Control of cyclin-dependent kinase 5 (Cdk5) activity byglutamatergic regulation of p35 stability

Fan-Yan Wei,* Kazuhito Tomizawa,� Toshio Ohshima,� Akiko Asada,* Taro Saito,*Chan Nguyen,§ James A. Bibb,§ Koichi Ishiguro,¶ Ashok B. Kulkarni,** Harish C. Pant,��Katsuhiko Mikoshiba,� Hideki Matsui� and Shin-ichi Hisanaga*

*Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Minami-osawa, Hachiohji, Tokyo,

Japan

�Department of Physiology, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan

�Laboratory for Developmental Neurobiology, Brain Science Institute, Wako, Saitama, Japan

§Department of Psychiatry, The University of Texas South-western Medical Center, Dallas, Texas, USA

¶Mitsubishi Kagaku Institute of Life Sciences, Machida, Tokyo, Japan

**Functional Genomics Unit, National Institute of Dental & Craniofacial Research and ��Laboratory of Neurochemistry,

National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland, USA

Abstract

Although the roles of cyclin-dependent kinase 5 (Cdk5) in

neurodevelopment and neurodegeneration have been stud-

ied extensively, regulation of Cdk5 activity has remained

largely unexplored. We report here that glutamate, acting via

NMDA or kainate receptors, can induce a transient Ca2+/

calmodulin-dependent activation of Cdk5 that results in

enhanced autophosphorylation and proteasome-dependent

degradation of a Cdk5 activator p35, and thus ultimately

down-regulation of Cdk5 activity. The relevance of this

regulation to synaptic plasticity was examined in hippocam-

pal slices using theta burst stimulation. p35–/– mice exhibited

a lower threshold for induction of long-term potentiation.

Thus excitatory glutamatergic neurotransmission regulates

Cdk5 activity through p35 degradation, and this pathway may

contribute to plasticity.

Keywords: calmodulin, cyclin-dependent kinase 5, protea-

some, glutamate, N-methyl-D-aspartate, long-term potentia-

tion.

J. Neurochem. (2005) 93, 502–512.

Cyclin-dependent kinase 5 (Cdk5) is a cdc2-like kinase thatrequires the neuronal-specific activator p35 or its homologuep39 for activity (Tang and Wang 1996; Dhavan and Tsai2001; Hisanaga and Saito 2003). The essential role of Cdk5in neuronal migration during brain development has beendocumented in Cdk5- and p35-deficient mice (Ohshima et al.1996, 1999; Chae et al. 1997). Furthermore, deregulation ofCdk5 by proteolytic conversion of p35 to p25 is suggested tobe involved in neuronal cell death (Kusakawa et al. 2000;Lee et al. 2000; Nath et al. 2000) and some neurodegener-ative diseases (Patrick et al. 1999; Nguyen et al. 2001; Buet al. 2002).

Emerging evidence suggests a role for Cdk5 in the synapticactivity of mature neurons. In the presynaptic compartment,Cdk5 may regulate neurotransmitter release through phos-phorylation of synapsin I (Matsubara et al. 1996) and the

a-subunit of the P/Q type voltage-dependent Ca2+ channel(Tomizawa et al. 2002). Cdk5 also regulates synaptic vesicleendocytosis through phosphorylation of dynamin I and

Received November 21, 2004; revised manuscript received December21, 2004; accepted December 22, 2004.Address correspondence and reprint requests to Shin-ichi Hisanaga,

Department of Biological Sciences, Graduate School of Science, TokyoMetropolitan University, Minami-osawa, Hachiohji, Tokyo 192–0397,Japan. E-mail: hisanaga-shinichi@c.metro-u.ac.jpAbbreviations used: APDC, 4-aminopyrolidine-2,4-dicarbonate;

CaMKII, Ca2+-calmodulin-dependent protein kinase II; Cdk5, cyclin-dependent kinase 5; CHX, cycloheminide; CKI, casein kinase I; DHPG,dihydroxyphenylglycine; EPSP, excitatory postsynaptic potential;L-AP4, L-(+)-2-amino-4-phosphonobutyric acid; LTP, long-term poten-tiation; PKC, protein kinase C; PP2B, protein phosphatase 2B; TBS,theta burst stimulation; ZLLH, carbobenzoxy-L-leucyl-L-leucinal.

Journal of Neurochemistry, 2005, 93, 502–512 doi:10.1111/j.1471-4159.2005.03058.x

502 � 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 502–512

amphiphysin I (Floyd et al. 2001; Nguyen and Bibb 2003; Tanet al. 2003; Tomizawa et al. 2003). In the postsynapticcompartment, Cdk5 has been shown to phosphorylate theNR2A subunit of the NMDA receptor (Li et al. 2001; Wanget al. 2003), PSD95 (Morabito et al. 2004), and proteinphosphatase inhibitor-1 (Bibb et al. 2001), aswell as its striatalhomologue, DARPP-32 (Bibb et al. 1999). Furthermore,NMDA stimulation increases the association of p35 withCa2+-calmodulin-dependent protein kinase IIa (CaMKIIa), amajor component of the postsynaptic density (Dhavan et al.2002). Despite this impressive list of downstream effectors, theidentities of upstream regulators of Cdk5 have remainedunknown. We report here that ionotropic glutamate receptorsregulate Cdk5 activity through the modulation of p35 stability.Evidence that this novel signal transduction pathway maycontribute to synaptic plasticity is also presented.

Materials and methods

Materials

Glutamate, NMDA, BAPTA-AM, calphostin C, KN-62, A23187,

and W7 were purchased from Sigma (St. Louis, MO, USA). Kainate

was obtained from Tocris (Avonmouth Bristol, UK). Lactacystin,

roscovitine, Suc-LLL-MCA and anti-Cdk5 antibody DC-17 were

from Calbiochem (San Diego, CA, USA). Carbobenzoxy-L-leucyl-

L-leucinal (ZLLH) was obtained from the Peptide Institute (Osaka,

Japan). Antibodies against p35 (C-19 and N-20), Cdk5 (C-8), and

inhibitor-1 (N-20) were obtained from Santa Cruz Biotechnology

(Santa Cruz, CA, USA). Phospho-Ser67 inhibitor-1 antibody was

provided by Paul Greengard (The Rockefeller University, NY,

USA). Cyclosporine A was provided by Kazuo Nagai (Tokyo

Institute of Technology, Yokohama, Japan). Calmodulin was

purified from porcine brains by the method of Yazawa et al. (1980).

