DYRK1A phosphorylates caspase 9 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 8 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-reg- ulated 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 9 at an inhibitory site, Thr125. DYRK1A- dependent phosphorylation of Thr125 is also blocked by harmine, confirm- ing 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 ⁄ threo- nine 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 9 auto-processing. Phosphorylation of caspase 9 by DYRK1A involves co-localization to the nucleus. These results indicate that DYRK1A sets a threshold for the activation of caspase 9 through basal inhibitory phos- phorylation of this protease. Regulation of apoptosis through inhibitory phosphorylation of caspase 9 may play a role in the function of DYRK1A during development and in pathogenesis.
protein
region’
behavioural abnormalities [4]. The human DYRK1A gene has been implicated as having a role in patho- genesis due to its location in the ‘Down’s syndrome critical (DSCR) on chromosome 21 [5,6], which is present in three copies in Down’s syndrome individuals.
DYRK1A is the most extensively characterized mem- the evolutionarily conserved dual-specificity ber of tyrosine-phosphorylation-regulated kinase (DYRK) family, which is distantly related to mitogen- activated protein kinases (MAPKs), cyclin-dependent protein kinases (CDKs), CDK-like kinases (CLKs) and glycogen synthase kinase 3 [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 neurogen- esis. Mutant flies with reduced expression of minibrain kinase, the Drosophila orthologue of DYRK1A, dis- play specific reductions in the size of the optic lobes and central brain hemispheres as well as distinctive
The molecular mechanisms underlying Down’s syn- drome 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 dos- age 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 calci- neurin, 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.
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Results
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.
Identification of DYRK1A as a Thr125 kinase in cells
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 9 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 9 into a high-molecular-mass multimeric complex, termed the apoptosome [9]. Apaf-1-induced dimerization of pro- caspase 9 leads to its activation and autocatalytic pro- cessing [10]. Active caspase 9 initiates a proteolytic cascade by processing and activating downstream effector caspases such as caspase 3 and caspase 7, lead- ing to the organized disassembly of the cell [11].
Previous studies have shown that caspase 9 is phosphor- ylated on a single major site, Thr125, catalysed by the proline-directed kinases ERK1 ⁄ 2 MAPKs and CDK1–cyclin B1 in response to growth factors and during mitosis, respectively. However, residual phos- phorylation when ERK1 ⁄ 2 and CDK1 are inhibited suggests that an additional kinase also targets this site [14,18]. In serum-starved U2.C9–C287A cells, a U2OS- derived cell line stably expressing catalytically inactive caspase 9 [18], the MEK1 inhibitors PD0325901 and U0126, which block ERK1 ⁄ 2 activation, did not reduce basal Thr125 phosphorylation (Fig. 1A). By contrast, both inhibitors blocked ERK1 ⁄ 2-dependent phospho- rylation 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, pur- valanol A or roscovitine (Fig. 1C), only roscovitine and purvalanol A, but not alsterpaullone, caused a reduc- tion 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 ⁄ 2 or CDK activity, but is sensitive to roscovitine and purvalanol A.
studies, we obtained evidence
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 the pathway is controlled down- [12]. In addition, stream of cytochrome c release at the level of the apoptosome [13]. Caspase 9 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 sur- vival signals, restrain caspase 9 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 9 on the same residue [18]. During these that Thr125 may be subject to phosphorylation by addi- tional protein kinase activities, because a basal level of Thr125 phosphorylation persists when ERK1 ⁄ 2 and CDK1–cyclin B1 are inhibited [14,18].
exist
DYRK1 has been reported to be inhibited by rosco- vitine 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 phosphory- lated serine or threonine residue [22,23], although [24,25]. DYRK1A in particular exceptions the )2 position favours an additional proline at [22,23], as found in caspase 9 (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. Transfec- tion of two distinct synthetic short interfering RNA (siRNA) duplexes targeting DYRK1A mRNA resulted
Here, we identify DYRK1A as an additional kinase that targets Thr125 of caspase 9 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 evi- dence that harmine, a potent and specific inhibitor of DYRKs in vitro [19], efficiently inhibits DYRK1A activity towards caspase 9 in cells and also blocks the co-translational activating tyrosine autophosphoryla- tion of DYRK1A, showing that this b-carboline alka- loid can be used to test proposed cellular targets of DYRK1A and potentially could be used to reverse the effects of DYRK1A overexpression.
