From the Laboratory of Molecular and Cellular
Neuroscience, The Rockefeller University,
New York, New York 10021-6399, the ¶ Department of
Physiology, Kurume University School of Medicine, Kurume,
Fukuoka, Japan 830-0011, the
Centers for Disease Control and
Prevention, National Institute for Occupational Safety and Health,
Morgantown, West Virginia 26505, and the ** Department of Biological
Sciences, Graduate School of Science, Tokyo Metropolitan University,
Tokyo, Japan 192-0397
Received for publication, August 8, 2000, and in revised form, January 2, 2001
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ABSTRACT |
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Protein phosphatase inhibitor-1 is a
prototypical mediator of cross-talk between protein kinases and protein
phosphatases. Activation of cAMP-dependent protein kinase
results in phosphorylation of inhibitor-1 at Thr-35, converting it into
a potent inhibitor of protein phosphatase-1. Here we report that
inhibitor-1 is phosphorylated in vitro at Ser-67 by the
proline-directed kinases, Cdk1, Cdk5, and mitogen-activated protein
kinase. By using phosphorylation state-specific antibodies and
selective protein kinase inhibitors, Cdk5 was found to be the only
kinase that phosphorylates inhibitor-1 at Ser-67 in intact striatal
brain tissue. In vitro and in vivo studies
indicated that phospho-Ser-67 inhibitor-1 was dephosphorylated by
protein phosphatases-2A and -2B. The state of phosphorylation of
inhibitor-1 at Ser-67 was dynamically regulated in striatal tissue by
glutamate-dependent regulation of
N-methyl-D-aspartic acid-type channels.
Phosphorylation of Ser-67 did not convert inhibitor-1 into an inhibitor
of protein phosphatase-1. However, inhibitor-1 phosphorylated at Ser-67
was a less efficient substrate for cAMP-dependent protein
kinase. These results demonstrate regulation of a
Cdk5-dependent phosphorylation site in inhibitor-1 and
suggest a role for this site in modulating the amplitude of signal
transduction events that involve cAMP-dependent protein
kinase activation.
Control of protein phosphorylation/dephosphorylation occurs
through regulation of protein kinase and protein phosphatase activities and is an integral component of intracellular signal transduction. Inhibitor-1 was the first endogenous molecule found to regulate protein
phosphatase activity (1). Inhibitor-1 purified from rabbit skeletal
muscle is an 18,700-kDa acid- and heat-stable protein composed of 166 amino acids that are highly conserved throughout phylogeny (2, 3). When
phosphorylated at Thr-35 by cAMP-dependent protein kinase
(PKA),1 inhibitor-1
selectively and potently inhibits type 1 protein phosphatase (protein
phosphatase-1, PP-1) with an IC50 value of ~1
nM (4-7). Phospho-Thr-35 inhibitor-1 is dephosphorylated
by Ca2+/calmodulin-dependent protein
phosphatase 2B (PP-2B, calcineurin) and protein phosphatase 2A (PP-2A),
with PP-2B activity predominating in the presence of Ca2+
(8-11). First messengers such as neurotransmitters (e.g.
dopamine and acetylcholine) and hormones (e.g. adrenaline)
that elevate intracellular cAMP levels promote
PKA-dependent phosphorylation of inhibitor-1 at Thr-35 in
various tissues. PP-1 inhibition by phospho-Thr-35 inhibitor-1 provides
substantial amplification of PKA-dependent signaling
cascades and modulates the intensity and duration of a number of
physiological responses including regulatory aspects of the cell cycle,
gene expression, carbohydrate and lipid metabolism, and synaptic
plasticity (12-17).
Inhibitor-1 is widely expressed in mammalian tissue with highest levels
occurring in the brain, skeletal muscle, adipose, and kidney tissues
(18-26). Within the brain, the highest levels of inhibitor-1
immunoreactivity are associated with the dentate gyrus of the
hippocampus and the neostriatum and substantia nigra of the basal
ganglia (21). Control of PP-1 by inhibitor-1 in the hippocampus is
thought to be an important component of the mechanisms underlying
learning and memory, including long term potentiation and long term
depression (15, 27). Mice lacking inhibitor-1 display deficits in long
term potentiation induction (25).
