuclear speckle staining caused by pThr125 antibody 6280 FEBS Journal 275 (2008) 6268–6280 ª 2008 The Authors Journal compilation ª 2008 FEBS ... Thr125 in cells (A) DYRK1A directly phosphorylates caspase at Thr125 Recombinant His6? ?caspase (His–C9) or His6? ?caspase 9( T125A) (both containing the catalytically inactivating C287A mutation) was incubated... inhibitor in vitro by Bain et al [ 19] Harmine inhibits purified DYRK1A in the nanomolar range, with DYRK2 and DYRK3 inhibited  10-fold less potently, and little or no inhibition by lm harmine of a panel... evidence that harmine, a potent and specific inhibitor of DYRKs in vitro [ 19] , efficiently inhibits DYRK1A activity towards caspase in cells and also blocks the co-translational activating tyrosine autophosphorylation