Culture and metabolic labeling of primary cortical neurons

Neurons from embryonic day 15–16 mouse (ICR, SLC, Tokyo,

Japan) brain cortices were plated at a density of 2 · 105 cells/cm2 in

polyethyleneimine-coated dishes in Dulbecco’s modified Eagle’s

medium and Ham’s F-12 (1 : 1) supplemented with 5% fetal bovine

serum and 5% horse serum (Saito et al. 1998). Cytosine arabinoside(20 lM) was added to the culture medium 3 days after plating to

inhibit the proliferation of glial cells. All experiments were

performed on day 7 of culture. When treating neurons with various

inhibitors, the inhibitors were added to the culture medium 30 min

before the neurons were stimulated with glutamate, kainate, or

NMDA. For metabolic phosphorylation of p35, neurons were

cultured in the presence of [32P]orthophosphate in phosphate-free

Dulbecco’s modified Eagle’s medium for 3 h (Wada et al. 1998).

Preparation of cell extracts and immunoprecipitation

Neurons were lysed by freezing and thawing in lysis buffer (10 mM

MOPS, pH 7.2, 1 mM MgCl2, 1 mM EGTA, 0.1 mM EDTA, 0.3 M

NaCl, 0.5% Nonidet P-40 (NP-40), 1 lg/mL leupeptin, 1 mM

dithiothreitol), and the supernatant (cell extract) was collected after

centrifugation at 10 000 g for 30 min. Anti-Cdk5 antibody C-8

(2 lL) was added to 100 lL of extract (corresponding to 50 lg

protein). After a 1-h incubation at 4�C, 20 lL protein A–Sepharose

CL-4B beads (50% slurry in lysis buffer; Amersham Pharmacia

Biotech., Tokyo, Japan) was added, and the sample was further

incubated for 1 h at 4�C. The beads were washed four times with

10 mM MOPS, pH 7.2, 1 mM EGTA, 0.1 mM EDTA, and 1 mM

MgCl2, then used for the histone H1 kinase assay (Kusakawa et al.2000).

For the detection of 32P-labeled p35, neurons were suspended in

RIPA buffer [10 mM Tris-HCl, pH 7.5, 1 mM EGTA, 0.15 M NaCl,

1% NP-40, 0.1% sodium dodecylsulfate, 10 mM b-glycerophos-phate, 5 mM NaF, 1 mM p-nitrophosphate, 0.2 mM Pefabloc SC

(Merck, Darmstadt, Germany), 1 lg/mL leupeptin, 1 mM dithio-

threitol] and lysed by freezing and thawing. The cell extract

(supernatant) was collected after centrifugation at 10 000 g for

30 min. p35 was isolated from the extract by immunoprecipitation

with the anti-p35 antibody, C-19, as described previously

(Kusakawa et al. 2000). 32P-labeled p35 prepared by immunopre-

cipitation was separated by sodium dodecyl sulfate–gel electrophor-

esis on a 12.5% polyacrylamide gel, and the 32P incorporated into

the p35 was detected by a BAS 2000 Image Analyzer (Fuji film,

Tokyo, Japan).

To detect p35 and Cdk5 in whole-cell extracts, neurons were

collected by centrifugation at 300 g for 3 min, immediately frozen

in liquid nitrogen, and lysed in 100 lL sodium dodecyl sulfate–gel

electrophoresis sample buffer (31.25 mM Tris-HCl, pH 6.8, 5%

glycerol, 1% sodium dodecyl sulfate, 2.5% b-mercaptoethanol) by

sonication and boiling for 5 min.

Detection of histone H1 kinase activity in Cdk5–p35

and the phosphorylation of p35

The kinase reaction of immunoprecipitated Cdk5–p35 was initiated

by adding 0.1 mM [c-32P]ATP to a reaction mixture containing

10 mM MOPS, pH 7.2, 1 mM MgCl2, and 0.3 mg/mL histone H1.

After 30-min incubation at 35�C, the reaction was stopped by the

addition of sodium dodecyl sulfate–gel electrophoresis sample

buffer, and the mixture was immediately boiled for 5 min. Samples

were separated by sodium dodecyl sulfate–gel electrophoresis on a

15% polyacrylamide gel, and the radioactivity associated with

histone H1 was quantified using a BAS2000 Image Analyzer.32P-Labeled p35 prepared by immunoprecipitation was separated

by sodium dodecyl sulfate–gel electrophoresis on a 12.5% poly-

acrylamide gel, and the 32P incorporated into the p35 was detected

by a BAS2000 Image Analyzer.

Preparation of mouse brain extract and incubation

with Ca2+-calmodulin

Brains of 7–8-week-old mice (ICR) were homogenized in 10 vol. of

20 mM MOPS, pH 7.4, 1 mM MgCl2, 0.1 M NaCl, 0.1 mM EDTA,

0.1 mM EGTA, 0.5% NP-40, 1 mM dithiothreitol, 0.1 mM Pefabloc,

and 10 lg/mL leupeptin at 4�C with a Teflon-pestle glass

homogenizer. The brain extract was obtained by centrifugation at

10 000 g for 15 min. The brain extract was incubated with 0.5 mM

CaCl2 and 50 lg/mL calmodulin at 35�C for 30 min. In some case,

0.1 mM W7 was added to inhibit calmodulin activity. Cdk5–p35 was

immunoprecipitated with anti-Cdk5 antibody (C8) or anti-p35

antibody (C19). The kinase activity was measured as described

above. p35, p25 and Cdk5 were detected by immunoblotting as

described below.