In the dopaminoceptive medium spiny neurons of the striatum,
inhibitor-1 is co-expressed with a homologous PP-1 inhibitor, DARPP-32
(28). Inhibitor-1 is ~10 times less abundant than DARPP-32 in the
striatum (21, 29, 30), which constitutes about 0.25% of total striatal
protein and is estimated to occur at a concentration of 50 µM. The NH2-terminal residues 9-50 of
inhibitor-1 and DARPP-32 display 60% identity (31) and phosphorylation
of the homologous residue on DARPP-32 (Thr-34) converts it into an
inhibitor of PP-1 with an IC50 value identical to that of
inhibitor-1 (7). The activity of DARPP-32 is regulated through
phosphorylation at other sites. Phosphorylation of Ser-102 by casein
kinase 2 and Ser-137 by casein kinase 1 potentiates PKA phosphorylation and attenuates PP-2B dephosphorylation of Thr-34, respectively (32-34). Phosphorylation of DARPP-32 by cyclin-dependent
kinase 5 (Cdk5) at Thr-75 prevents DARPP-32 from serving as a PKA
substrate and converts DARPP-32 into a competitive inhibitor of PKA
(35). Following residue 50, very little primary sequence homology
exists between inhibitor-1 and DARPP-32, and inhibitor-1 does not
contain phosphorylation sites homologous to those found in DARPP-32
other than Thr-35. Therefore, it seemed possible that the activity of inhibitor-1 is differentially regulated by phosphorylation of other
putative sites.
In this report we show that inhibitor-1 is phosphorylated on Ser-67 by
MAP kinase and by two members of the cyclin-dependent protein kinase family in vitro but serves as a substrate
only for Cdk5 in striatal neurons. We demonstrate that this site of phosphorylation is predominantly dephosphorylated by PP-2A and PP-2B.
Kinetic analyses indicate that phosphorylation at Ser-67 reduces the
ability of inhibitor-1 to serve as a substrate for PKA but has no
effect on PP-1 inhibition.
Materials--
Oligonucleotides were obtained from Operon
Technologies, Inc. Restriction and DNA-modifying enzymes and
electrocompetent bacteria were from Life Technologies, Inc. Protease
inhibitors, proteolytic enzymes, dithiothreitol, and ATP were from
Roche Molecular Biochemicals. [ Preparation of Inhibitor-1--
Inhibitor-1 was purified from
rabbit skeletal muscle as reported previously (3) or purified as a
recombinant protein from Escherichia coli. For recombinant
inhibitor-1, a cDNA fragment encompassing the nucleotide sequence
for rat inhibitor-1 (2) was excised from a pUC120 subclone and purified
by agarose gel chromatography. The inhibitor-1 sequence was amplified
by PCR. The NH2-terminal oligonucleotide primer,
5'-GCGGCCATGGAGCCCGACAAC-3', included an NcoI
restriction enzyme cleavage site containing the ATG start codon
(inhibitor-1 sequence underlined). The COOH-terminal primer,
5'-GCGCTCGAGTCAATGATGATGATGATGATGGACCAAGCTGGCTCCTTG-3', encoded 6 histidine residues, a stop codon, and an
XhoI restriction enzyme site following the COOH-terminal
valine codon. A PCR product of the correct size was ligated into the
NcoI/XhoI restriction enzyme sites of the
bacterial expression vector pET 15B (Novagen). Conservation of the
inhibitor-1 ORF sequence was confirmed by automated fluorescent DNA
sequencing using the Big Dye Terminator Ready Reaction kit (PerkinElmer
Life Sciences/Applied Biosystems, Inc.) and an ABI 377 XL Prism DNA Sequencer.
