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The Journal of Neuroscience, September 15, 2002, 22(18):7879-7891
The Cyclin-Dependent Kinase 5 Activators p35 and p39 Interact
with the 
-Subunit of Ca2+/Calmodulin-Dependent Protein
Kinase II and -Actinin-1 in a Calcium-Dependent Manner
Rani
Dhavan1, *,
Paul L.
Greer1, *,
Maria A.
Morabito1,
Lianna R.
Orlando2, and
Li-Huei
Tsai1, 3
1 Department of Pathology, Harvard Medical School,
Boston, Massachusetts 02115, 2 Department of Neurology,
Massachusetts General Hospital, Boston, Massachusetts 02114, and
3 Howard Hughes Medical Institute, Boston, Massachusetts
02115
 |
ABSTRACT |
Cyclin-dependent kinase 5 (Cdk5) is a critical regulator of
neuronal migration in the developing CNS, and recent studies
have revealed a role for Cdk5 in synaptogenesis and regulation of
synaptic transmission. Deregulation of Cdk5 has been linked to the
pathology of neurodegenerative diseases such as Alzheimer's disease.
Activation of Cdk5 requires its association with a regulatory subunit,
and two Cdk5 activators, p35 and p39, have been identified. To gain further insight into the functions of Cdk5, we identified proteins that
interact with p39 in a yeast two-hybrid screen. In this study we report
that 
-actinin-1 and the -subunit of
Ca2+/calmodulin-dependent protein kinase II
(CaMKII), two proteins localized at the postsynaptic density,
interact with Cdk5 via their association with p35 and p39. CaMKII
and -actinin-1 bind to distinct regions of p35 and p39 and
also can interact with each other. The association of CaMKII
and -actinin-1 to the Cdk5 activators, as well as to each other, is
stimulated by calcium. Further, the activation of glutamate receptors
increases the association of p35 and p39 with CaMKII, and the
inhibition of CaMKII activation diminishes this effect. The
glutamate-mediated increase in association of p35 and CaMKII is
mediated in large part by NMDA receptors, suggesting that cross talk
between the Cdk5 and CaMKII signal transduction pathways may be a
component of the complex molecular mechanisms contributing to synaptic
plasticity, memory, and learning.
Key words:
Cdk5; p35; p39; CaMKII; actinin; synapses; calcium; yeast
two-hybrid; NMDA
| |
INTRODUCTION |
Cyclin-dependent kinase 5 (Cdk5) is
a proline-directed serine/threonine kinase that has emerged as a
crucial regulator of neuronal migration in the developing brain (Dhavan
and Tsai, 2001
). Cdk5-deficient mice die at birth and display
widespread disruptions in neuronal layering of many brain structures,
indicating impairment in directing neuronal positioning (Ohshima et
al., 1996). Activation of Cdk5 requires binding to one of two
regulatory subunits, p35 or p39 (Lew et al., 1994; Tsai et al., 1994;
Tang et al., 1995). The phenotype of the p35/p39 double knock-out mice
is identical to the Cdk5-deficient mice, suggesting that together the
two activators of Cdk5 are necessary and sufficient for Cdk5 function
(Ko et al., 2001). Over 20 functionally diverse proteins involved in cytoskeleton dynamics, cell adhesion, transport, and membrane trafficking have been identified as Cdk5 substrates, elucidating the
molecular mechanisms of Cdk5 function (Dhavan and Tsai, 2001; Smith et
al., 2001).
Recently, synaptic functions of Cdk5 have come to light. Cdk5 and its
activators are localized at synapses and are present in presynaptic and
postsynaptic subcellular fractions, including postsynaptic density
(PSD) fractions (Humbert et al., 2000b; Niethammer et al., 2000). Cdk5
and p35 also are enriched at the neuromuscular junction, where Cdk5
activity is required for the neuregulin-induced transcription of
acetylcholine receptors (Fu et al., 2001). Cdk5-mediated phosphorylation of Synapsin 1, Munc18, Amphiphysin 1, and the -subunit (1A) of P/Q-type voltage-dependent calcium
(Ca2+) channels has revealed a role for
this kinase in synaptic vesicle trafficking and neurotransmitter
release (Dhavan and Tsai, 2001; Tomizawa et al., 2002). Cdk5 also
appears to play a role in postsynaptic transmission, and
phosphorylation of DARPP32 by Cdk5 modulates dopamine signaling in
striatal neurons (Bibb et al., 1999). Stimulation of metabotropic
glutamate receptors (mGluRs) in neostriatal slices causes a transient
increase in Cdk5 activity, and Cdk5 activity has been implicated in the
mGluR-mediated enhancement of voltage-dependent Ca2+ currents (Liu et al., 2001). Finally,
Cdk5 phosphorylates the NR2A subunit of the NMDA receptor, and
pharmacological inhibition of Cdk5 modulates NMDA receptor conductance
(Li et al., 2001).
To gain further insight into the functions of Cdk5, we identified
proteins that interact with its activator p39 in a yeast two-hybrid
screen. Two of the proteins identified in this screen, -actinin-1
and Ca2+/calmodulin-dependent protein
kinase II (CaMKII), are enriched at the PSD and interact with the
NMDA receptor (Wyszynski et al., 1997; Walikonis et al., 2000; Lisman
et al., 2002). We report that -actinin-1 and CaMKII interact with
p35, p39, Cdk5, and each other in a
Ca2+-dependent manner. Furthermore, the
activation of glutamate receptors increases the association of p35 and
p39 with CaMKII, suggesting that synaptic activity may influence the
cross talk between Cdk5 and CaMKII.
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MATERIALS AND METHODS |
Constructs. Mouse p39 cDNA was amplified by PCR, and
the fragment was inserted into the SalI and NotI
sites of the pPC97 vector (pDBLeu; Invitrogen, Carlsbad, CA) for
expression as a GAL4 DNA binding domain fusion protein (p39-pPC97).
Identical cloning strategy was used to generate p35-pPC97, p25-pPC97
(residues 99-307 of human p35), p29-pPC97 (residues 115-367 of mouse
p39), p10-pPC97 (residues 1-98 of human p35), and p19-pPC97 (residues
1-113 of mouse p39).
The cDNAs encoding the N-terminal (residues 1-316) and C-terminal
(residues 314-478) domains of CaMKII were generated by PCR from a
CaMKII-containing plasmid (a gift from Dr. H. Schulman, Stanford
University, CA) and subcloned into the SalI and
NotI sites of pPC97 and pPC86 vectors (Invitrogen) for
expression as GAL4 DNA binding or activation domain fusion proteins,
respectively. A cDNA encoding full-length -actinin-1 (I.M.A.G.E.
Consortium) was used as a template to generate truncation mutants of
-actinin-1 by PCR (residues are indicated in Fig. 1) and cloned into
the SalI and NotI sites of pPC97 and pPC86
vectors. The cDNAs encoding p39, p29, p35, p25, Cdk5, and the
-actinin-1 cDNA clone identified in the screen were subcloned into
the SalI and NotI sites of PGEX4T2 (Amersham
Pharmacia, Uppsala, Sweden) for the expression of GST fusion proteins.