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Fig. 1. Small-molecule inhibitors suggest DYRK1A as a potential kinase that phosphorylates caspase 9 on Thr125. (A) U2.C9–C287A cells stably expressing caspase 9(C287A) were serum-starved and treated with protein kinase inhibitors for 30 min as indicated. (B) U2.C9–C287A cells were serum-starved and preincubated with inhibitors 30 min prior to addition of TPA for 15 min or (C) incubated with nocodazole for 16 h before addition of inhibitors for 15 min. 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 9 with the reported DYRK and DYRK1A consensus phosphorylation sequences [22,23]. Phosphorylated residues are highlighted in bold.
– – A p-Casp9 (T125) Casp9 H.sapiens M.musculus R.norvegicus Actin
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
member DYRK1B (85% identical amino acids in the catalytic domain) [3] when this kinase was expressed as a green fluorescent protein (GFP) fusion protein, indi-
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Fig. 2. DYRK1A depletion inhibits phosphorylation of caspase 9 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.
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conclude that DYRK1A is required for basal phosphor- ylation of caspase 9 at Thr125 and we can exclude DYRK1B as a significant Thr125 kinase in U2OS cells.
Direct phosphorylation of caspase 9 by DYRK1A
not
impinge
on ERK1 ⁄ 2
In contrast
cating their specificity for DYRK1A (Fig. 2B). Deple- tion 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 phos- phorylation of caspase 9 in cells. DYRK1A depletion did phosphorylation (Fig. 2C,D) nor did it prevent the phosphorylation of caspase 9 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 9. 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
To establish whether DYRK1A can phosphorylate cas- pase 9 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 cas- pase 9 mutant in which Thr125 was mutated to alanine (T125A) was not phosphorylated (Fig. 3A). Thus, DYRK1A phosphorylates caspase 9 directly in vitro
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Fig. 3. DYRK1A interacts with caspase 9 and induces its phosphorylation on Thr125 in cells. (A) DYRK1A directly phosphorylates caspase 9 at Thr125. Recombinant His6–caspase 9 (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 9 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.
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and that Thr125 is the sole phosphorylation site on caspase 9 targeted by DYRK1A.
the reduction of Thr125 phosphorylation is due to defective ERK1 ⁄ 2 activation (Fig. 4A). In agreement, we also found no effect of 1 lm harmine on TPA- induced ERK1 ⁄ 2 activation and the subsequent phos- phorylation of Thr125 (Fig. 4B).
against
another
Expression of exogenous FLAG-tagged DYRK1A in U2.C9–C287A cells caused a strong increase in the phosphorylation of caspase 9 on Thr125 (Fig. 3B), confirming the ability of DYRK1A to target caspase 9 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 activa- tion-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 cataly- sing Thr125 phosphorylation even though it is not responsible for basal Thr125 phosphorylation in U2OS cells.
phosphorylation
induced
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 cyclin-dependent kinase, CDK2–cyclin A, at 1 lm harmine. Consistent with the latter study, we observed that 1 lm harmine had no effect on CDK1–cyclin B1-dependent Thr125 phos- phorylation induced by nocodazole (Fig. 4C). These results confirm that harmine selectively inhibits the basal phosphorylation of caspase 9 at Thr125 that is the ERK1 ⁄ 2-dependent due to DYRK1A, but not phosphorylation induced by mitogens or the CDK1– cyclin B1-dependent by mitotic arrest. When the phosphorylation of Thr125 in caspase 9 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 1 lm harmine on the phosphorylation of Thr125 on endogenous caspase 9 immunoprecipitated from HeLa cells. Immunoblots showed a reduction of Thr125 phosphorylation in response to harmine, confirming that endogenous caspase 9 is also phosphorylated in a DYRK1A-depen- dent manner (Fig. 4E).
Many protein kinases interact with their substrates in complexes that can be co-precipitated. For example, caspase 9 associates with CDK1–cyclin B1 in cells dur- ing G2 and mitosis [18]. To test for the interaction between caspase 9 and DYRK1A, U2OS cells were transiently co-transfected with expression vectors encoding caspase 9 and FLAG–DYRK1A or FLAG– DYRK1A(K188R). Both wild-type and K188R mutant FLAG–DYRK1A were able to precipitate caspase 9, indicating caspase 9 and DYRK1A do indeed asso- ciate in cells, but DYRK1A kinase activity is not required. Furthermore, caspase 9(C287A ⁄ T125A) lack- ing Thr125 still precipitated with FLAG–DYRK1A (Fig. 3D), showing that Thr125 and its phosphoryla- tion are dispensable for the interaction. Taken together with the ability of DYRK1A to catalyse the phospho- rylation of caspase 9 at Thr125 in vitro, these results strongly indicate that DYRK1A targets caspase 9 directly in cells.