Glutamatergic regulation of Cdk5–p35 activity 503

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 502–512

Fluorogenic peptide substrate assay for proteasome activity

For the measurement of the proteasome activity, neurons were lysed

in ice-cold 20 mM HEPES, pH 7.2, 0.1 mM EDTA, 1 mM ATP, 20%

glycerol, and 0.04% NP-40. The cell extract was collected after

centrifugation at 10 000 g for 15 min. The extract was incubated at

37�C with Suc-LLL-MCA (100 lM) in reaction buffer (50 mM Tris-

HCl, pH 8.0, 5 mM EGTA) for 30 min. The reaction was stopped by

adding 1% sodium dodecyl sulfate. Cleavage of peptides was

measured by excitation at 380 nm and emission at 460 nm.

Hippocampal slice pharmacology

Mouse hippocampal slices were acutely prepared essentially as

previously described (Caporaso et al. 2000). Microdissected hippo-

campal slices (400 lm thick) were treated with 100 lM NMDA for

the indicated times. All slices were incubated in Kreb’s buffer for

equal amounts of time (60 min). Homogenates were prepared by

sonication in boiling 1% sodium dodecyl sulfate with 50 mM NaF.

Equal amounts of protein as determined by BCA assay were loaded

in each lane.

For the histone H1 kinase assay, slices were lysed by sonication

in lysis buffer, and the supernatant was collected after centrifugation

at 10 000 g for 30 min. Immunoprecipitation of Cdk5/p35 was

carried out using anti-p35 antibody (N-20) according to the method

described above.

Injection of kainate into mice and preparation of the brain

extract

Seven-week-old mice (ICR) were injected subcutaneously with

50 mg kainate per kg body weight, or the same volume of saline.

Mice were killed 30 min after injection, and the cerebral cortices

were dissected and immediately frozen in liquid nitrogen. The

cerebral cortices were homogenized with a Teflon homogenizer in

lysis buffer. The brain extract was collected as the supernatant after

centrifugation at 10 000 g for 30 min.

Long-term potentiation induction and electrophysiological

recording in hippocampus slices

p35 knockout mice were generated, maintained in 129/

Sv · C57BL/6J backgrounds, and genotyped as described previ-

ously (Ohshima et al. 2001). All mice were handled in accordance

with institutional guidelines and housed in as pathogen-free

environment on a 12 : 12 h light : dark cycle.

Electric stimulation was carried out in 6–8-week-old mice as

described previously (Lu et al. 1999; Tomizawa et al. 2003).

Briefly, a glass micropipette filled with artificial CSF (aCSF, 1–

5 MW resistance) was placed in the stratum radiatum of the CA1

region to record the field excitatory postsynaptic potentials

(fEPSPs), and a bipolar stimulating electrode was placed along the

Schaffer collateral fibers. The intensity of the stimulation was

adjusted to produce an EPSP with a slope between 35% and 50% of

the maximum. The test stimulation was delivered once per minute

(0.017 Hz). Long-term potentiation (LTP) was induced by theta-

burst stimulation (TBS), a strong stimulation paradigm. TBS

protocols consisted of four pulses at 100 Hz repeated for 15 times

with an interval of 200 ms between each burst. Slices were lysed by

sample buffer 15 min after stimulation. Data are shown as mean

(± SEM) percentage of the intensity of p35–/– signal in unstimulated

control slices.

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and

immunoblotting

Protein extracts (10 lg) were separated by 12.5% polyacrylamide

gel sodium dodecyl sulfate–gel electrophoresis and transferred to

polyvinylidene difluoride membranes (Millipore, Bedford, MA,

USA). After probing with primary antibodies and then anti-rabbit or

anti-mouse IgG secondary antibodies (DAKO, Glostrup Denmark),

the reaction was detected with either the BCIP/NBT phosphatase

substrate system (KLP, Gaithersburg, MD, USA) or the enhanced

chemiluminescence system (ECL; Amersham Pharmacia Biotech).

The amount of p35 was quantified using NIH Image Analyzer after

incorporation of the blot images into Adobe Photoshop (Adobe

Systems Inc., San Jose, CA, USA) using a scanner.

All experiments in this study were performed multiple times with

similar results, and representative results are shown in the figures.

Data were analyzed using the Student t-test to compare the two

conditions and p < 0.05 was considered to be significant.

Results

Stimulation of NMDA or kainate receptors reduces p35

levels in primary cortical neurons

Long-term stimulation with excitotoxic glutamate has beenshown to induce cleavage of p35 to p25 by calpain duringneuronal cell death (Fig. 1a) (Lee et al. 2000). In character-izing this effect, it was observed that exposure of primarycortical cultured neurons to glutamate for shorter periods (upto 1 h) decreased levels of p35 to �25% that of untreatedneurons, but without the generation of p25, as assessed byimmunoblot analysis (Fig. 1b). Cortical neurons treated withglutamate for 1 h did not show any signs of cell death andwere able to survive for, at least, several days after theremoval of glutamate, indicating that this decrease in p35 is aprocess occurring in living neurons.

To determine which glutamate receptor classes wereinvolved in this novel effect, neuronswere treated with variousspecific agonists. Two ionotropic glutamate receptor agonists,NMDAand kainate, induced a dose-dependent decrease in p35levels in a time course similar to that of glutamate (Figs 1c andd). This effect was similarly observed by immunfluorescentanalysis of neuronal cultures (data not shown). Furthermore,an NMDA receptor antagonist (MK801) and a kainate/AMPAreceptor antagonist (CNQX) suppressed the effects of kainateand NMDA on p35 levels (Fig. 1e). In contrast, group I, II andIII metabotropic glutamate receptors agonists (DHPG, dihy-droxyphenylglycine; APDC, 4-aminopyrolidine-2,4-dicar-bonate; L-AP4, L-(x)-2-amino-4-phosphonobutyric acid,respectively) did not decrease the levels of p35, but some ofthem rather increased slightly (Fig. 1f). A small amount of p25was detected in some cultures, but the short-term treatmentwith NMDA or kainite resulted in no further increase. Theseresults indicate that the glutamate-induced reduction in p35levels occurred via specific activation of ionotropic NMDAand kainate receptors.