The pET 15B vector containing the rat
inhibitor-1/His6 sequence served as a template for
site-directed mutagenesis using the polymerase chain reaction. The
primers were
5'-CTGGAACTCTTCATGGTGGGTGTAGTCCTTGTCATCTTCTTCCGTTGCCGTGGAGCCATTG-3' and 5'-TACCATGGAGCCCGACAAC-3' where the mutated
nucleotide is in bold and restriction sites are underlined. The PCR
product was gel-purified, digested with SstI and
NcoI, and substituted for the inhibitor-1 cDNA in the
rat inhibitor-1/His6 construct. The presence of the
mutation was confirmed by DNA sequencing.
Recombinant inhibitor-1/His6 was expressed in
bacteria and was affinity-purified (~5 mg/l culture) using
nickel-nitrilotriacetic acid-agarose resin (Qiagen) according to the
manufacturer's instructions. Peak elution fractions were pooled and
dialyzed against 10 mM HEPES, pH 7.4. A sample of material
was subjected to SDS-PAGE and estimated to be greater than 95% pure by
Coomassie Blue staining. Protein concentrations were determined by
quantitative amino acid analysis.
In Vitro Phosphorylation and Dephosphorylation
Reactions--
The MAP kinase reaction solution included 10 µM inhibitor-1 in 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 20 mM EGTA, 200 µM ATP, and 0.6 mCi/ml [
For kinetic analysis of PKA-dependent phosphorylation of
dephospho- and phospho-Ser-67 inhibitor-1, purified recombinant rat protein was phosphorylated to a stoichiometry of greater than 0.9 mol/mol using native Cdk1/cyclin B. Phospho-Ser-67 inhibitor-1 was
precipitated by addition of trichloroacetic acid to 20% in the
presence of 1 mg/ml bovine serum albumin. Inhibitor-1 was resuspended
in 1 M Tris-HCl, pH 8, and samples were dialyzed overnight in 10 mM HEPES, pH 7.4, with two changes of buffer.
Dephospho-inhibitor-1 was treated identically except for omission of
protein kinase from the in vitro phosphorylation reactions.
Dephospho- and phospho-Ser-67 inhibitor-1 were phosphorylated by PKA in
the presence of [
Phosphatase assays, in which soluble extracts from mouse striatal
homogenates were used as the source of phosphatase, were conducted as
described previously (43, 44). Striatal homogenates to which 5 mM EDTA and 1 mM EGTA were added were used to
observe basal levels of PP-1 and PP-2A phosphatase activity in a
30-µl reaction mixture containing 5 µM
[32P]phospho-Ser-67 inhibitor-1 as substrate. To assess
the contribution of PP-1 to the observed dephosphorylation, 100 nM thio-phospho-DARPP-32 was added. Okadaic acid, 2 nM and 1 µM, was added to examine the contribution of PP-2A alone and PP-2A/PP-1 together, respectively. PP-2B activity was assessed by the addition of 2.5 µM
Ca2+ and 1 µM calmodulin. PP-2C activity was
assessed by the addition of 1 µM MgCl2. PP-1
inhibition assays were conducted under linear conditions as described
previously (7) using recombinant rat inhibitor-1. Inhibitor-1 was
phosphorylated to a stoichiometry of ~1 mol/mol by PKA or Cdk1
followed by repurification by trichloroacetic acid precipitation and
dialysis as described above.
Identification of the Phosphorylation Sites on Inhibitor-1 and
Generation of Phosphorylation State-specific
Antibodies--
Two-dimensional phosphopeptide map and phosphoamino
acid analyses were performed as described (45). Two methods were
employed for identification of the sites of phosphorylation by Cdk1 and MAP kinase. For identification of the Cdk1 site, a 250-µg sample of
inhibitor-1 was phosphorylated in the presence of trace amounts of
[
To determine the site of MAP kinase phosphorylation, a smaller scale
sequencing procedure was used. A 10-µg aliquot of inhibitor-1 was
phosphorylated by MAP kinase in the presence of
[
Polyclonal phosphorylation state-specific antibodies for phospho-Ser-67
inhibitor-1 were generated and affinity-purified using a phosphopeptide
corresponding to residues 64-72 of both rabbit and rat inhibitor-1
essentially as described (48). Ser-67 is conserved between rat and
rabbit species, but at the Regulation of Phosphorylation of Inhibitor-1 at Ser-67 in
Striatal Slices--
C57BL/6 mouse striatal slices were acutely
microdissected and treated as described (49). Following equilibration
in Krebs bicarbonate buffer oxygenated by continuous aeration with 95% O2, 5% CO2, slices were incubated with 100 µM NMDA for 5 min, 10 µM cyclosporin A for
60 min, or 1 µM okadaic acid for 60 min, or incubated in
Krebs bicarbonate buffer, without any additions, for a comparable
period. In other experiments, slices were incubated in buffer
containing varying concentrations of roscovitine or PD 98059 for 60 min. Following incubation, slices were transferred to
microcentrifuge tubes, snap-frozen in liquid nitrogen, and then
homogenized by sonication in boiling 1% SDS, 50 mM NaF.