Yeast two-hybrid screen. A yeast two-hybrid screen was
performed in yeast strain MaV203 containing HIS3,
LacZ, and URA reporter genes under the control of
the GAL4-activating sequences as described previously (Vidal et al.,
1996a,b; Niethammer and Sheng, 1998). Briefly, >10 million
cotransformants of p39-pPC97 and a human fetal (third trimester) brain
cDNA library (Invitrogen) were screened for interacting proteins by
growth on synthetic dropout Leu
Trp His
plates containing 20 mM 3-amino-1,2,4-triazole.
Positive colonies were tested further for expression of the
LacZ reporter gene by 
-galactosidase assay (Clontech
yeast protocol handbook, Palo Alto, CA) and for induction of the
URA reporter gene that allowed growth on
Leu Trp
Ura plates and conferred sensitivity to
5-fluoroorotic acid (Vidal et al., 1996a,b). Plasmids were isolated
from positive yeast colonies as described previously (Niethammer and
Sheng, 1998), and the interacting proteins were identified by
sequencing. The p35 yeast two-hybrid screen has been described
previously (Kwon et al., 2000).
Mapping domains of interaction. pPC86--actinin-1 or
pPC86-CaMKII identified in the screen was cotransformed into MaV203 yeast strain with pPC97-p39, p35-pPC97, p29-pPC97, p25-pPC97, p19-pPC97, or p10-pPC97. -Galactosidase assay was used to compare the strength of interaction among the proteins encoded by cotransformed cDNA. We judged colonies that turned blue within 1 hr to contain strongly interacting proteins (++++). The +++, ++, and + symbols were
used to designate colonies that turned blue within 1-4 hr, 4-8 hr,
and 8-24 hr, respectively. The 
symbol was used to indicate colonies that did not turn blue within 24 hr. To map the binding region
of CaMKII, we cotransformed the CaMKII truncation mutants in pPC86 vector with p39-pPC97, p35-pPC97, or -actinin-1-pPC97. Deletion constructs of -actinin-1 in pPC86 vector were cotransformed with p39-pPC97, p35-pPC97, or CaMKII-pPC97 and were assayed as described above.
GST pulldown assay. GST fusion proteins were expressed,
purified, and cross-linked to glutathione-Sepharose 4B (Amersham
Pharmacia) as described previously (Tarricone et al., 2001). Adult rat
brain was lysed in RIPA buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 1% NP-40, 0.5% sodium deoxycholate,
0.1% SDS) with protease and phosphatase inhibitors [containing (in
mM) 1 PMSF, 1 DTT, 1 Na3VO4, 5 NaF, 2 sodium
pyrophosphate, and 50 -glycerophosphate plus 1 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 nM okadaic acid] in a
homogenizing Dounce and then was spun at 13,000 rpm for 15 min at
4°C. Two milligrams of lysate were incubated with 5 µg of
cross-linked GST fusion proteins in RIPA buffer for 2 hr at 4°C. The
beads were washed three times with RIPA buffer, and the associated
proteins were resolved by SDS-PAGE and analyzed by immunoblotting.
Antibodies for immunoblotting. For Western blot analysis we
used an affinity-purified p35 polyclonal antibody (1:1000) (Ko et al.,
2001), an affinity-purified p39 polyclonal antibody (1:1000) (Humbert
et al., 2000a), a monoclonal Cdk5 antibody DC-17 (1:10) (Tsai et al.,
1993), a monoclonal CaMKII antibody (1:2000; Chemicon, Temecula,
CA), a monoclonal -actinin-1 antibody (1:1000; Sigma-Aldrich, St.
Louis, MO), an affinity-purified polyclonal Lis1 antibody (1:1000)
(Smith et al., 2000), or a polyclonal actin antibody (1:1000;
Sigma-Aldrich).
Coimmunoprecipitation. Two milligrams of adult rat brain
lysate (described above) were incubated with 1 µg of p35, p39, Cdk5 (C8; Santa Cruz Biotechnology, Santa Cruz, CA), or HA (Santa Cruz Biotechnology) polyclonal antibodies or -actinin-1 or HA (Santa Cruz
Biotechnology) monoclonal antibodies for 1 hr at 4°C in RIPA buffer.
Then 100 µl of a 10% slurry of protein A-Sepharose (Amersham Pharmacia), or IgM-Sepharose (Sigma-Aldrich) for -actinin-1 IPs, was
added for 1 hr at 4°C. The beads were washed three times with RIPA
buffer, and bound proteins were analyzed by SDS-PAGE and Western
blotting. In some cases the indicated concentration of CaCl2, MgCl2, EDTA, or EGTA
was added to adult rat brain lysate and incubated for 20 min before coimmunoprecipitation.
Immunostaining. Dissociated hippocampal neurons from
embryonic day 18 (E18) rat brains were plated on coverslips coated with poly-L-lysine (Sigma-Aldrich) at a density of 5 × 104/coverslips and maintained in culture
in Neurobasal medium (Invitrogen) supplemented with B27 (Invitrogen)
and L-glutamine (Sigma-Aldrich) as described previously
(Brewer et al., 1993) for 3 weeks. Neurons were fixed in 4%
paraformaldehyde/5% sucrose in PBS for 30 min at room temperature
(RT), followed by 5 min in cold methanol at -20°C. Cells were
permeabilized in blocking buffer [10% normal goat serum (NGS), 0.2%
Triton X-100 in PBS] for 1 hr at RT. Primary antibodies in staining
buffer (3% NGS, 0.1% Triton X-100 in PBS) were applied for 16 hr at
4°C. The CaMKII, p35, p39, Cdk5 (C8; Santa Cruz Biotechnology),
and -actinin-1 antibodies described above were used at a dilution of
1:250 for immunostaining. Oregon green-conjugated anti-rabbit and Texas
Red-conjugated anti-mouse secondary antibodies (Molecular Probes,
Eugene, OR) were used at a dilution of 1:500 in staining buffer for 1 hr at RT. For triple staining, a Texas Red-conjugated IgM-specific
anti-mouse secondary antibody was used with Oregon green-conjugated
anti-rabbit and Cy5-conjugated IgG-specific anti-mouse secondary
antibodies (Molecular Probes) to visualize -actinin-1, p35 (or p39
or Cdk5), and CaMKII, respectively. All images were acquired by
using a 60× or 100× oil-immersion objective on a Nikon inverted
microscope linked to a Delta vision deconvolution imaging system
(Applied Precision, Issaquah, WA). Images of double- and triple-labeled cells were combined and colorized with Adobe Photoshop software.