Harmine is a potent inhibitor of DYRK1A in cells
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 pur- valanol A [21], inhibits the autophosphorylation of DYRK1A on tyrosine in addition to the phosphoryla- tion of exogenous substrates. Using an assay developed by Lochhead et al. translated FLAG– [21], we 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 1 lm (Fig. 4F).
Inhibition of caspase 9 auto-processing by DYRK1A
shown an inhibitory effect of Previous work has Thr125 phosphorylation on caspase 9 activation, thereby blocking downstream caspase 3 activation and
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 (cid:2) 10-fold less potently, and little or no inhibition by 1 lm harmine of a panel of 67 other protein kinases the basal phosphorylation of [19]. We found that Thr125 in U2.C9–C287A cells that is due to DYRK1A was potently inhibited by harmine, with a partial inhi- bition even at a concentration of 0.01 lm and almost complete inhibition at 1 lm (Fig. 4A). We did not observe any impairment of the basal phosphorylation of ERK1 ⁄ 2 by harmine, excluding the possibility that
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Fig. 4. Harmine inhibits DYRK1A-dependent phosphorylation of caspase 9 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 min. (B) Harmine does not inhibit ERK1 ⁄ 2 activation or TPA-induced Thr125 phosphorylation. Serum-starved U2.C9–C287A cells were incubated with harmine for 15 min prior to addition of TPA for further 15 min. (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 min. (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 min. (E) HeLa cells were incubated in the presence of 1 lM harmine where indicated for 30 min, followed by immunoprecipitation of endogenous caspase 9 using a caspase 9-spe- cific antibody. Mock immunoprecipitations were carried out using an anti-Myc IgG. (F) FLAG–DYRK1A was in vitro translated in rabbit reticulo- cyte 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.
1 – – EV FLAG-DYRK1A p-Casp9(T125) IP Harmine PA Casp9 – – Inhibitor ( M) Casp9 p-Tyr Input FLAG-DYRK1A Actin
Thr125 on caspase 9. This result indicates that phos- phorylation of Thr125 by DYRK1A inhibits caspase 9 activation, consistent with previous studies on the effects of phosphorylation of this site by other kinases [14,18].
DYRK1A phosphorylates caspase 9 in the nucleus
co-expression
DYRK1A has been reported as a predominantly nuclear kinase [2]. We therefore wished to determine whether DYRK1A and caspase 9 co-localize. As anti- cipated, DYRK1A expressed as a fusion protein with GFP was found to be predominantly nuclear by con- focal microscopy (Fig. 6A). When caspase 9 was expressed as the catalytically inactive mutant C287A in
apoptosis [14,18]. Although proteolytic processing of caspase 9 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 catalyt- ically active caspase 9 in U2OS cells, generation of the p35 auto-processed form was significantly reduced by co-expression of DYRK1A (Fig. 5). Auto-processing of caspase 9 was not antagonized by DYRK1A if the Thr125 residue of caspase 9 was converted to alanine (Fig. 5A). Furthermore, inhibition of caspase 9 auto- processing required the kinase activity of DYRK1A, because the DYRK1A–K188R of mutant did not block caspase 9 auto-processing (Fig. 5B). Thus, DYRK1A inhibits the auto-processing of caspase 9 in cells through the phosphorylation of
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To test if caspase 9 phosphorylation takes place in the nucleus where DYRK1A is localized, we engi- neered GFP–caspase 9 fusion constructs tagged with either a nuclear localization signal (NLS) or a nuclear the export signal NLS–GFP–caspase 9 and NES–GFP–caspase 9 pro- teins localized to the nucleus and cytoplasm, respec- tively (Fig. 6B). When co-expressed with DYRK1A, GFP–caspase 9 and NLS–GFP–caspase 9 exhibited a higher level of Thr125 phosphorylation than NES– In agreement with this GFP–caspase 9 (Fig. 6C). result, we also found that the endogenous basal Thr125 kinase had a stronger preference for nuclear caspase 9 than for cytoplasmic caspase 9 (Fig. 6D). This nuclear kinase activity towards caspase 9 was sen- sitive to harmine and thus due to DYRK1A (Fig. S2). these results demonstrate that DYRK1A Together, and caspase 9 co-localize to the nucleus and that DYRK1A phosphorylates caspase 9 in the nuclear compartment.