Equal amounts of protein (as determined by BCA assay) from homogenates were subjected to SDS-PAGE (either 15 or a 10-20% gradient of acrylamide), and proteins were electrophoretically transferred to PVDF
membranes. Immunoreactive proteins were detected by chemiluminescence (ECL, Amersham Pharmacia Biotech) and quantified using NIH Image software on laser-scanned x-ray film exposures that provided optimal linearity of signal intensity.
Phosphorylation of Inhibitor-1 by Proline-directed Protein
Kinases--
In initial experiments, phosphoamino acid analysis of
radiolabeled inhibitor-1 immunoprecipitated from
32P-prelabeled striatal slices indicated that basal
phosphorylation was due to the presence of phosphoserine. Furthermore,
it has been reported previously that inhibitor-1 purified from rabbit skeletal muscle was phosphorylated at Ser-67 to a stoichiometry of
0.5-0.7 mol/mol (50). The amino acid sequence of inhibitor-1 contains
consensus phosphorylation sites for proline-directed kinases including
members of the cyclin-dependent and MAP kinase families.
In vitro phosphorylation reactions indicated that
inhibitor-1 was an efficient substrate for Cdk1, Cdk5, and MAP kinase.
In typical experiments, inhibitor-1 isolated from rabbit skeletal muscle was phosphorylated by Cdk1 and MAP kinase to a stoichiometry of
1.15 and 0.82 mol/mol, respectively (Fig.
1, A, C and D).
Recombinant rat inhibitor-1 was phosphorylated by Cdk5 to a
stoichiometry of 0.86 (Fig. 1, B and D). In
kinetic studies conducted under initial rate conditions, the apparent
Km values for recombinant inhibitor-1
phosphorylation by sea star Cdk1, baculovirus-derived Cdk5, and sea
star MAP kinase were determined to be 11.4, 5.5, and 14.1 µM, respectively. The apparent
Vmax values for Cdk1 and Cdk5 were determined to
be 1.5 and 0.9 µmol/min/mg, respectively. Velocity parameters for MAP
kinase were not defined due to the unknown proportion of the kinase
preparation in the active state.
Identification of Phosphorylation Sites and Generation of
Phosphorylation State-specific Antibodies--
To determine if Cdk1
and Cdk5 phosphorylated the same site, a sample of
32P-labeled phospho-inhibitor-l from each kinase reaction
was subjected to phosphoamino acid analysis and phosphopeptide mapping
(Fig. 2A). For both of these
kinases, the predominant phosphorylated residue was serine. Tryptic
phosphopeptide maps of rabbit inhibitor-1 phosphorylated by Cdk1 and
Cdk5 generated were very similar, with two major and three minor
phosphopeptides migrating to the same positions. By using conventional
methodology (51), phosphopeptides corresponding to spots 1 and 3 were
isolated, and microsequencing analysis indicated a single site of
phosphorylation, Ser-67. These results were supported by
matrix-assisted laser desorption ionization/time of flight mass
spectrometry analysis of the same purified phosphopeptides (data not
shown).
Phosphoamino acid analysis indicated that MAP kinase phosphorylated
inhibitor-1 on serine, and phosphopeptide maps of recombinant rat
inhibitor-1 phosphorylated with Cdk1 and MAP kinase were identical (data not shown). A novel methodology was used to identify the site of
rat recombinant inhibitor-1 phosphorylation by MAP kinase (47) (Fig.