Glutamate treatment of hippocampal neurons. As described
above, 3 × 106 hippocampal neurons
were plated on poly-L-lysine-coated 10 cm plates. After 2 weeks in culture the medium was replaced with KRH buffer [containing
(in mM) 128 NaCl, 5 KCl, 1 NaH2PO4, 2.7 CaCl2, 1.2 MgSO4, 10 glucose, and 20 HEPES, pH 7.4] for 30 min at 37°C. Neurons were
treated with 100 µM glutamate/10 µM glycine in Mg2+-free KRH buffer for 5 min and then
were Dounce homogenized in ice-cold sucrose buffer [0.32 M
sucrose plus (in mM) 4 HEPES, pH 7.4, 1 MgCl2, 1 NaHCO3, and 0.5 CaCl2] containing all of the protease and
phosphatase inhibitors listed above. The homogenized cells were
centrifuged at 1000 × g for 10 min. The resulting
supernatant was centrifuged at 3000 × g for 15 min.
The pellet was lysed in RIPA buffer (described above) by 10 strokes in
a glass-glass Dounce homogenizer and centrifuged at 13,000 rpm for 15 min at 4°C; then the supernatant was collected. One milligram of
protein was used per immunoprecipitation, as described above.
Treatment of hippocampal slices. Hippocampal slices were
prepared from 3-week-old Sprague Dawley rats with a McIlwain tissue chopper and placed in KRH buffer aerated with 95%
O2/5% CO2. After equilibration at 37°C for 30 min the slices were stimulated in Mg2+-free KRH buffer for 5 min with 100 µM glutamate/10 µM glycine, 100 µM NMDA/10 µM glycine, or 100 µM AMPA. In some cases the slices were pretreated with 10 µM each KN-62, KN-93, KN-92, and MK801, 1 µM H-89, 100 µM CNQX, 100 µM
AIDA, or 5 mM EDTA for 10 min before the stimulations.
Treatments were stopped on ice, and the drug was removed. Slices were
homogenized in ice-cold sucrose buffer. Lysates were prepared and
immunoprecipitations were performed as described above.
Cdk5 kinase activity. One milligram of neuronal lysate was
immunoprecipitated with p35 antibody as described above. The
Sepharose-bound complexes were washed two times with kinase buffer (20 mM MOPS, pH 7.2, 5 mM
MgCl2). Kinase buffer (30 µl) containing 500 µM Histone H1 peptide (PKTPAKAKKL), 100 µM
ATP, and 5 µCi [
-32P]ATP was
incubated with the immunocomplexes for 30 min at RT. Samples were
centrifuged at 13,000 rpm for 2 min, and 10 µl of the supernatant was
spotted on phosphocellulose disc paper. The discs were washed four
times with 0.3% phosphoric acid and counted in 10 ml of scintillation fluid.
CaMKII activity from hippocampal slices. CaMKII-dependent
activity was measured by using a
Ca2+/calmodulin-dependent protein kinase
assay system (Invitrogen) according to the manufacturer's
instructions. Briefly, slices were Dounce homogenized in cold
extraction buffer [containing (in mM) 20 PIPES, pH 7.0, 0.5 EGTA, 1 EDTA, 10 sodium pyrophosphate, 2 DTT, and 0.4 ammonium
molydate plus 10 µg/ml leupeptin and 0.1% Triton X-100]. Then 20 µl of the extract was incubated in a buffer containing (in
mM) 50 PIPES, pH 7.0, 20 MgCl2, 0.05 ATP, and 15 µM autocamtide-3 plus 200 mg/ml BSA and 50 µCi/ml [-32P]ATP in a final volume
of 50 µl either in the presence of 1 mM EGTA
(Ca2+/CaM-independent activity) or 1 mM CaCl2/2.4 µM
calmodulin (total activity). The reaction was incubated for 2 min at
30°C and stopped by the addition of 10 µl of ice-cold 20% TCA.
Reactions were centrifuged at 13,000 rpm for 2 min. Then 30 µl of
supernatant was spotted on phosphocellulose discs and counted as
described above. Autonomous CaMKII activity is expressed as the
percentage of total activity that is
Ca2+/CaM-independent.
Statistic analysis. The Western blot images on film were
digitized by a calibrated ImageScanner scanner and LabScan 3.0 software (Amersham Pharmacia). The volumes of image spots were quantified by
using the ImageMaster 3 software (Amersham Pharmacia). Data were
analyzed by paired Student's t test or by one-way ANOVA, followed by Newman-Keuls multiple comparison tests that used GraphPad Prism (version 3.02; GraphPad, San Diego, CA). Differences were considered significant at p < 0.05.
| |
RESULTS |
p39 interacts with -actinin-1 and CaMKII in a yeast
two-hybrid screen
Full-length p39 was used as bait in a yeast two-hybrid screen of a
human brain cDNA library. Two clones identified in this screen were
identical cDNA inserts encoding the full-length -subunit of CaMKII
(Fig. 1A). Both
CaMKII cDNAs contained 120 bp of 5'-untranslated region (UTR), the
1434 bp coding region, and a 1495 bp 3'-UTR.
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Figure 1.
CaMKII and -actinin-1 interact
with the Cdk5 activators p39 and p35, and with each other, in a yeast
two-hybrid system. A, A schematic of full-length
CaMKII and -actinin-1 proteins is presented above a diagram of
the protein encoded by the cDNA clones identified in the screen. The
numbers in parentheses indicate the
number of copies of CaMKII and -actinin-1 cDNAs isolated in the
screen. CaMKII contains an N-terminal kinase domain, a
Ca2+/calmodulin binding domain (CBD),
and an association domain (AD). Both CaMKII cDNAs
identified in the screen contain the entire open reading frame (amino
acid residues 1-478). -Actinin-1 is composed of an N-terminal actin
binding domain, a central region containing four spectrin repeats
(I-IV), and a C terminus with two EF-hand
motifs. The cDNA identified in the screen encodes amino acid residues
363-892 of -actinin-1. B, cDNAs encoding full-length
p39 (residues 1-369) and p35 (residues 1-307) or N- and C-terminal
truncation mutants of p39 and p35 were tested for association with the
CaMKII and -actinin-1 clones identified in the yeast two-hybrid
screen. Strength of an interaction is indicated by ++++, +++, ++, or + on the basis of -galactosidase assays, whereas - denotes no
interaction (see Materials and Methods). C, Full-length
or truncation mutants of CaMKII were tested for their ability to
interact with full-length p35 and p39 in a yeast two-hybrid
-galactosidase assay. A schematic of the proteins encoded by the
CaMKII cDNAs is indicated. D, Full-length or
truncation mutants of -actinin-1 were tested for their interaction
with p35 and p39. A schematic representation of the proteins encoded by
-actinin-1 cDNAs is presented. The N terminus contains an actin
binding domain (ABD); the central region contains four
spectrin-like repeats (SRI-IV). The C terminus
contains a pair of EF-hand motifs (EF-H) depicted
by two vertical lines. E, Full-length or
truncation mutants of CaMKII were tested for their ability to
interact with full-length -actinin-1 in a yeast two-hybrid assay.
F, Full-length or truncation mutants of -actinin-1
were tested for their ability to interact with full-length CaMKII in
the yeast two-hybrid assay.