Processed Casp9 Procaspase9 Processed Casp9
p-Casp9(T125)
DYRK1A
Discussion
Fig. 5. Phosphorylation of Thr125 by DYRK1A inhibits caspase 9 activation. (A) U2OS cells were transfected with pcDNA3 vectors encoding catalytically active caspase 9 (wild-type; WT) or catalyti- cally active caspase 9(T125A) and FLAG–DYRK1A or empty vector (EV). (B) U2OS cells were transfected with pcDNA3 vectors encod- ing catalytically active caspase 9 (wild-type) and wild-type (WT) or K188R FLAG–DYRK1A or empty vector (EV). Cell lysates were prepared 7 h after transfection and blotted with the indicated anti- bodies.
Actin
The dual-specificity tyrosine phosphorylation-regulated protein kinase DYRK1A plays important roles during development and in human pathologies. However, lit- tle is currently known about the substrates through which it exerts these effects. Here, we have identified the apoptotic protease caspase 9 as a substrate for site, phosphorylation by DYRK1A at a critical Thr125. Previously we have shown that phosphoryla- tion of this site inhibits the activation of caspase 9 and restrains apoptosis in human cells [14,18]. We propose that basal phosphorylation of Thr125 in caspase 9 by DYRK1A sets a threshold in the response to apoptotic stimuli that is augmented in proliferating cells through the activities of ERK1 ⁄ 2 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-depen- dent regulation of DYRK activity [31], and DYRK1A may also be regulated through alterations of its expres- sion during development and the cell cycle in mamma- lian cells [32,33]. Thus, the level of phosphorylation of caspase 9 catalysed by DYRK1A and its significance for cell survival is likely to be modulated by changes in DYRK1A expression in vivo.
(Fig. 6A). The
increase
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 9 (Fig. 6A). These speckles were not removed by siRNA-mediated depletion of endogenous caspase 9 (Fig. S1); therefore the speckles are not likely to corre- spond to phosphorylated caspase 9 and probably origi- nate from another epitope. However, in cells in which caspase 9 was co-expressed with DYRK1A, the signal strongly detected by the pThr125 antibody was increased in the nucleus, and pThr125 epitopes also appeared in the cytoplasm. The increased signal gener- ated by DYRK1A expression was due entirely to the phosphorylation of caspase 9 at Thr125, because no increased signal was detected when cells were co-trans- fected with a non-phosphorylatable mutant of cas- pase 9(T125A) in Thr125 phosphorylation also required the kinase activity of DYRK1A, because it was not induced by the ATP- binding site mutant K188R (data not shown).
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
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DNA
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Fig. 6. Phosphorylation of caspase 9 on Thr125 by DYRK1A in the nucleus. (A) Immunofluorescence staining of U2OS cells transiently trans- fected with vectors encoding caspase 9(C287A), caspase 9(T125A ⁄ C287A), GFP or GFP–DYRK1A. Cells were fixed 20–24 h after transfec- tion and stained with antibodies directed against total caspase 9 and caspase 9 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 9. 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 9. 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).
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[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: muta- tion 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 9 also has an essential function in mouse brain development [36,37].
[28,44], but inhibition of DYRK1A at low (sub-micro- molar) 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–ser- ine ⁄ 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 cell- permeable DYRK1A inhibitor is anticipated to facili- tate 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
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 9 (Fig. S1). This study shows that caspase 9 is partially localized to the nucleus and its phosphoryla- tion by DYRK1A is promoted by nuclear targeting and diminished by cytoplasmic targeting. Previously, although caspase 9 was reported to be mainly localized to mitochondria and cytosol when analysed by subcel- lular fractionation of Jurkat cells [39], GFP–caspase 9 was found partially localized to nuclei in Jurkat and HEK293 cells [40]. Nuclear caspase 9 has also been observed in mammary epithelial cells [41]. Our results show that caspase 9 is distributed in both the nucleus and cytoplasm in U2OS cells. Caspase 9 would encounter DYRK1A in the nucleus and become phos- phorylated, before being redistributed to the cyto- plasm. In this way, a nuclear kinase, DYRK1A, can regulate the cytoplasmic activity of caspase 9 as an ini- tiator of apoptosis. It does, however, remain possible that caspase 9 has a distinct function within the nucleus that is controlled by DYRK1A.
