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EMBO Rep. Feb 2004; 5(2): 161–166.
Published online Jan 16, 2004. doi:  10.1038/sj.embor.7400072
PMCID: PMC1298983
Scientific Report

Involvement of ASK1 in Ca2+-induced p38 MAP kinase activation


The mammalian mitogen-activated protein (MAP) kinase kinase kinase apoptosis signal-regulating kinase 1 (ASK1) is a pivotal component in cytokine- and stress-induced apoptosis. It also regulates cell differentiation and survival through p38 MAP kinase activation. Here we show that Ca2+ signalling regulates the ASK1–p38 MAP kinase cascade. Ca2+ influx evoked by membrane depolarization in primary neurons and synaptosomes induced activation of p38, which was impaired in those derived from ASK1-deficient mice. Ca2+/calmodulin-dependent protein kinase type II (CaMKII) activated ASK1 by phosphorylation. Moreover, p38 activation induced by the expression of constitutively active CaMKII required endogenous ASK1. Thus, ASK1 is a critical intermediate of Ca2+ signalling between CaMKII and p38 MAP kinase.


Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein (MAP) kinase kinase kinase family member and is activated in response to various cytotoxic stresses, including TNF, Fas and reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) (Ichijo et al, 1997; Chang et al, 1998; Nishitoh et al, 1998; Saitoh et al, 1998). Overexpression of wild-type or constitutively active ASK1 induces apoptosis in various cells through mitochondria-dependent caspase activation (Ichijo et al, 1997; Saitoh et al, 1998; Hatai et al, 2000), and ASK1 is required for apoptosis induced by oxidative stress, TNF and endoplasmic reticulum (ER) stress (Tobiume et al, 2001; Nishitoh et al, 2002). Conversely, ASK1 induces neuronal differentiation and survival in PC12 cells (Takeda et al, 2000) and keratinocyte differentiation (Sayama et al, 2000), both of which are largely dependent on p38 MAP kinase activation. Thus, ASK1 is implicated in a variety of biological activities.

A series of recent studies has suggested the potential roles for p38 MAP kinases in diverse neuronal function. It has been shown that p38 is involved in the induction of long-term depression (LTD) of synaptic transmission at hippocampal CA3–CA1 synapses (Bolshakov et al, 2000). p38 has also been reported to play a major role when Rap, a member of the Ras superfamily of small GTPases, mediates removal of synaptic AMPARs (glutamate receptors of the AMPA subtype) that occurs during LTD (Zhu et al, 2002). Moreover, activation of presynaptic P2X7 receptors, which form ATP-gated Ca2+-permeable cation channels, appears to depress mossy-fibre-CA3 synaptic transmission through p38 (Armstrong et al, 2002). These neuronal functions of p38 are thought to be commonly regulated by Ca2+ signals. In the nervous system, increases in intracellular Ca2+ following activation of voltage-dependent Ca2+ channels (VDCC), ligand-gated channels or release of Ca2+ from intracellular stores control diverse biological functions including neurotransmitter release and many forms of synaptic plasticity (Berridge, 1998). However, whether and how Ca2+ signals regulate p38 remains to be elucidated. In this study, we demonstrate that Ca2+ signals regulate the ASK1–p38 MAP kinase cascade and that Ca2+/calmodulin-dependent protein kinase type II (CaMKII) serves as an activator of the ASK1–p38 axis in Ca2+ signalling.

Results and Discussion

To examine the possible involvement of ASK1 in Ca2+ signalling, we first treated mouse embryonic fibroblasts (MEFs) with maitotoxin (MTX), which induced influx of extracellular Ca2+ even in nonexcitable cells (Escobar et al, 1998). The activation states of ASK1 and p38 were monitored by specific antibodies that detect the activating phosphorylation of each molecule (Tobiume et al, 2002). Activations of p38 as well as ASK1 were readily detected within 5 min of treatment with MTX in MEFs (Fig 1A, left panels). MTX-induced activations of ASK1 and p38 were abolished in the presence of EGTA in the culture medium of MEFs (see supplementary information online). These results suggest that Ca2+ influx activates ASK1 and p38. Next, we analysed the requirement of ASK1 for Ca2+-induced p38 activation by using MEFs derived from ASK1-deficient (ASK1−/−) mice (Tobiume et al, 2001). Interestingly, only the early (within 5 min) but not the relatively late (10 or 15 min) response of p38 to MTX was impaired in ASK1−/− MEFs (Fig 1A, right panels), suggesting that ASK1 contributes to an early-phase activation of p38 by Ca2+ influx in MEFs. To confirm the role of ASK1 in Ca2+-induced activation of p38, we reintroduced wild-type ASK1 cDNA into ASK1−/− MEFs using an adenovirus vector. Reintroduction of ASK1 clearly restored the MTX-induced early activation of p38 in ASK1−/− MEFs (Fig 1B), indicating that ASK1 is specifically required for the early activation of p38 by Ca2+ influx in MEFs. These results contrasted with our previous finding that ASK1 is selectively required for TNF- and oxidativestress-induced, sustained (late-phase) activation of p38 in MEFs (Tobiume et al, 2001). These differences may account for the distinct roles of ASK1 in each signalling pathway, in that the duration of activation of MAP kinases, that is transient (early-phase) versus sustained (late-phase), has been proposed to be a critical factor for the decision of cell fate (Marshall, 1995; Takeda et al, 2003).