2B). 32P-Labeled phospho-inhibitor-1 from
in vitro MAP kinase phosphorylation reaction mixtures was
purified by SDS-PAGE chromatography and transferred to PVDF membrane.
This material was proteolytically digested, eluted from the membrane,
and subjected to small scale capillary reversed phase HPLC. Eluted
peptides were directly spotted onto PVDF strips that were used to
generate autoradiograms. Small pieces of PVDF containing pure
radiolabeled peptides, based on A210 absorbance
profiles (Fig. 2B, left), were excised and subjected to mass
spectrometry analysis and microsequencing (Fig. 2B,
right). Analysis of the major radiolabeled peptide yielded
the amino acid sequence, IPNPLLKSTLSMSPR. The predicted mass of this
peptide (1654 daltons plus 16 daltons for an oxidized methionine and 80 additional Da for the PO3 group) is 1750. The observed mass
was determined to be 1750 by matrix-assisted laser desorption
ionization/time of flight analysis. Analysis of a minor phosphopeptide
yielded the same sequence minus the first isoleucine. These results
indicated that Ser-67 was the single site of inhibitor-1 phosphorylated by MAP kinase.
The identity of Ser-67 as the site of phosphorylation by Cdk1, MAP
kinase, and Cdk5 was confirmed using site-directed mutagenesis to
generate purified recombinant Ser-67
To directly monitor phosphorylation in vitro and in
vivo, a phosphorylation state-specific antibody was
generated that detected inhibitor-1 only when phosphorylated at Ser-67
(Fig. 3B). Immunoblot analyses of time course in
vitro phosphorylation reaction mixtures with Cdk1, MAP kinase, and
Cdk5 demonstrated that the antibody did not detect
dephospho-inhibitor-1 (0-min time point). Signal intensity increased
with the period of incubation of the phosphorylation reactions. In
contrast, no signal was detected at any time point if the recombinant
Ser-67 Expression of Cyclin-dependent Protein Kinases in the
Striatum during Development--
In order to determine which of the
proline-directed protein kinase/inhibitor-1 phosphorylation reactions
could be physiologically relevant, an analysis was conducted with
antibodies to detect various kinases, cofactors, and inhibitor-1 in the
striatum. Immunoblots of striatal tissue taken at different stages of
development indicated that inhibitor-1 is expressed at substantial
levels in adult post-mitotic striatal neurons (Fig.
4) as reported previously (18). Cdk1 is
involved in cell cycle regulation and is not expressed in
end-differentiated neurons. Cdk5 is a neuronal
cyclin-dependent-like kinase that is regulated by the
neuron-specific activating cofactor, p35 (52, 53). Immunoblots of
striatal homogenates with antibodies to Cdk1, Cdk2, Cdk4, and Cdk5
indicated that, while all kinase species were expressed during early
stages of development, only Cdk5 was expressed in detectable levels in
adult mouse striatum, as was p35 (Fig. 4). Furthermore, both ERK1 and
ERK2 isoforms of MAP kinase and the upstream MAP kinase-activating
kinase, MEK-1, are expressed throughout development and in adult
striatum (data not shown). These results indicated that the
physiologically relevant kinases that might phosphorylate Ser-67 of
inhibitor-1 in adult brain are Cdk5 and/or MAP kinase.
Regulation of Phospho-Ser-67 Inhibitor-1 by Selective Protein
Kinase Inhibitors in Striatal Slices--
In homogenates prepared from
acutely dissected striatal slices, phospho-Ser-67 inhibitor-1 was
detected with the phosphorylation state-specific antibody, and the
basal stoichiometry of phosphorylation was determined to be 0.34 mol/mol, based on a comparison with standard curves constructed using
in vitro phosphorylated material (data not shown). To
determine if MAP kinase, Cdk5, or both could be responsible for
catalyzing this phosphorylation in intact neurons, striatal slices were
treated with various concentrations of the selective inhibitor of
MEK/MAP kinase activation, PD 98059 or the selective Cdk5 inhibitor,
roscovitine. Homogenates from these slices were analyzed by
immunoblotting for phospho-Ser-67 inhibitor 1, total inhibitor-1,
phospho-Thr-75 DARPP-32, and phospho-Thr-202/phospho-Tyr-204 MAP kinase
(Fig. 5). PD 98059 did not affect
phospho-Ser-67 inhibitor-1 levels. Similarly, there was no effect upon
levels of phospho-Thr-75 DARPP-32, which has previously been shown to
be a Cdk5-dependent phosphorylation site (35).