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An additional cDNA clone identified in the screen represented the
C-terminal two-thirds of human -actinin-1 (also called nonskeletal
muscle -actinin). The -actinins are a family of closely related
proteins that are composed of three domains: an N-terminal actin
binding domain, a central region composed of four spectrin-like
repeats, and a C-terminal region containing two
Ca2+-binding EF-hand motifs (Blanchard et
al., 1989). Our cDNA insert began at nucleotide 1087 (amino acid 363)
and extended for 1.8 kb. The encoded protein contains the last three
spectrin-like repeats and continues to the C-terminal end of the
protein (Fig. 1A).
-Actinin-1 and CaMKII bind distinct regions of p39
and p35
Seven cDNA clones of -actinin-1 also had been identified
previously in a yeast two-hybrid screen by using full-length p35 as
bait and an embryonic mouse (E12.5) cDNA library (M. Nikolic and L.-H.
Tsai, unpublished observations). The -actinin-1 protein encoded by
all seven clones contained the last three spectrin-like repeats and
continued to the C-terminal end of the protein. Moreover, p35 is 46%
identical to p39, with >70% identity in the C-terminal Cdk5 binding
region (Tang et al., 1995). p35 could bind to -actinin-1, and with
greater affinity than p39, in the yeast two-hybrid assay (Fig.
1B). p35 also could interact with CaMKII in the
yeast two-hybrid system, although the association was weaker than with
p39 (Fig. 1B).
The domains of p35 and p39 required for association with full-length
-actinin-1 and CaMKII were investigated in a yeast two-hybrid
system by using N- and C-terminal truncation mutants of p35 and p39.
The N terminus of p35 and p39 contains a myristoylation signal and is
thought to be critical for the membrane localization of the two
activators (Patrick et al., 1999; Patzke and Tsai, 2002). The
C-terminal two-thirds of p35 and p39 adopt a cyclin-box tertiary
structure that is sufficient for binding and activating Cdk5 (Qi et
al., 1995; Tarricone et al., 2001). The N-terminal regions of p39 and
p35 could interact with -actinin-1 with equivalent or greater
efficiency than the respective full-length proteins, whereas the Cdk5
binding domains did not display any association with -actinin-1 in
the yeast two-hybrid assay (Fig. 1B). This suggests
that the N terminus of p35 and p39 is necessary and sufficient for
association with -actinin-1. In contrast, CaMKII could bind to
the C terminus of p35 and p39 but did not interact with the N-terminal
domains of these two proteins (Fig. 1B). Thus
CaMKII and -actinin-1 bind to different regions of both p35 and p39.
The N terminus of CaMKII is required for the association with
p35 and p39
All CaMKII subunits contain an N-terminal catalytic domain, a
central regulatory region, and a C-terminal association domain (Fig.
1A) (Soderling et al., 2001). The regulatory region
contains an overlapping autoinhibitory domain and a
Ca2+/calmodulin binding motif, whereas
individual subunits interact via their association domains to form a
multimeric holoenzyme. The N-terminal 316 residues of CaMKII,
containing the catalytic domain and the regulatory region, were
necessary and sufficient for binding to the Cdk5 activators (Fig.
1C).
p35 and p39 recognize two independent binding sites
on -actinin-1
The -actinin-1 clone identified in our yeast two-hybrid screen
contains the last three spectrin repeats and the EF-hand-containing C-terminal domain. To narrow further the residues of -actinin-1 that
are required for association with the Cdk5 activators, we assessed the
ability of truncation mutants of -actinin-1 to bind to full-length
p39 and p35 in a yeast two-hybrid assay (Fig. 1D). The N-terminal actin binding domain of -actinin-1 did not interact with p39 or p35 (Fig. 1D). A C-terminal truncation
mutant of -actinin-1, lacking the C-terminal EF-hand-containing
domain, demonstrated reduced binding to p35 and p39 compared with the
full-length protein (Fig. 1D). This suggests that,
although the C-terminal region of -actinin-1 is not necessary for
association, residues in this domain may contribute to the interaction
with p35 and p39. In fact, the C-terminal region of -actinin-1 alone
was sufficient for association with p35 and p39 (Fig.
1D). A truncation mutant of -actinin-1 lacking
both the C-terminal domain and the N-terminal actin binding domain
could also interact with p39 and p35 (Fig. 1D).
Larger N-terminal deletions did not reduce binding further, and
residues encoding just the spectrin-like repeat IV domain were
sufficient for association with both Cdk5 activators (Fig. 1D). Thus p35 and p39 can recognize at least two
distinct regions of -actinin-1, one contained in the spectrin-like
repeat IV and the other in the C-terminal region of the protein.
-Actinin-1 interacts with CaMKII
Walikonis and colleagues have shown that -actinin-4, another
member of the -actinin family, interacts with both the - and -subunits of CaMKII at a site just downstream of its EF-hands, between residues 806 and 871 (Walikonis et al., 2001). -Actinin-1 and -actinin-4 are 88% identical and 97% similar in this region (Honda et al., 1998). The authors suggest that -actinin-1, which has
been localized to the PSD (Walikonis et al., 2000), also may be a
physiologically relevant binding partner of CaMKII. We therefore investigated this possibility directly. In a yeast two-hybrid assay the
full-length CaMKII and -actinin-1 indeed did display a very
strong interaction (Fig. 1E,F).
The N terminus of CaMKII, encompassing the kinase domain and the
regulatory region, was sufficient for interaction with -actinin-1, whereas the C-terminal association domain of CaMKII did not display any binding (Fig. 1E). Thus, consistent with the
observations for -actinin-4 (Walikonis et al., 2001), the N terminus
of CaMKII is necessary and sufficient for association with
-actinin-1. Deletion of the C-terminal domain of -actinin-1
completely abolished binding to CaMKII (Fig. 1F).
Further, the C-terminal domain of -actinin-1 alone could interact
with CaMKII with the same strength as the full-length protein (Fig.
1F). As is the case for -actinin-4, the
EF-hand-containing C terminus of -actinin-1 is therefore necessary
and sufficient for binding to CaMKII (Walikonis et al., 2001).
CaMKII and -actinin-1 interact with p35/Cdk5 and
p39/Cdk5 complexes
We used GST pulldown assays to confirm the association of
-actinin-1 and CaMKII with p39 and p35 independently of the yeast two-hybrid system. For these experiments we expressed recombinant p39,
p25, and Cdk5 as GST fusion proteins in bacteria. Full-length p35
cannot be expressed as a GST fusion protein because of severe degradation (Qi et al., 1995). Therefore, GST-p25, the C-terminal Cdk5
binding and activating region of p35, was used in these assays. All GST
fusion proteins were used to pull down interacting proteins from adult
rat brain lysate, and associated proteins were identified by
immunoblotting. As expected, GST-p39 and GST-p25 both could bind to
Cdk5 in this assay (Fig.