504 F.-Y. Wei et al.

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 502–512

NMDA or kainate-induced reduction of p35 levels occurs

via proteasomal degradation

Either inhibition of synthesis or enhancement of degradationcould result in a rapid reduction of p35 levels. The formerpossibility was evaluated by comparing the rate of p35reduction in neurons treated with NMDA or kainate or aninhibitor of protein synthesis, cycloheximide (CHX)(Fig. 2a). Since the rate of p35 reduction induced by NMDAor kainate was significantly faster than that of neurons treatedwith CHX, the effect of NMDA and kainate must bemediated, at least in part, through an enhancement of p35degradation.

p35 is degraded by two distinct proteolytic mechanisms.Cleavage of p35 to p25 by calpain occurs during neuronalcell death (Kusakawa et al. 2000; Lee et al. 2000; Nath et al.

2000; Kerokoski et al. 2002), whereas the turnover of p35 inhealthy neurons is dependent on the ubiquitin–proteasomesystem (Patrick et al. 1998; Saito et al. 1998). To examinewhether the decrease in p35 resulted from proteasome-dependent degradation, neurons were treated with NMDA orkainate in the presence of a proteasome inhibitor, lactacystin,or a calpain inhibitor, ZLLH (Fig. 2b). Lactacystin com-pletely inhibited the decrease in p35, indicating the reductionin p35 was due to proteasomal activity.

To ensure that the enhanced p35 degradation was specificand not due to an overall increase in proteasomal activityafter NMDA or kainate treatment, proteasomal activity wasmeasured using a fluorogenic peptide (Fig. 2c). The protea-some inhibitor MG132 was included as a control (MG inFig. 2c). Overall proteasomal activity was not enhanced afterstimulation with NMDA or kainate, indicating that theseagents cause a reduction in p35 by a process of degradationsignal tagging, such as ubiquitination.

Cyclin-dependent kinase 5 phosphorylates p35 in

response to NMDA or kainate receptor activation

NMDA and kainate receptors are Ca2+-permeable cationchannels (Ozawa et al. 1998; Craig and Boudin 2001). TheCa2+-activated enzymes, protein kinase C (PKC), Ca2+-

(b)

(c)

(d)

NMDA

0 1 5 15 30 60 (min)

0 0.01 0.1 0.5 1 (mM)

0 1 5 15 30 60 (min)

0 0.01 0.1 0.5 1 (mM)

0 1 5 15 30 60 (min)

0 0.01 0.1 0.5 1 (mM)

MK80

1

CNQX

+ + +

_

_

_

DHPG

APDC

L-AP40 1 10 100

(f)

0 1 6 12 (h)

(a)

(e)

p3

5le

ve

l(%

)

100

75

50

25

0

Tim e (m in)0 15 30 60

Glutamate Kainate

Glutamate

Antagonist

Agonist

NMDA

Kainate

(µM)

Fig. 1 Ionotropic glutamate receptors regulate p35 levels in cultured

mouse cortical neurons. Cultured cortical neurons were treated with

1 mM glutamate for 0, 1, 6, 12 h (a), with 0.1 mM glutamate for 0, 1, 5,

15, 30, 60 min (upper panel of b), or at indicated concentrations for 1 h

(lower panel of b). p35 and cyclin-dependent kinase 5 (Cdk5) were

detected by immunoblotting whole-cell lysates. Quantification of the

amount of p35 in the upper panel is shown in the middle panel of (b).

Cultured neurons were similarly treated with 0.1 mM NMDA (c) or

0.1 mM kainate (d). Black arrowheads and white arrows indicate p35

and Cdk5, respectively. White arrowheads indicate p25 or the position

of p25. (e) Cortical neurons were treated with 0.1 mM NMDA or kainate

in the presence of 2 lM MK801 or 20 lM CNQX for 1 h. (f) p35 levels in

cortical neurons treated with 0–100 lM DHPG, APDC, or L-AP4 for

1 h.

0 30 60 (min)

__

+ +

30 (min)

(a) (b)

(c)

0

50

100

600N K N KMG_

+_CHX

NMDA

Kainate

p35

leve

ls (

%)

100

75

50

25

00 30 60

Time (min)

Inhibitor

Agonist

NMDA

Kainate

LactaZLLH

Z-L

LL-M

CA

Cle

avag

e A

ct. (

%)

Fig. 2 NMDA and kainate stimulate the proteasome-dependent deg-

radation of p35. (a) Cultured neurons were treated with 10 lg/mL

cycloheximide (CHX, squares in lower graph) or 0.1 mM NMDA (black

circles) or kainite (white circles). p35 levels were detected by immu-

noblotting with anti-p35 antibody (upper panels) and quantitated by

densitometric analysis (lower graph). This is one of two independent

experiments with the similar results. (b) Neurons were treated with

0.1 mM NMDA or 0.1 mM kainate for 1 h in the presence of 10 lM

ZLLH, 20 lM lactacystin (Lacta), or no inhibitors (–). The lysates were

probed by immunoblotting with anti-p35 antibody. (c) Proteasome

activity in neurons treated with 0.1 mM NMDA (N) or 0.1 mM kainate

(K) for 30 min and 60 min. The neuron extracts were incubated with

the specific proteasome substrate (Z-LLL-MCA), together with 5 lM

MG132 (MG) in one case, for 30 min. Cleavage activity is expressed

as a percentage of the activity of control neurons.

Glutamatergic regulation of Cdk5–p35 activity 505

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 502–512

calmodulin-dependent protein kinase II (CaMKII), andprotein phosphatase 2B (PP2B), are known to mediatesignals originating from NMDA or kainate receptor activa-tion. To examine the role of these enzymes in mediating p35degradation, a CaMKII inhibitor (KN-62), a PKC inhibitor(calphostin C), and a PP2B inhibitor (cyclosporine A) wereemployed (Fig. 3). However, when added to the culturemedium, none of these inhibitors suppressed the NMDA orkainate-induced degradation of p35, suggesting the existenceof a novel signaling from NMDA and kainite receptors.