Caspase 9 cDNA in pcDNA3 (Invitrogen, Carlsbad, CA, USA) or pET28a (Novagen, Madison, WI, USA) has been described previously [14]. To generate an expression con- struct for GFP–caspase 9 fusion protein, caspase 9 cDNA was subcloned into pEGFP(C2). Vectors encoding NLS– GFP–caspase 9 and NES–GFP–caspase 9 were constructed by insertion of the SV40T NLS or the NES from the pro- tein kinase A inhibitor between the initiating ATG and the second codon of EGFP using the QuikChange(cid:2) site- directed 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 QuikChange(cid:2) kit (Stratagene, Cedar Creek, TX, USA) according to the manufacturer’s instructions. Recom- binant DYRK1A, expressed as a fusion protein with gluta- thione 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 proto- cols: caspase 9 mAb (Chemicon, Temecula, CA, USA), phospho-ERK1 ⁄ 2, phospho-Tyr-100 (both Cell Signalling Technology, Beverly, MA, USA), ERK1 ⁄ 2 (Millipore), GFP, DYRK1A G-19 (both Santa Cruz Biotechnology, Santa Cruz, CA), Actin, FLAG-M2 (both Sigma-Aldrich,
Identification of caspase 9 as a bona fide substrate for DYRK1A in cells has enabled us to confirm the b-carboline alkaloid harmine as an intracellular inhibi- tor of DYRK1A, as suggested by its identification as a potent and selective inhibitor of DYRKs in vitro [19]. in Peganum harmala and b-Carbolines are present other plants which have been used as medicinal pre- parations as well as hallucinogens in traditional rituals. Harmine has a long history of use as a chemothera- peutic drug for a number of diseases, including malar- ial 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
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St Louis, MO, USA), myc-9E10 (Cancer Research UK, London, UK). Generation and characterization of rabbit anti-[phospho-caspase 9(T125)] IgG was described previ- ously [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
following siRNA duplexes were used to deplete DYRK1A (sense strands): 5¢-UAAGGAUGCUUGAUUAUGAdTdT-3¢ (DYRK1A#1), 5¢-AAACUCGAAUUCAACCUUAdTdT-3¢ siRNAs used were 5¢-CGUACG (DYRK1A#2). Other (Luciferase), 5¢-CGACCU CGGAAUACUUCGAdTdT-3¢ 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).
[32P]ATP[cP]
Caspase 9 and FLAG immunoprecipitations
Recombinant His6–caspase 9 (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, 1 mm dithiothreitol) (from a 10 mCiÆmL)1 containing 1.5 lCi stock with specific activity 3000 CiÆmmol)1). The kinase assay was initiated by adding 30 ng of active recombinant GST–DYRK1A. Reaction mixtures were incubated at 30 (cid:3)C for 30 min. Reactions were stopped by boiling in SDS ⁄ PAGE sample buffer and half the volume of a reac- tion was analysed by SDS ⁄ PAGE, followed by autoradio- graphy.
Cell culture, DNA transfections and treatments
Cells were lysed in IP buffer (20 mm Tris pH 7.6, 137 mm NaCl, 2 mm EDTA, 1 mm Na3VO4, 50 mm NaF, 5 mm b-glycerophosphate, 1% Triton X-100, 1 lm okadaic acid, 1 mm phenylmenthanesulfonyl fluoride, 1 lgÆmL)1 each aprotinin, leupeptin and pepstatin A). Immunoprecipitation of endogenous caspase 9 from HeLa cells was carried out as described previously [14]. For co-immunoprecipitation of FLAG–DYRK1A and caspase 9 from U2OS cells, cell lysate (0.5 mg) was incubated with 15 lL anti-(FLAG aga- rose) (Sigma) for 1 h at 4 (cid:3)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
alsterpaullone
(10 lm),
DYRK1A contains an internal stretch of 13 consecutive histidine residues, enabling the endogenous DYRK1A pro- tein 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 5 mm imidazole for 5 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 buf- fer 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% Tri- ton X-100). For elution, beads were boiled in SDS ⁄ PAGE sample buffer. Supernatant was analysed by western blot- ting and endogenous DYRK1A was detected with a DYRK1A-specific antibody.