2A). GST-p39 and
GST-p25, but not GST alone, also could precipitate CaMKII from adult
rat brain lysate (Fig. 2A). GST-p29, containing the C
terminus of p39, also can interact with CaMKII (data not shown).
These results are consistent with the yeast two-hybrid data that
indicate that the Cdk5-activating domain of p35 and p39 is necessary
and sufficient for interaction with CaMKII. The specificity of the
GST pulldowns is demonstrated by showing that Lis1, a protein expressed
at high levels in the adult brain (Smith et al., 2000), does not
interact with the GST fusion proteins under these conditions.
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Figure 2.
CaMKII, -actinin-1, and Cdk5 and its
activators interact in adult rat brain. A, GST fusion
proteins of p39 (GST-p39), p25 (GST-p25),
Cdk5 (GST-Cdk5), or GST alone were incubated with adult
rat brain lysate. Then 10% of the lysates used for the pulldowns
(10% Input) and the associated proteins were resolved
by SDS-PAGE and detected by immunoblotting with CaMKII,
-actinin-1, Cdk5, and Lis1 antibodies, as indicated on the
right. B, GST alone and a GST fusion
protein encoding residues 363-892 of actinin-1 (GST--actinin-1)
were incubated with adult rat brain lysate, and the associated proteins
were detected by Western blotting with p39, p35, Cdk5, and CaMKII
antibodies, as indicated on the right. The p35 antibody
recognizes both full-length p35 and the N-terminal truncation product termed p25, as
indicated by the arrows on the left.
C, CaMKII and -actinin-1 coimmunoprecipitate with
Cdk5 and its activators. p35, p39, Cdk5, and HA antibodies (indicated
at the top of the lanes) were used for
immunoprecipitations from adult rat brain lysate. Then 5% of the
lysate used for the immunoprecipitation (5% Input) and
the immunoprecipitated proteins were resolved by SDS-PAGE and detected
by immunoblotting with the antibodies indicated on the
right. D, -Actinin-1 or HA antibody
was used for immunoprecipitations from adult rat brain lysate. Then
10% of the proteins used for the immunoprecipitations (10%
Input) and the associated proteins were resolved by SDS-PAGE and
detected by immunoblotting with the antibodies indicated on the
right.
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Although full-length p39 could bind to -actinin-1 in our pulldown
assay, GST-p25 and GST alone did not interact with -actinin-1 (Fig.
2A). GST-p29 also did not pull down -actinin-1 in
these assays (data not shown), confirming that the N terminus of p35 and p39 is necessary for interaction with -actinin-1. GST-Cdk5 could
pull down both CaMKII and -actinin-1, but not Lis1, from brain
lysate, presumably via its interaction with p35 and p39 (Fig.
2A).
The -actinin-1 clone obtained in the screen (residues 363-892) was
expressed as a recombinant GST fusion protein in bacteria and was used
to pull down interacting proteins from adult rat brain lysate. In this
assay GST--actinin-1, but not GST alone, could interact with p39,
p35, and Cdk5 (Fig. 2B). Immunoreactivity representative of p25 was not detected in the -actinin-1 pulldowns, consistent with our previous GST pulldown and yeast two-hybrid results
that the N terminus of p35 and p39 is necessary for association with
-actinin-1 (Fig. 2B). Together, the results from
the GST pulldown assays suggest that CaMKII and -actinin-1 can
interact with the p35/Cdk5 and p39/Cdk5 complexes in brain. Consistent with the yeast two-hybrid interaction results, GST--actinin-1 also
bound CaMKII from adult rat brain lysate (Fig.
2B).
CaMKII, -actinin-1, and the Cdk5 activators interact
in vivo
To determine whether p39 and p35 interact with CaMKII and
-actinin-1 in vivo, we performed immunoprecipitations
from adult rat brain lysate by using antibodies against p35, p39, and
Cdk5, and we detected associated proteins via immunoblotting (Fig.
2C). Both CaMKII and -actinin-1 were detected in p35,
p39, and Cdk5 immunoprecipitates, but not in control
immunoprecipitations with HA antibody (Fig. 2C). Under these
conditions Lis1 did not interact with p35, p39, or Cdk5, demonstrating
the specificity of the coimmunoprecipitations. Reverse
immunoprecipitations with the -actinin-1 antibody also precipitated
p35, p39, Cdk5, and CaMKII from adult rat brain lysate (Fig.
2D). Together, these results suggest that CaMKII and -actinin-1 interact with each other and also form a complex with
p35/Cdk5 and p39/Cdk5 in vivo. p39, p35, Cdk5, or
-actinin-1 was not detected in CaMKII immunoprecipitations (data
not shown). This could be attributable to the fact that CaMKII is a
very abundant protein in brain, and only a small portion of it
interacts with the Cdk5 activators and -actinin-1.
CaMKII, -actinin-1, and Cdk5 and its activators colocalize in
hippocampal neurons
CaMKII is a major component of excitatory synapses and is
enriched particularly at the PSD (Lisman et al., 2002). -Actinin also has been localized at the PSD and in dendritic spines (Wyszynski et al., 1997, 1998; Walikonis et al., 2000). We used
immunocytochemistry to determine whether Cdk5 and its activators
colocalize with CaMKII and -actinin-1 at synaptic sites in
neurons (Fig. 3). Dissociated hippocampal
neurons were maintained in culture for 3 weeks, during which time they
develop extensive processes and synaptic junctions and then were used
for immunocytochemistry.
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Figure 3.
CaMKII, -actinin-1, and Cdk5 and
its activators colocalize in hippocampal neurons. Hippocampal neurons
maintained in culture for 3 weeks were immunostained as described in
Materials and Methods. A-L, Images were acquired by
using a 60× oil-immersion objective. Scale bar in A, 10 µM (also applies to A-C,
E-G, and I-K).
M-P, Image was acquired by using a 100× oil-immersion
objective. Scale bar in M, 10 µM (also
applies to N-P). A-D, Colocalization of
Cdk5 and CaMKII. Hippocampal neurons were double stained for Cdk5
(A, green) and CaMKII (B,
red). C, The images shown in
A and B are combined to reveal the
colocalization of Cdk5 (green) and CaMKII
(red) in yellow. The boxed
area in C is presented in D at
3.5-fold magnification. E-H, Colocalization of p35 and
CaMKII. Hippocampal neurons were double stained for p35
(E, green) and CaMKII
(F, red). G, The images
were merged to indicate colocalization between p35
(green) and CaMKII (red) in
yellow. The boxed region of the image in
G is presented at 3.5-fold magnification in
H. I-L, Colocalization of p35 and
-actinin-1. Hippocampal neurons were double stained for p35
(I, green) and -actinin-1
(J, red). L, The
images were merged to indicate colocalization of p35
(green) and -actinin-1 (red) in
yellow. The boxed region of the image in
K is presented at 3.5-fold magnification in
L. M-P, Triple colocalization of Cdk5,
CaMKII, and -actinin-1. IgG- and IgM-specific secondary
antibodies were used to triple-stain hippocampal neurons with Cdk5,
CaMKII, and -actinin-1 antibodies (see Materials and Methods).