Phosphorylation of p35 is required for degradation ofp35 via the proteasome pathway (Patrick et al. 1998; Saitoet al. 1998), and it has been shown that p35 can bephosphorylated by casein kinase I (CKI) and Cdk5 in vitro(Tsai et al. 1994; Patrick et al. 1999; Liu et al. 2001; Saitoet al. 2003). The possible involvement of these kinases wasexamined by using inhibitors specific for each (Fig. 3).Neurons were treated with NMDA or kainate in thepresence of either a CKI inhibitor (CKI-7) or a Cdk5inhibitor (roscovitine). Roscovitine, but not CKI-7, sup-pressed the degradation of p35. Furthermore, roscovitinealone did not change the level of p35 during this incubationperiod (data not shown).

To further evaluate the role that Cdk5 plays in thedegradation of p35, kinase activity was determined usingCdk5 immunoprecipitated from cell culture lysates beforeand after stimulation with NMDA or kainate (Figs 4a and b).The kinase activity of Cdk5/p35, as assessed by phosphory-lation of histone H1, increased transiently 1–2 min after theaddition of NMDA or kainate, and then decreased graduallyover the next hour. To examine whether this curious biphasicregulation of Cdk5 activity was reflected in levels of p35phosphorylation, cortical neurons were cultured in the

presence of [32P]orthophosphate. p35 was isolated fromneuronal extracts before and after stimulation with NMDA orkainate, and its phosphorylation was detected by autoradi-ography. Indeed, phosphorylation of p35 increased at 1 minafter the addition of NMDA or kainate (Figs 4c and d) andthis effect was obliterated by roscovitine (Fig. 4c). Thus,transient activation of Cdk5 by NMDA or kainate results inautophosphorylation and then degradation of p35.

The activation of cyclin-dependent kinase 5 and

subsequent degradation of p35 is Ca2+-calmodulin

dependent

There are three known mechanisms by which the kinaseactivity of Cdk5 can be stimulated: an increase in the level ofp35, the conversion of p35 to p25, and the phosphorylationof Cdk5 (Patrick et al. 1999; Sharma et al. 1999; Zukerberg

++

___

C

0

50

100

KN-62 CalC CsACtrl

p35

leve

ls (

%)

Inhibitor CKI-7Ros

+

__

CsACalCKN-62

+++

CKI-7 Ros

*

AgonistNMDA

Kainate

Fig. 3 The degradation of p35 is mediated by cyclin-dependent kinase

5 (Cdk5) but not Ca2+-calmodulin-dependent protein kinase II (CaM-

KII), protein kinase C (PKC), protein phosphatase 2B (PP2B), or

casein kinase I (CKI). Cultured neurons were treated with 0.1 mM

NMDA or 0.1 mM kainate in the presence of 50 lM KN-62, 1 lM cal-

phostin C (CalC), 2 lM cyclosporine A (CsA), 20 lM casein kinase

inhibitor (CKI-7) or 50 lM roscovitine for 1 h. p35 was detected by

immunoblotting with anti-p35 antibody (upper panel) and quantitated

by densitometric analysis (black bars for NMDA and white bars for

kainate in lower panel). *p < 0.001.

(a) (b)

(c) (d)

_

Ros

Cntl

NMDA

Kainat

e

0 1 5 15 30 60 (min) 0 1 5 15 30 60 (min)

0 15 30 60 (min)

Cdk

5 A

ctiv

ity (

%) 150

100

50

0

150

100

50

0

Cdk

5 A

ctiv

ity (

%)

0 15 30 60 (min)

NMDA Kainate0 1 5 15 30 60 (min) 0 1 5 15 30 60 (min)

H1 H1

Pho

spho

ryla

tion

of p

35 (

%)

150

100

50

0Ctrl NMDA Kainate

**

C

dk5

Act

ivity

(%

)

125

100

750 5 10 15

(min)

Fig. 4 NMDA or kainate transiently activates cyclin-dependent kinase

5 (Cdk5) kinase activity and stimulates phosphorylation of p35. (a) and

(b) cyclin-dependent kinase 5 (Cdk5)–p35 was isolated by immuno-

precipitation from cultured neurons treated with 0.1 mM NMDA (a) or

0.1 mM kainate (b) for 0, 1, 5, 15, 30, or 60 min. The kinase activity

was measured using histone H1 as the substrate (H1 in upper panels

of a and b). Quantification of kinase activity is shown in the middle

panels of (a) and (b) (n ¼ 3). An inset in (a) represents the activation

of Cdk5 at 1 and 2 min after NMDA application. Black and white

arrowheads in lower panel indicate p35 and the position of p25,

respectively. (c) and (d) Cortical neurons cultured in the presence of

[32P]orthophosphate were stimulated by 0.1 mM NMDA or 0.1 mM

kainate for 1 min in the presence or absence of 50 lM roscovitine (Ros

in c). p35 was immunoprecipitated and its phosphorylation status was

detected by autoradiography (c) and quantified (d). *p < 0.05.

506 F.-Y. Wei et al.

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 502–512

et al. 2000; Hashiguchi et al. 2002). We examined these aspossible causes of the immediate activation of Cdk5.However, there was neither an increase in the amount ofp35 nor in its conversion to p25 in the first few minutes afterstimulation with NMDA or kainate (Figs 4a and b, lowerpanels). In addition, we did not detect phosphorylation ofCdk5 when the Cdk5–p35 complex was prepared fromNMDA- or kainate-treated cortical neurons cultured in thepresence of 32P (data not shown).

In an effort to identify the novel signaling mechanismresponsible for the observed up-regulation, focus was givento the ionotropic glutamate receptors. First, the transient up-regulation of Cdk5 activity by NMDA and kainate receptorswas confirmed using specific receptor antagonists (Figs 5aand b). MK801 and CNQX inhibited both the transient up-regulation and the subsequent down-regulation of the Cdk5kinase activity. A major function of these receptors is theconductance of Ca2+ current into neurons. Although no Ca2+-dependent downstream kinases and phosphatases were foundto mediate NMDA- or kainate-induced p35 degradation(Fig. 3), Ca2+ entry could still be required for transient Cdk5activation and subsequent p35 degradation. Indeed, bothwere suppressed when neurons were treated with BAPTA-AM, a membrane-permeable Ca2+ chelator (Figs 5a–c). Incontrast, when intracellular Ca2+ levels were increased withthe Ca2+ ionophore A23187, p35 was cleaved to p25

(Fig. 5d). These results suggest that the transient activationof Cdk5 leading to the subsequent degradation of p35 occursat physiological Ca2+ concentrations induced by treatmentwith NMDA or kainate. At higher concentrations of Ca2+,calpain cleavage of p35 to p25 predominates.