Figure 1
ASK1 is required for Ca2+-induced activation of p38. (A) Lack of Ca2+-induced early activation of p38 in ASK1−/− MEFs. ASK1+/+ and ASK1−/− MEFs were treated with 0.5 nM maitotoxin (MTX) for ...

Since the induction of robust Ca2+ influx by MTX in MEFs is less physiological, we evoked Ca2+ influx through VDCC by membrane depolarization in primary neurons from ASK1+/+ and ASK1−/− embryonic day 14.5 (E14.5) mice. Both the basal activity and the activation of p38 induced by membrane depolarization were considerably impaired in ASK1−/− neurons (Fig 1C). This result suggested that, in neurons, ASK1 was required not only for Ca2+-induced p38 activation but also for the basal activity of p38. Nevertheless, since partial activation of p38 was still observed in ASK1−/− neurons (Fig 1C), an ASK1-independent pathway that mediates a depolarization-induced signal to p38 may exist in cultured neurons. We next examined the contribution of ASK1 to Ca2+ signalling in a more compartmentalized region within neurons. Subcellular fractionations of mouse brains disclosed that ASK1 existed in the synaptosomal fraction (Fig 1D). When purified synaptosomes from ASK1+/+ mouse brains were treated with 50 mM KCl in the presence of Ca2+, activation of p38 was detected (Fig 1E, top panel), indicating that all the required signalling components leading to p38 were contained in synaptosomes. In the synaptosomes derived from ASK1−/− mouse brains, membrane depolarization-induced activation of p38 was abolished (Fig 1E), although weak activation below the level of detection might be induced. In the same experiment, response of ERK to Ca2+ influx was intact in ASK1−/− synaptosomes (Fig 1F), excluding the possibility that the synaptosomes derived from ASK1−/− mice were qualitatively different from those derived from ASK1+/+ mice. Activation of JNK was undetectable in cultured neurons and synaptosomes derived from both ASK1+/+ and ASK1−/− mice (data not shown). These results suggest that ASK1 selectively mediates p38 activity in Ca2+ signalling in synapses, and subcellular localization of ASK1 may be an important factor to determine the response of neurons to Ca2+ signals.

In Caenorhabditis elegans, Ca2+ signalling through VDCC and UNC-43/CaMKII determines asymmetric expression of the candidate odorant receptor gene str-2 in AWC olfactory neurons (Troemel et al, 1999). Recently, nsy-1, one of the complementation genes that affect str-2 asymmetry, has been found to encode the C. elegans homologue of ASK1 and to function downstream of UNC-43/CaMKII (Sagasti et al, 2001). We thus examined the involvement of VDCC and CaMKII in Ca2+ signalling in mammalian cells. Nifedipine, the antagonist of VDCC, and KN-93, the inhibitor of CaMKs, both effectively inhibited the ASK1 activation induced by membrane depolarization in PC12 cells (Fig 2A,B), while the VDCC agonist FPL64176 activated ASK1 in a KN-93sensitive manner (Fig 2A). Ca2+ influx also induced activation of p38, which was inhibited by KN-93 in PC12 cells (Fig 2B) and primary neurons (Fig 2C). When the expression of endogenous CaMKII was reduced in HeLa cells transfected with a small interfering RNA (siRNA), MTX-induced activations of ASK1 and p38 were partially impaired (Fig 2D). Although we cannot exclude the possibility that other KN-93sensitive CaMKs such as CaMKI and CaMKIV are also involved in Ca2+-induced activation of ASK1 (Hook & Means, 2001), these results together with genetic evidence in C. elegans strongly suggest that CaMKII activated by Ca2+ influx through VDCC in turn activates the ASK1–p38 pathway in mammalian cells. Since H2O2 (an activator of ASK1 through inactivation of the ASK1 inhibitor thioredoxin; Saitoh et al, 1998)-induced activation of ASK1 was insensitive to KN-93 (Fig 2E), CaMK activity appeared to be required for Ca2+-induced but not oxidativestress-induced activation of ASK1.