M, Colocalization of Cdk5 (green)
and CaMKII (blue) in triple-stained neurons is revealed
by the blue-green color. N,
Colocalization of Cdk5 (green) and -actinin-1
(red) in triple-stained hippocampal neurons is visible
as yellow. O, Colocalization of CaMKII
(blue) and -actinin-1 (red) in
triple-stained hippocampal neurons is visible as pink.
P, Triple colocalization of Cdk5
(green), -actinin-1 (red), and
CaMKII (blue) in triple-stained neurons is visible as
white.
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In these hippocampal neurons the punctate staining of CaMKII along
processes was readily detectable (Fig. 3B,F).
Discrete spots of Cdk5 immunostaining were visible along dendrites
(Fig. 3A), and an overlay of these images revealed punctate
colocalization of Cdk5 and CaMKII immunoreactivity along dendrites
(Fig. 3C,D). Like Cdk5, p35 immunoreactivity was visible in
neuronal shafts as well as in discrete puncta along processes (Fig.
3E,I). Merged images of p35 and CaMKII
immunoreactivity revealed a punctate pattern of colocalization of these
proteins (Fig. 3G,H). p39 also colocalized with
CaMKII in these neurons (data not shown).
-Actinin-1 immunoreactivity was detected primarily in dendritic
spines, with lower levels in the cytoplasm of neuronal processes (Fig.
3J). In addition to staining along the length of
neurites, p35 also was present in very fine spiny protrusions, and
these p35 positive spines also were immunoreactive for -actinin-1
(Fig. 3I-L). Similar colocalization with -actinin-1 also
was observed for Cdk5 (Fig. 3N) and p39 in dendritic
shafts and spines (data not shown).
We used IgG- and IgM-specific secondary antibodies to determine whether
triple colocalization of CaMKII, -actinin-1, and Cdk5 was
detectable in mature hippocampal neurons. High-magnification images
confirm the colocalization of Cdk5 with CaMKII (Fig.
3M) and -actinin-1 (Fig. 3N).
Overlap of CaMKII and -actinin-1 immunoreactivity indicates that
these two proteins also colocalize in discrete spots along dendrites
(Fig. 3O). Triple labeling indicates that a subset of
-actinin-1/CaMKII sites also contains Cdk5 immunostaining, as
indicated by the white color in the merged images (Fig. 3P). Triple colocalization of CaMKII and -actinin-1 with either p35 or
p39 also was detectable in these neurons (data not shown). This
suggests that CaMKII, -actinin-1, and Cdk5 along with its activators all can colocalize in mature hippocampal neurons, supporting the possibility that they can exist in a complex. The partial overlap
between Cdk5/-actinin-1 and Cdk5/CaMKII coimmunostaining suggests
that, although a complex of Cdk5, CaMKII, and -actinin-1 may
exist at synapses, exclusive complexes of Cdk5/CaMKII,
Cdk5/-actinin-1, and CaMKII/-actinin-1 are also probably present.
Ca2+ stimulates the association of p35 with
CaMKII and -actinin-1
Changes in intracellular Ca2+ levels
have been shown to regulate the binding and signaling properties of
both -actinin-1 and CaMKII (Blanchard et al., 1989; Krupp et al.,
1999; Soderling et al., 2001; Lisman et al., 2002). To determine
whether Ca2+ affects the association of
CaMKII or -actinin-1 with the Cdk5 activators, we tested the
ability of p35, p39, or -actinin-1 antibodies to coimmunoprecipitate
associated proteins from adult rat brain lysate in the presence of
increasing Ca2+ concentrations (Fig.
4).
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Figure 4.
Ca2+-dependent association of
CaMKII, -actinin-1, and the Cdk5 activators. A,
Ca2+ enhances the coimmunoprecipitation of CaMKII
and -actinin-1 with p35. EDTA (1 mM) or
CaCl2 was added to adult rat brain lysate to a final
concentration indicated above the lanes (0-5 mM). p35 antibody (p35
IP) or no antibody (no Ab) was used for
immunoprecipitations from these lysates, and the immunoprecipitated
samples were resolved by SDS-PAGE. The amounts of associated
-actinin-1 (top), CaMKII (middle),
and Cdk5 (bottom) were analyzed by Western blots. Then
5% of the lysates used for the immunoprecipitations
(Lysate) was analyzed by Western blotting for the levels
of p35, -actinin-1, CaMKII, and Cdk5. B,
Ca2+ enhances the coimmunoprecipitation of CaMKII
and -actinin-1 with p39. EGTA (1 mM) or
CaCl2 was added to adult rat brain lysate to a final
concentration indicated above the lanes. p39 antibody
(p39 IP) was used for immunoprecipitations from
these lysates; the amounts of associated -actinin-1
(top), CaMKII (middle), and Cdk5
(bottom) were analyzed by Western blots.
C, Ca2+ enhances the
coimmunoprecipitation of p35 and CaMKII with -actinin-1. The same
samples described in A were used for
immunoprecipitations with the -actinin-1 antibody
(-actinin-1 IP) or no antibody (no
Ab). The amounts of associated p35 (top),
CaMKII (middle), and -actinin-1
(bottom) were analyzed by Western blots.
D, Magnesium does not enhance the coimmunoprecipitation
of CaMKII and -actinin-1 with p35. CaCl2
(Ca2+) or MgCl2
(Mg2+) was added to adult rat brain
lysate to a final concentration indicated above the lanes.
p35 antibody was used for immunoprecipitations from these lysates. Then
10% of the lysates used for the immunoprecipitations (10%
Input) and the immunoprecipitated samples (p35
IP) were resolved by SDS-PAGE. The amounts of associated
-actinin-1 (top), CaMKII (middle),
and Cdk5 (bottom) were analyzed by Western blots.
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In the absence of exogenously added Ca2+,
we could detect coimmunoprecipitation of both -actinin-1 and
CaMKII with p35 (Figs. 2C, 4A). The
addition of 10 µM
Ca2+ to brain lysates stimulated a
detectable increase in the coimmunoprecipitation of both -actinin-1
and CaMKII with p35 (Fig. 4A). The addition of
10-50 µM Ca2+ to
brain lysates resulted in a twofold increase in the
coimmunoprecipitation of -actinin-1 with p35 and a threefold to
fourfold increase in the association of CaMKII with p35. The
-actinin-1 and CaMKII that coimmunoprecipitated with p35 at high
Ca2+ concentrations was not attributable
to nonspecific precipitation of protein aggregates because no protein
was detected in the non-IgG controls (Fig. 4A,C). The
addition of 1 mM EDTA reduced the
coimmunoprecipitation of -actinin-1 and CaMKII with p35 (Fig.
4A). The addition of calcium to brain lysates
similarly induced coimmunoprecipitation of actinin-1 and CaMKII with
p39, whereas the addition of EGTA inhibited the associations (Fig.