The effect of Ca2+ on the histone H1 kinase activity ofCdk5/p35 purified from Sf9 cells or immunoprecipitatedfrom mouse brain extracts was next assessed, but additionof Ca2+ had no effect (data not shown), ruling out a directactivation of Cdk5 by Ca2+. The possibility of a Ca2+-dependent Cdk5 activator was also investigated by meas-uring Cdk5 kinase activity immunoprecipitated from brainextract preincubated with Ca2+ (Fig. 5e). Preincubation withCa2+ stimulated the histone H1 kinase activity of Cdk5.Furthermore, when calmodulin was added to the brainextract during preincubation, Cdk5 displayed even higherkinase activity. Moreover, the addition of a calmodulininhibitor (W-7) negated the activation of Cdk5 by Ca2+. Toensure that the observed histone H1 kinase activity in theimmunoprecipitates was due to Cdk5–p35 and not anotherCa2+- or Ca2+-calmodulin-activated protein kinase, roscovi-

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Fig. 5 The activation of cyclin-dependent kinase 5 (Cdk5) and sub-

sequent degradation of p35 is Ca2+-calmodulin-dependent. (a) and (b)

Neurons were stimulated with 0.1 mM NMDA (a) or 0.1 mM kainate (b)

for 1 min and 60 min in the presence of the antagonists MK801 and

CNQX, respectively, or 0.2 mM BAPTA-AM (BAPTA). Histone H1

phosphorylation by immunoprecipitated Cdk5/p35 is shown by auto-

radiograph in upper panel. The kinase activity was measured and

expressed in relation to the activity just before NMDA/kainate addition

(0 min) in lower panel. *p < 0.05 and **p < 0.001. (c) Cultured neurons

were treated with 0.1 mM NMDA or 0.1 mM kainate in the presence of

0.2 mM BAPTA-AM (BAPTA) for 1 h. p35 was detected by immuno-

blotting with anti-p35 antibody. (d) Cultured neurons were treated with

0.1 mM NMDA, 0.1 mM kainate or 5 lM A23187 for 1 h. The lysates

were probed by immunoblotting with anti-p35 antibody. Black and

white arrowheads indicate p35 and p25, respectively. (e) The histone

H1 kinase activity of Cdk5 immunoprecipitated from brain extract

preincubated with 0.05 mg/mL calmodulin (CaM) or 0.1 mM W-7 in the

presence (+) or absence (–) of 0.5 mM CaCl2 for 60 min on ice. In one

case, 50 lM roscovitine was included in the assay to show that histone

H1 phosphorylation was due to Cdk5 activity. (f) Inhibition of Ca2+-

calmodulin-activated histone H1 kinase activity by roscovitine. Histone

H1 kinase activity of immunoprecipitates prepared as described in (e)

was measured in the presence or absence of 50 lM roscovitine. (g)

Immunoblotting of anti-p35 immunoprecipitates with anti-Cdk5 anti-

body. (h) Immunoblottings of the brain extract incubated with Ca2+ or

Ca2+-calmodulin with anti-p35 antibody (upper panel) and anti-Cdk5

antibody (lower panel). (i) Cortical neurons cultured in the presence of

[32P]orthophosphate were stimulated by 0.1 mM NMDA for 1 min in the

presence or absence of 0.2 mM W-7. p35 was immunoprecipitated and

its phosphorylation status was detected by autoradiography and

quantified. *p < 0.05. (j) Primary cultured neurons were treated with

0.1 mM NMDA or 0.1 mM kainate in the presence or absence of

0.2 mM W-7 for 1 h. The level of p35 was determined by immuno-

blotting.

Glutamatergic regulation of Cdk5–p35 activity 507

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 502–512

tine was used as a control. Roscovitine inhibited both thebasal and Ca2+-calmodulin-activated H1 kinase activity ofCdk5/p35 (Figs 5e and f). Further, the increased kinaseactivity of Cdk5 was neither due to the increased immu-noprecipitation of Cdk5–p35 after incubation (Fig. 5g) nordue to the cleavage of p35 to p25 during the incubationwith Ca2+ or Ca2+-calmodulin (Fig. 5h, white arrowhead).Inclusion of protease inhibitors suppressed the p25 genera-tion. These biochemical studies were extended to primarycortical neurons (Figs 5i and j). W-7 inhibited NMDA/kainate-induced transient phosphorylation (Fig. 5i) andsubsequent degradation (Fig. 5j) of p35, supporting thehypothesis that calmodulin activates Cdk5 leading to p35degradation.

In vivo stimulation of NMDA and kainate receptors

reduces p35 levels, ultimately leading to a decrease in

cyclin-dependent kinase 5 activity

The physiologic relevance of this novel NMDA/kainatesignaling pathway was next assessed by theta-burst stimu-lation (TBS), a strong stimulation paradigm, of the Schaffercollateral/CA1 pathway in hippocampal slices followed byimmunoblot analysis to determine the level of p35 after30 min of stimulation (Fig. 6a). The level of p35 in TBSslices was decreased to 74.2 ± 10% (n ¼ 7, p < 0.05) ofthe controls (lower panel of Fig. 6a). Furthermore, in threeof nine slices, an upward mobility shift suggestive ofphosphorylation of p35 was observed when sodiumdodecyl sulfate–gel electrophoresis was run for longerlength (for example, see lane 2 of TBS in Fig. 6a). Theupward shift of p35 by autophosphorylation with Cdk5was observed in the process of p35 degradation in brainextracts and cultured neurons (Saito et al. 2003), but it isunclear why only one-third of slices displayed this upwardshift.