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 UÆmL)1 penicillin, 50 lgÆmL)1 streptomycin and 2 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 ngÆmL)1, Calbiochem). Where indi- cated, 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 (Qia- gen, Valencia, CA, USA) according to manufacturer’s pro- tocol. To arrest cells in mitosis, cells were treated with 100 ngÆmL)1 nocodazole for 16 h. To activate ERK1 ⁄ 2 MAPK signalling, cells were incubated with 1 lm TPA for 15 min. The protein kinase inhibitors PD0325901 (0.1 lm), (10 lm), purvalanol A U0126 (10 lm), roscovitine (20 lm) or harmine (routinely, 1 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.
RNA interference
In vitro translation and immunoprecipitation of FLAG–DYRK1A from rabbit reticulocyte lysate
siRNA transfections,
cells were
For 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 cou- pled transcription ⁄ translation protocol (Promega, Madison, WI, USA) and pcDNA3–FLAG–DYRK1A as template.
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DYRK1B, a novel member of the DYRK family of protein kinases. Biochem Biophys Res Commun 254, 474–479.
4 Tejedor F, Zhu XR, Kaltenbach E, Ackermann A,
Following incubation of the reaction mixture at 30 (cid:3)C for 1 h, IP buffer containing 2 mm Na3VO4 and washed anti- (FLAG–M2) agarose beads (Sigma) were added. Immuno- precipitation was carried out as outlined below. Protein was eluted in SDS ⁄ PAGE sample buffer containing 2 mm Na3VO4 at 37 (cid:3)C.
Baumann A, Canal I, Heisenberg M, Fischbach KF & Pongs O (1995) Minibrain: a new protein kinase family involved in postembryonic neurogenesis in Drosophila. Neuron 14, 287–301.
Immunofluorescence
5 Song WJ, Sternberg LR, Kasten-Sportes C, Keuren ML, Chung SH, Slack AC, Miller DE, Glover TW, Chiang PW, Lou L et al. (1996) Isolation of human and murine homologues of the Drosophila minibrain gene: human homologue maps to 21q22.2 in the Down syndrome ‘critical region’. Genomics 38, 331–339.
6 Guimera J, Casas C, Pucharcos C, Solans A, Domenech A, Planas AM, Ashley J, Lovett M, Estivill X & Prit- chard MA (1996) A human homologue of Drosophila minibrain (MNB) is expressed in the neuronal regions affected in Down syndrome and maps to the critical region. Hum Mol Genet 5, 1305–1310.
7 Arron JR, Winslow MM, Polleri A, Chang CP, Wu H, Gao X, Neilson JR, Chen L, Heit JJ, Kim SK et al. (2006) NFAT dysregulation by increased dosage of DSCR1 and DYRK1A on chromosome 21. Nature 441, 595–600.
8 Gwack Y, Sharma S, Nardone J, Tanasa B, Iuga A,
Cells were transfected on coverslips and fixed with 3% para- formaldehyde, 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 1 h at room temperature. Samples were incubated overnight at 4 (cid:3)C with anti-(caspase 9) (1 : 100) and anti-[phospho-caspase 9(T125)] (1 : 50) purified sera, followed by incubation with AlexaFlu- (1 : 100; or647-conjugated anti-mouse Invitrogen) and (1 : 20; DAKO) IgG for TRITC-conjugated anti-rabbit 45 min 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
Srikanth S, Okamura H, Bolton D, Feske S, Hogan PG et al. (2006) A genome-wide Drosophila RNAi screen identifies DYRK-family kinases as regulators of NFAT. Nature 441, 646–650.
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We thank P. Cohen (Dundee, UK) for reagents and helpful discussion of this work. We are also grateful to W. Becker (Aachen, Germany) for providing GFP– DYRK1A and GFP–DYRK1B DNA constructs, and to D. Meek (Dundee, UK) for the pcDNA3–FLAG vector. We thank the members of the Clarke labora- tory, A. Cole, M. Soutar, C. Sutherland and S. Keyse for their help. This work was supported by a Cancer Research UK Studentship (AS), the Association for International Cancer Research (LAA) and a Royal Society–Wolfson Research Merit Award (PRC).
family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 9, 47–59.
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Supporting information
The following supplementary material is available: Fig. S1. Deletion of caspase 9 by siRNA does not reduce nuclear speckle staining caused by pThr125 antibody.
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