4B). In contrast, the coimmunoprecipitation of Cdk5
with p35 or p39 was not affected by changing calcium, EDTA, or EGTA
concentration in brain lysates (Fig. 4A,B).
Reverse immunoprecipitations with -actinin-1 antibody also revealed
the increased coimmunoprecipitation of p35 with -actinin-1 after the
elevation of Ca2+ concentrations in brain
lysates (Fig. 4C). Interestingly, increasing the
Ca2+ concentration in brain lysates also
enhanced the coimmunoprecipitation of CaMKII with -actinin-1,
with a twofold increase in the coimmunoprecipitation of these proteins
after the addition of 10 µM
Ca2+ (Fig. 4C). The addition of
MgCl2 to brain lysates did not increase the
coimmunoprecipitation of CaMKII or -actinin-1 with p35 (Fig. 4D), indicating that this effect is specific for
Ca2+ and is not the result of increasing
concentrations of general divalent cations. Our results suggest that
Ca2+ can stimulate the association of
CaMKII and -actinin-1 with p35 as well as with each other.
Glutamate treatment of neurons can regulate the association of p35
with CaMKII
The stimulation of glutamate receptors in neurons can lead to
increases in cytosolic Ca2+ (Nakanishi et
al., 1998). We therefore investigated whether the treatment of neurons
with glutamate could modulate the association of the Cdk5 activators
with CaMKII and -actinin-1. Dissociated hippocampal neurons were
maintained in culture for 2 weeks and then were treated with 100 µM glutamate/10 µM glycine in a
Mg2+-free buffer for 5 min. After
treatment, p35 and -actinin-1 were immunoprecipitated from lysates
of these neurons, and the associated proteins were detected by Western
blotting (Fig. 5A,B). Both
CaMKII and -actinin-1 coimmunoprecipitated with p35 under basal
conditions, and the treatment of hippocampal neurons with glutamate
induced a significant increase in the amount of CaMKII that
coimmunoprecipitated with p35 (Fig. 5A). Surprisingly, no
increase in the coimmunoprecipitation of -actinin-1 and p35 was
detected under the same treatment conditions (Fig. 5A,B).
Glutamate treatment also did not affect CaMKII coimmunoprecipitation with -actinin-1 significantly (Fig. 5B). In fact, a
slight decrease in association between CaMKII and -actinin-1 was
seen in some experiments, but this effect did not prove to be
significant in repeated experiments (Fig. 5B).
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Figure 5.
Glutamate treatment of dissociated hippocampal
neurons enhances the association of CaMKII and p35.
A, Dissociated hippocampal neurons in culture for 2 weeks were treated
(Glu/Gly) with buffer () or 100 µM
glutamate/10 µM glycine (+) for 5 min. Crude synaptosome
fractions prepared from these neurons were used for the
immunoprecipitation of p35. Then 10% of the proteins used for the
immunoprecipitations (10% Input) and the p35-associated
proteins (p35 IP) were resolved by SDS-PAGE. The
amounts of CaMKII (top), -actinin-1
(middle), and Cdk5 (bottom) were analyzed
by immunoblotting. Quantitative analysis from three independent
experiments is presented in the histogram. The amount of CaMKII,
-actinin-1, and Cdk5 immunoprecipitated with p35 is presented as a
percentage of the values that were measured in control treatments and
is the mean ± SEM of three independent experiments.
*Statistically different (p < 0.05) from
control values by Student's t test. B,
The same lysates described in A were used for
immunoprecipitation with -actinin-1 antibody. Then 10% of the
proteins used for the immunoprecipitations (10% Input)
and the -actinin-1-associated proteins (-actinin-1
IP) were resolved by SDS-PAGE; the amounts of p35
(top) and CaMKII (bottom) were
analyzed by immunoblotting. Quantitative analysis from three
independent experiments is presented in the histogram. The amount of
CaMKII and p35 immunoprecipitated with -actinin-1 is presented as
a percentage of the values that were measured in control treatments and
is the mean ± SEM of three independent experiments.
C, Cdk5 kinase activity is unaltered by glutamate
treatment of hippocampal neurons. Dissociated hippocampal neurons were
treated with either buffer (Control) or 100 µM glutamate/10 µM glycine
(Glu/Gly), as described in A. The
Cdk5/p35 complex was immunoprecipitated with p35 antibody, and Cdk5
kinase activity was measured in an in vitro kinase assay
toward Histone H1. The Cdk5 kinase activity is presented as a
percentage of the values that were measured from control-treated
neurons and is the mean ± SEM from three independent
experiments.
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We also wanted to determine whether glutamate treatment could result in
altered Cdk5 kinase activity. Toward this end we used the p35 antibody
to immunoprecipitate the p35/Cdk5 complex from lysates of control and
glutamate-stimulated hippocampal neurons. We detected no change in Cdk5
kinase activity after comparing the Cdk5 kinase activity of these
precipitates in an in vitro kinase assay by using Histone H1
as a substrate (Fig. 5C).
CaMKII activation is required for the glutamate-dependent increase
in the association of p35 with CaMKII
Our data suggest that the stimulation of glutamate receptors in
neurons can enhance the association of CaMKII with the Cdk5 activators. To determine whether Ca2+
influx was required for this effect, we treated hippocampal slices with
100 µM glutamate/10 µM glycine for 5 min in
the presence or absence of 5 mM EDTA. As in the dissociated
hippocampal neurons, the treatment of hippocampal slices with glutamate
enhanced the coimmunoprecipitation of CaMKII with p35 (Fig.
6A). Pretreatment of
the slices with 5 mM EDTA to reduce extracellular
free calcium blocked the glutamate effect, suggesting that calcium
influx is critical (Fig. 6A). The treatment of
hippocampal slices with glutamate also resulted in an increase in the
coimmunoprecipitation of CaMKII with p39, which could be abolished
by pretreatment with EGTA (Fig. 6B).
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Figure 6.
Ca2+ influx and
CaMKII activity are required for the glutamate-mediated enhanced
association of the Cdk5 activators and CaMKII. *Statistically
different (p < 0.05) from control
treatments; **statistically different (p < 0.05) from Glu/Gly treatment by one-way ANOVA. A,
Hippocampal slices were treated with either buffer or 100 µM glutamate/10 µM glycine
(Glu/Gly) in the absence or presence of 5 mM
EDTA for 5 min. Crude synaptosome fractions prepared from these neurons
were used for the immunoprecipitation of p35. The amounts of CaMKII
(top) and Cdk5 (bottom) associated with
p35 were analyzed by immunoblotting. Quantitative analysis from three
independent experiments is presented in the histogram. The amount of
CaMKII immunoprecipitated with p35 is presented as a percentage of
the values that were measured in control treatments and is the
mean ± SEM of three independent experiments. B,
Hippocampal slices were treated with 100 µM glutamate/10
µM glycine (Glu/Gly) in the absence or
presence of 5 mM EGTA for 5 min. The lysates were used for
the immunoprecipitation of p39, and the amounts of CaMKII
(top) and Cdk5 (bottom) associated with
p39 were analyzed by immunoblotting. Quantitative analysis from three
independent experiments is presented in the histogram.