Figure 2
Ca2+-induced activation of the ASK1–p38 pathway through CaMKII. (A) Activation of ASK1 by Ca2+ signals through VDCC and CaMKs. PC12 cells were pretreated with or without 20 μM each of either the VDCC antagonist nifedipine ...

The kinase activity of CaMKII is tightly regulated by the autoinhibitory domain in its C-terminal region (Soderling et al, 2000; Lisman et al, 2002), and deletion of this domain generates a constitutively activated enzyme that is independent of Ca2+/calmodulin regulation (Zou & Cline, 1996; Zhu et al, 2002). We transfected Myc-tagged C-terminally truncated active CaMKII (tCaMKII) together with HA-tagged p38 into MEFs derived from ASK1+/+ or ASK1−/− mice. tCaMKII activated p38 in ASK1+/+ MEFs but not in ASK1−/− MEFs (Fig 2F), suggesting that ASK1 was indispensable for CaMKII-induced p38 activation. Taken together, these results suggest that CaMKII is an activator of the ASK1–p38 pathway in mammalian Ca2+ signalling.

We next examined the mechanism by which CaMKII activates ASK1. When GST-tagged kinase negative ASKI (GST-ASK1KN) was incubated with purified CaMKII in the presence of Ca2+/calmodulin, [γ-32P]ATP was incorporated into GST-ASK1 (Fig 3A), indicating that CaMKII directly phosphorylates ASK1. Since Thr 845 is the activating phosphorylation site that is essential for ASK1 activation (Tobiume et al, 2002), we examined the phosphorylation state of Thr 845 by anti-phospho-ASK1 antibody. Phosphorylation of Thr 845 of GST-ASK1KN was not increased by the incubation with purified CaMKII (Fig 3B), indicating that CaMKII can directly phosphorylate ASK1 but only at sites other than Thr 845. To investigate further the activation mechanism of ASK1 by CaMKII in vivo, we transfected Flag-tagged CaMKII (Flag-CaMKII) together with HA-tagged kinase negative ASK1 (HA-ASK1KN) into HEK-293 cells and immunoprecipitated CaMKII with anti-Flag antibody. The immune complex was incubated in vitro in the presence or absence of Ca2+/calmodulin together with ATP (Fig 3C). HA-ASK1KN was found to be co-immunoprecipitated with Flag-CaMKII (Fig 3C, second panel, lanes 5 and 6). Co-purified ASK1KN was phosphorylated at Thr 845 only in the presence of Ca2+/calmodulin (Fig 3C, top panel), suggesting that the immune complex from cell lysate includes co-purified endogenous kinase(s), which directly phosphorylates Thr 845 of ASK1 on the activation of CaMKII. In contrast, ASK1KN co-purified with Flag-tagged kinase negative CaMKII (Flag-CaMKIIKN) was no longer phosphorylated at Thr 845 even in the presence of Ca2+/calmodulin (Fig 3D, lanes 5 and 6). These results suggest that kinase activity of CaMKII in the immune complex is indirectly required for the activating phosphorylation of ASK1 at Thr 845 and that the co-purified endogenous kinase(s) is further required to phosphorylate ASK1 at Thr 845. One candidate kinase that directly phosphorylates Thr 845 is ASK1 per se, since Thr 845 of ASK1 can be auto-phosphorylated within a homo-oligomer of ASK1 in a trans-molecular manner (Tobiume et al, 2002). If this is the case, the candidate kinase supposed to phosphorylate Thr 845 of ASK1KN in Fig 3C,D appears to be endogenous ASK1. Alternatively, another unidentified kinase may act as an intermediary kinase for Thr 845 of ASK1 between CaMKII and ASK1 in Ca2+ signalling. In either case, phosphorylation of ASK1 by CaMKII at sites other than Thr 845 may be an important step to initiate the phosphorylation of Thr 845 by other kinases in vivo.

Figure 3
CaMKII activates ASK1. (A) CaMKII phosphorylates ASK1. GST-tagged kinase negative ASK1 (GST-ASK1KN) and/or CaMKII purified from rat forebrain (purchased from Upstate Bio) were incubated in a kinase buffer in the presence of 2 mM CaCl2/2.4 μM calmodulin ...

In this study, we show that the CaMKII–ASK1–p38 MAP kinase cascade constitutes a novel Ca2+ signal mediator. On the basis of our finding that ASK1 was required for Ca2+-induced activation of p38 in synaptosomes, we propose a synaptic role for the CaMKII–ASK1–p38 pathway in Ca2+ signalling. This signalling module may have important roles in the recently identified function of p38 for synaptic plasticity. Thus, the identification of target molecules of p38 in Ca2+ signalling should shed new light on neuronal regulation.


ASK1−/− mice ASK1−/− mice were generated as described (Tobiume et al, 2001). All ASK1−/− and ASK1+/+ mice used for the preparation of MEFs and synaptosomes were generated as F2 littermates of a strain backcrossed in a C57BL/6J background for six generations.