In another in vivo system, seven-week-old mice weresubcutaneously administered 50 mg of kainate/kg bodyweight. As kainate is a known epileptogenic agent (Borto-lotto et al. 1999), the mice exhibited mild seizures 30 minafter injection as expected. At this point, the mice were killedand levels of p35 in the cerebral cortex were compared withthose of saline-injected control mice by immunoblotting(Fig. 6b). p35 was reduced to 75.5 ± 6.88% (n ¼ 10,p < 0.05) of control levels in kainate-injected mice. We didnot observe p25 in a brain of mice treated with kainite for30 min.

Finally, to confirm the ultimate inactivation of Cdk5 byNMDA in intact neuronal tissue, NMDA modulation of aCdk5-specific site (Ser67) (Bibb et al. 2001) on proteinphosphatase inhibitor-1 was examined in hippocampal slices(Fig. 6c, P-Ser67 Inhibitor-1 in upper panel and black circlesin lower graph). Phosphorylation of Ser67 was greatlyreduced in NMDA-treated slices, in which the Cdk5 activity(H1 in upper panel and white circles in lower graph) and the

protein amount of p35 (p35 in upper panel) were alsodecreased, though a little bit delayed after the dephospho-rylation of Ser67.

p35–/– mice have a lower threshold for long term

potentiation

Down-regulation of Cdk5/p35 activity by NMDA may haveimportant consequences for synaptic plasticity. This possi-bility was investigated using the Schaffer collateral/CA1pathway of p35–/– mice, which possess the ultimate down-regulation of p35. In this paradigm, a difference might beexpected to be observed between wild-type and p35–/– miceunder a weak stimulation protocol. As induction of LTP byweak stimulation does not result in p35 degradation, a

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Fig. 6 In vivo stimulation of NMDA and kainate receptors reduces p35

levels, ultimately leading to a decrease in cyclin-dependent kinase 5

(Cdk5) activity. (a) Hippocampal slices were treated with theta burst

stimulation (TBS), and after 30 min p35 was analyzed by immuno-

blotting (TBS in upper panel). p35 of unstimulated slices is shown in

control (Ctrl) of upper panel. Levels of p35 were quantified and

expressed in relation to those of unstimulated (Ctrl) slices (lower

panel). Values represent the mean ± SEM for seven slices after the

largest and smallest data were removed. *p < 0.05. (b) Mice were

killed 30 min after the subcutaneous injection of kainate (50 mg/kg) or

saline. Levels of p35 in the cerebral cortex were determined by

immunoblotting. Values represent the mean ± SEM of 10 mice.

*p < 0.05. (c) Hippocampal slices were treated with 0.1 mM NMDA for

0, 5 10, 20, 40 and 60 min. Cdk5, p35, inhibitor-1 and phosphorylated

inhibitor-1 were examined by immunoblotting with anti-Cdk5, anti-p35,

anti-inhibitor-1 and anti-phospho-Ser67 inhibitor-1 antibodies (upper

panel). The kinase activity of Cdk5–p35 was measured with immu-

noprecipitated Cdk5–p35 using histone H1 as substrate. Autoradio-

graph is shown in upper panel (H1) and quantification is white circles in

lower panel. The level of phosphorylated inhibitor-1 normalized to total

inhibitor-1 protein was quantified (black circles in lower panel, n ¼ 4).

508 F.-Y. Wei et al.

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 502–512

difference in the level of LTP induction between wild typeand p35–/– mice would represent the contribution of p35down-regulation of LTP. Stronger stimulation protocolswould be expected to negate this result, because the wild-type mice would degrade their p35 to a level closer to that ofthe p35–/– mice. Indeed, LTP induction by a weak TBSprotocol consisting of two bursts of four pulses (2 · 4) wassignificantly different between wild-type and p35–/– mice(Fig. 7). This weak stimulation failed to induce LTP in wild-type mice (109 ± 6.2%, n ¼ 7), whereas it induced stableLTP in p35–/– mice (138 ± 8.5%, n ¼ 8). However, theeffects of stronger TBS protocols consisting of four (4 · 4),six (4 · 6), or eight bursts (4 · 8) of four pulses at thetarhythm (Fig. 7b) and tetanic stimulation (data not shown)

were indistinguishable between the groups. These resultsindicate that the threshold for LTP induction is lowered in thehippocampus of p35–/– mice.

Discussion

Cdk5 is active only when associated with an activationsubunit (Tang et al. 1997; Amin et al. 2002). Thus, the levelof p35 is a primary determinant of Cdk5 kinase activity,although phosphorylation of Cdk5 has been suggested tomodulate its kinase activity as well (Sharma et al. 1999;Zukerberg et al. 2000; Sasaki et al. 2002). Consequently,one approach to the function of Cdk5/p35 is to determinewhat factors control the synthesis and degradation of p35.The synthesis of p35 can be stimulated by neurotrophicfactors (Tokuoka et al. 2000; Harada et al. 2001) orextracellular matrix components (Paglini et al. 1998; Liet al. 2000). We show here that glutamate, the majorexcitatory neurotransmitter in the central nervous system(Ozawa et al. 1998; Craig and Boudin 2001), induces p35degradation. This antithetical regulation of Cdk5 activity byneurotrophins and a neurotransmitter via p35 synthesis anddegradation may prove to be important in a variety ofprocesses.

Glutamate receptors consist of two major superfamilies ofreceptors, usually referred to as ionotropic and metabotropicglutamate receptors, both of which are further classified intoseveral subgroups (Ozawa et al. 1998; Craig and Boudin2001). Each glutamate receptor has respective functions invarious neuronal processes that depend on the stage ofdevelopment and brain regions involved. In these studies wehave demonstrated that degradation of p35 was mediated byNMDA and kainate ionotropic glutamate receptors, but notby metabotropic glutamate receptors. After 1 h of treatment,p35 was decreased to �25% of control levels. Althoughprolonged exposure to glutamate can result in cytotoxiceffects, our cortical neurons did not display any evidences forneuronal dell death after the 1 h treatment, indicating that thep35 degradation occurred in viable neurons. Althoughtreatment of neurons for 1 h resulted into degradation ofthe majority of p35 in neurons, its decrease was detectableeven after 5 min treatment (Fig. 1). Because Cdk5–p35 islocalized throughout in neurons including cell body, axonand dendrites (Nikolic et al. 1996), it is not unexpected thattreatments of �1 h would be required in order to observe adecrease of p35. However, clearly the shorter exposure wassufficient to cause degradation, possibly by initially targetingp35 localized in postsynaptic region where glutamateionotropic receptors are concentrated.