C, Hippocampal slices were treated with either buffer or
100 µM glutamate/10 µM glycine
(Glu/Gly) in the presence or absence of 10 µM each KN62, KN93, and KN92 or 0.5 µM H-89
for 5 min. Crude synaptosome fractions prepared from these neurons were
used for the immunoprecipitation of p35. The amounts of CaMKII
(top) and Cdk5 (bottom) associated with
p35 were analyzed by immunoblotting. Quantitative analysis from three
independent experiments is presented in the middle
histogram. The amount of CaMKII immunoprecipitated with p35 is
presented as a percentage of the values that were measured in control
treatments and is the mean ± SEM of three independent
experiments. In the bottom histogram crude synaptosome
fractions prepared from the treated slices described above were assayed
for total and Ca2+/CaM-independent CaMKII
kinase activity (see Materials and Methods). Autonomous CaMKII activity
is defined as the Ca2+/CaM-independent activity
expressed as a percentage of the total CaMKII activity. The autonomous
CaMKII activity in slices is expressed as a percentage of the
autonomous CaMKII activity in control slices and is the mean ± SEM from three independent experiments.
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A relationship between CaMKII activation and its association with the
Cdk5 activators was investigated by the treatment of hippocampal slices
with kinase inhibitors. Pretreatment with the CaMKII inhibitors KN62
and KN93 reduced the glutamate-mediated increase in the association of
p35 with CaMKII (Fig. 6C). The structurally similar but
inactive analog of KN93 (KN92) and the PKA inhibitor H-89 had no
effects. None of these treatments altered the association between p35
and Cdk5, also measured by coimmunoprecipitation. Consistent with
previous reports (Fukunaga et al., 1992; Tan and Chen, 1997), glutamate
treatment of the slices stimulated CaMKII autonomous activity, which
was abolished by the pretreatment of slices with KN62 and KN93, but not
by KN92 or H-89 (Fig. 6C). This suggests that the
glutamate-mediated activation of CaMKII may regulate the formation or
maintenance of the p35/CaMKII complex. Both KN62 and KN93, although
commonly used as CaMKII inhibitors, have been reported to inhibit other
members of the Ca2+/CaM-dependent family,
and we cannot rule out that the inhibition of other family members in
our assay may contribute to the regulation of the CaMKII/p35 complex.
Activation of NMDA receptor enhances the association of p35
and CaMKII
To identify the specific neurotransmitter receptors involved in
the glutamate-mediated stimulation of p35/CaMKII coimmunoprecipitation, we treated hippocampal slices with glutamate in the presence of glutamate receptor antagonists (Fig. 7).
Ionotropic glutamate receptor antagonists were effective in reducing
the glutamate-stimulated increase in p35/CaMKII association. A
combination of antagonists, used to block all glutamate receptors,
completely abolished the glutamate effect, but the NMDA receptor
antagonist MK801 alone reduced the association virtually to basal
levels (Fig. 7). The non-NMDA receptor antagonist CNQX also caused a
smaller but statistically significant reduction, whereas treatment with
the mGluR antagonist AIDA had no effect (Fig. 7). These results suggest
that the CaMKII/p35 association triggered by glutamate treatment
occurs predominantly via NMDA receptor signaling.
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Figure 7.
Activation of the NMDA receptor enhances the
association of CaMKII and p35. Hippocampal slices were treated with
either buffer or 100 µM glutamate/10 µM
glycine in the absence (Glu) or presence of 10 µM MK801 (Glu/MK801), 100 µM
CNQX (Glu/CNQX), 100 µM AIDA
(Glu/AIDA), and 10 µM MK801 plus 100 µM CNQX plus 100 µM AIDA
(Glu/MK801+CNQX+AIDA) for 5 min. Hippocampal slices also
were treated with either 100 µM NMDA in the absence
(NMDA) or presence of 10 µM MK801
(NMDA/MK801) or 10 µM KN62
(NMDA/KN62) or 100 µM AMPA in the absence
(AMPA) or presence of 100 µM CNQX
(AMPA/CNQX) or 10 µM KN62
(AMPA/KN62) for 5 min. Crude synaptosome fractions
prepared from these neurons were used for the immunoprecipitation of
p35. The amounts of CaMKII (top) and Cdk5
(bottom) associated with p35 were analyzed by
immunoblotting. Quantitative analysis from three independent
experiments is presented in the histogram. The amount of CaMKII
immunoprecipitated with p35 is presented as a percentage of the values
that were measured in control treatments and is the mean ± SEM of
three independent experiments. aStatistically different
(p < 0.05) from control treatment;
bstatistically different (p < 0.05) from Glu treatment; cstatistically different
(p < 0.05) from NMDA treatment;
dstatistically different (p < 0.05) from AMPA treatment by one-way ANOVA.
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In further support, the treatment of hippocampal slices with NMDA
dramatically increased the association of CaMKII with p35, mimicking
the effect of glutamate (Fig. 7). The NMDA-mediated association could
be abolished by pretreatment with the receptor antagonist MK801 or with
the CaMKII inhibitor KN62. Stimulation with AMPA also enhanced
the association between p35 and CaMKII, but not as efficiently as
with NMDA. Together, our data suggest that the activation of CaMKII by
NMDA receptor favors the association of CaMKII with p35.
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DISCUSSION |
Several presynaptic and postsynaptic proteins have been identified
as Cdk5 substrates, and a role for Cdk5 in synaptogenesis and synaptic
transmission is beginning to emerge (Dhavan and Tsai, 2001). In this
study we report that CaMKII and -actinin-1 interact with Cdk5 via
its activators p35 and p39. CaMKII and -actinin-1 bind distinct
domains of the Cdk5 activators and can also interact with each other.
Triple colocalization of CaMKII and -actinin-1 with Cdk5 and its
activators is detected in neurons, suggesting that these proteins can
exist together in a complex. Ca2+ augments
the association of CaMKII and -actinin-1 with p35 and p39 and
also enhances their interaction with each other. Stimulation of
glutamate receptors also could increase the association of the Cdk5
activators with CaMKII, and this effect is mediated primarily by the
NMDA receptors. Together, these results suggest that Cdk5, via its
activators, forms a complex with CaMKII and -actinin-1 that may
be modulated by synaptic activity.
Interaction of Cdk5 and CaMKII with -actinin-1
The -actinin family members are actin cross-linking proteins
that bind and tether cell surface receptors, ion channels, and adhesion
complexes to the actin cytoskeleton (Blanchard et al., 1989; Otey et
al., 1990; Knudsen et al., 1995; Wyszynski et al., 1997; Cukovic et
al., 2001). Both -actinin-1 and -actinin-2 are present in the PSD
and in dendritic spines (Wyszynski et al., 1997, 1998; Walikonis et
al., 2000). -Actinin-2 binds directly to both the
TOP
ABSTRACT
INTRODUCTION