Cell culture MEFs were prepared from E12.5 mice and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and 100 U ml−1 penicillin G. For primary neurons, telencephalons from E14.5 mice were triturated in Hank's balanced salt solution by mild and frequent pipetting. Dissociated cells were cultured for 3 days in N2-supplemented DMEM-F-12 medium on six-well culture plates precoated with poly-L-ornithine and fibronectin.

Immunoblotting Cell lysates were resolved on SDS–PAGE and electroblotted onto PVDF membranes. After blocking with 5% skim milk in TBS-T (50 mM Tris–HCl (pH 8.0), 150 mM NaCl and 0.05% Tween 20), the membranes were probed with antibodies. The antibody–antigen complexes were detected using the ECL system (Amersham). The antibodies to phospho-ASK1 (Tobiume et al, 2002), ASK1 (Saitoh et al, 1998), phospho-p38 (Cell Signaling), p38 (Santa Cruz), HA (3F10, Roche), Flag (M2, Sigma), myc (9E10, Calbiochem), GST (Amersham), phospho-CaMKII (Upstate Bio) and synaptophysin (Progen) were used.

RNA interference Double-stranded siRNA targeting the 5′-AAGGGAGCCAUCCUCACCACC-3′ sequence of human CaMKIIβ mRNA or a control scrambled 5′-AAGCGCGCUUUGUAGGAUUCG-3′ sequence (Dharmacon) were transfected into HeLa cells using Oligofectamine (Invitrogen) according to the manufacturer's instructions. Expression of CaMKII was detected using the monoclonal antibodies to CaMKIIβ (Zymed; CBβ-1) and to all four isoforms and their respective splicing variants of human CaMKII (BD Pharmingen).

Purification of synaptosomes Cerebral tissue of 8-week-old mice was homogenized in 0.32 M sucrose containing 1 mM p-APMSF, 1 mM NaHCO3, 1 mM MgCl2 and 0.5 mM CaCl2. The homogenate was centrifuged for 10 min at 1,400g to yield a pellet (P1) and a supernatant (S1). S1 was further centrifuged for 10 min at 13,800g, yielding a crude synaptosomal pellet (P2) and a supernatant (S2). P2 was resuspended in 0.32 M sucrose containing 1 mM NaHCO3 and layered on top of a discontinuous sucrose gradient (0.8 and 1.2 M). After centrifugation for 2 h at 82,500g, the gradient was collected in three fractions: the fraction on top (P2A), the synaptosomal fraction between 0.8 and 1.2 M sucrose (P2B), and the pellet (P2C). Sucrose (0.32 M) was added to P2B followed by centrifugation for 10 min at 13,800g. The pellet was resuspended in phosphate-buffered saline (PBS) including 2 mM CaCl2 and subjected to treatment with KCl.

In vitro kinase assay GST-tagged kinase negative ASK1 (GST-ASK1KN) produced in bacteria and/or CaMKII purified from rat forebrain (Upstate Bio) were incubated for 20 min at 30°C in a kinase buffer containing 20 mM Tris–HCl (pH 7.5), 20 mM MgCl2 and 100 μM ATP in the presence of 2 mM CaCl2/2.4 μM calmodulin and 0.3 μCi of [γ-32P]ATP. Samples were subjected to SDS–PAGE followed by immunoblotting and autoradiography. For detection of the Thr 845 phosphorylation of GST-ASK1KN, GST-ASK1 bound to glutathione sepharose beads (Amersham) was treated with λPPase (NEB) according to the manufacturer's instructions to reduce the basal phosphorylation of Thr 845. After washing with PBS including PPase inhibitor cocktails (Sigma), the beads were incubated with purified CaMKII in the same kinase buffer in the presence of 2 mM CaCl2/2.4 μM calmodulin. Samples were subjected to SDS–PAGE followed by immunoblotting.

Immune complex kinase assay The immune complex was incubated in a kinase buffer containing 20 mM Tris–HCl (pH 7.5), 20 mM MgCl2 and 100 μM ATP in the presence of either 2 mM EGTA or 2 mM CaCl2/2.4 μM calmodulin for 20 min at 30°C and then subjected to SDS–PAGE followed by immunoblotting.

Supplementary information is available at EMBO reports online (http://www.nature.com/embor/journal/vaop/ncurrent/extref/7400072s1.pdf).

Supplementary Material

Supplementary Information


We are grateful to A. Sagasti and C.I. Bargmann for valuable discussions. We also thank all the members of the Cell Signaling Laboratory for their critical comments. This work was supported in part by Grants-in-Aid for scientific research from the Ministry of Education, Science and Culture in Japan and by CREST, Japan Science and Technology Corporation.


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