Activation of ionotropic glutamate receptors stimulated theproteasomal degradation of p35 unassociated with anincrease in p25. Proteolysis of p35 to p25 was observedonly after extended treatment (6–12 h) with glutamate,consistent with the results of Lee et al. (2000). In contrast,

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Fig. 7 p35–/– mice exhibit a lower threshold for long-term potentiation

(LTP) induction. (a) LTP induction by theta burst stimulation (TBS),

two bursts of four pulses at 100 Hz. After a stable baseline had been

recorded for 30 min, TBS was delivered (arrow). Data are the

mean ± SEM as percentages of the average baseline excitatory

postsynaptic potential (EPSP) slope. Insets: representative tracings

from wild-type and p35–/– mice of baseline and 60 min post-TBS.

Calibration bars are 1.0 mV and 5.0 ms. (b) Comparison of LTP

magnitude induced by different TBS protocols. Data are the

mean ± SEM at 60 min post-TBS as percentages of the average

baseline EPSP slope. White bar, wild-type; gray bar, p35–/–. *p < 0.01.

Glutamatergic regulation of Cdk5–p35 activity 509

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 502–512

Kerokoski et al. (2002) reported that p35 is cleaved to p25within 30 min of glutamate or NMDA administration. Thisdiscrepancy may be due to differences in the experimentalconditions. Kerokoski et al. (2002) treated hippocampalneurons with glutamate or NMDA in the presence of 2.5 mM

CaCl2, whereas we treated cortical neurons with glutamate orNMDAwithout further addition of Ca2+ to the medium (Saitoet al. 1998). A larger Ca2+ influx due to higher concentra-tions of Ca2+ in the medium may induce more activation ofcalpain, resulting in the cleavage of p35 to p25, as wasshown with the Ca2+ ionophore-treated neurons. In contrast,the novel signaling pathway discussed here functions underphysiological Ca2+ concentrations.

In this novel pathway, glutamate, acting via ionotropicNMDA or kainate receptors, leads to the inactivation ofCdk5 by a pathway involving the proteasomal degradation ofp35. Ca2+ entry induced by NMDA or kainate triggers thedegradation of p35 through a transient activation of Cdk5/p35 that results in phosphorylation of p35. Thus, transientactivation of Cdk5 by NMDA ultimately results in inhibitionof Cdk5 activity, which would result in decreased level ofphosph-Ser67 inhibitor-1. NMDA-induced reduction in thelevel of phospho-Ser67 inhibitor-1 in striatal slices waspreviously attributed to stimulation of calcineurin (PP2B),the phosphatase responsible for dephosphorylation of thissite (Bibb et al. 2001). The present results suggest that thedecrease in Ser67 phosphorylation may be the result ofcoordinated action between decreased kinase activity andenhanced phosphatase activity. Although metabotropic glu-tamate receptor agonists did not induce the inactivation ofCdk5/p35 in cortical neurons, one report claims that DHPG,a group I mGluR agonist, increases Cdk5 activity inneostriatal slices via casein kinase I-dependent phosphory-lation of p35 (Liu et al. 2001). Glutamate may differentiallyregulate Cdk5 activity depending on the expression pattern ofthe various types of glutamate receptors in neurons.

NMDA- and kainate-induced Ca2+ signaling is known tobe mediated by the Ca2+-activated enzymes PKC, CaMKII,and PP2B (Platenik et al. 2000). However, none of theseenzymes was found to be involved in the transient activationof Cdk5 or the degradation of p35. Instead, the Ca2+-bindingprotein calmodulin was implicated in this process. Theregulation of Cdk5 activity by Ca2+ is very complex, as p35was recently found to bind in a Ca2+-dependent manner toCa2+-CaMKII and a-actinin, both of which are enriched inthe postsynaptic region (Dhavan et al. 2002). It will beinteresting to investigate the relationship between theregulation of Cdk5–p35 kinase activity by Ca2+ and theCa2+-dependent interaction of Cdk5–p35 with Ca2+-CaMKIIand a-actinin. Cdk5 activity may be regulated by thesebinding partners.

The importance of the regulation of Cdk5 activity byCa2+ and glutamate may lie in the relationship betweenCdk5 and synaptic plasticity. Indeed, Cdk5 has been

implicated in associative and aversive learning by animalbehavioral studies (Fischer et al. 2003). Support for thephysiological relevance of the novel signaling describedherein is provided by several in vivo models ranging fromTBS or NMDA treatment of hippocampal slices to kainatetreatment of whole animals. A critical observation furthersupporting the physiological relevance of this pathway isthat p35–/– mice exhibit a lower threshold for LTP thanwild-type mice. However, this finding must be consideredcarefully, because the Cdk5 signaling system could bereorganized compensatory in p35–/– mice or neuronal cellconnections could potentially be disorganized in p35–/–

mice as was implicated by difference in baseline fEPSPbetween p35–/– and wild-type mice. In actuality, neuronalcell layers were only mildly or marginally affected in theCA1 region of the hippocampus (Chae et al. 1997 andToshio Ohshima. et al., in preparation). All together, ourdata strongly implicate the down-regulation of Cdk5 inNMDA-dependent LTP induction.

Acknowledgements

We thank Dr Satoru Tahakashi at the National Institute of Dental and

Craniofacial Research, National Institutes of Health, for the critical

reading, Dr Atsuko Uchida for helpful advice on experiments, Dr

Kazuo Nagai for cyclosporine A, and Dr Paul Greengard at The

Rockefeller University for the phospho-Ser67 inhibitor-1 antibody.

This work was supported in part by Grants-in-Aid for Scientific

Research on Priority Areas (C), Advanced Brain Science Project

from the Ministry of Education, Culture, Sports, and Science and

Technology, of Japan (SH) and and the National Institute of Drug

Abuse (JAB).

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