Phosphorylation of MAP kinase was reduced to near undetectable levels
by treatment of striatal slices with 100 µM PD 98059. Conversely, treatment of slices with roscovitine caused a reduction in
the levels of phospho-Ser-67 inhibitor-1 and phospho-Thr-75 DARPP-32
without affecting phospho-MAP kinase levels. These data indicate that
Cdk5, but not MAP kinase, is responsible for phosphorylation of
inhibitor-1 on Ser-67 in intact neurons of the adult striatum.
Dephosphorylation of Phospho-Ser-67 Inhibitor-1 in
Vitro--
To identify the endogenous protein phosphatase(s)
responsible for dephosphorylation of phospho-Ser-67 inhibitor-1,
reactions were conducted using striatal homogenate as the source of
phosphatase activity and 32P-labeled phospho-Ser-67
inhibitor-1 prepared in vitro using Cdk1 (Fig.
6A). Thio-phospho-Thr-34
DARPP-32 (100 nM), a potent and selective inhibitor of PP-1
(54), had no effect on basal phosphatase activity. Selective inhibition
of PP-2A activity by the addition of 2 nM okadaic acid (55)
resulted in a greater than 95% loss of phosphatase activity.
Inhibition of both PP-1 and PP-2A activities by the addition of 1 µM okadaic acid had the same effect. Activation of PP-2B
by the addition of Ca2+ and calmodulin resulted in a marked
increase in phosphatase activity. Activation of PP-2C by the addition
of Mg2+ had no detectable effect upon basal phosphatase
activity. These results indicate that both PP-2A and PP-2B may
contribute to the dephosphorylation of phospho-Ser-67 inhibitor-1 in
striatal homogenates.
Regulation of Phospho-Ser-67 Inhibitor-1 by NMDA and Protein
Phosphatase Inhibitors in Striatal Slices--
The state of
phosphorylation of inhibitor-1 at Ser-67 was characterized in striatal
slices prepared from wild type and genetically altered
(PP-2B
Dephosphorylation of phospho-Ser-67 inhibitor-1 was further
characterized by treatment of striatal slices from wild type mice with
selective protein phosphatase inhibitors (Fig. 6C).
Treatment with cyclosporin A, an inhibitor of PP-2B, caused a 1.8-fold
increase. We have previously reported that pretreatment of striatal
slices with 1 µM okadaic acid inhibited PP-2A activity
completely (IC50 ~100 nM) and PP-1 activity
by about 30% (43). Okadaic acid (1 µM) caused an
increase in the level of phospho-Ser-67 inhibitor-1 (2.1-fold) similar
to that seen using cyclosporin A. Together with the results from the
in vitro studies, these results indicate that both PP-2A and
PP-2B can dephosphorylate phospho-Ser-67 in the striatum.
Analysis of the Effect of Phospho-Ser-67 upon Inhibitor-1
Function--
To determine the effect of phosphorylation at Ser-67
upon the ability of inhibitor-1 to function as an inhibitor of
PP-1 activity, protein phosphatase inhibition assays were
performed in vitro in the presence of various concentrations
of either dephospho-, phospho-Thr-35, phospho-Ser-67, or
phospho-Thr-35/phospho-Ser-67 inhibitor-1 (Fig.
7). Neither dephospho- nor phospho-Ser-67
inhibitor-1 inhibited PP-1 activity at any of the concentrations tested
(0-1 µM). Phospho-Thr-35 inhibitor-1 potently inhibited
the activity of PP-1 with an IC50 value of 3.0 nM. Phosphorylation of Ser-67 had no significant effect on
the inhibitory activity of phospho-Thr-35 inhibitor-1.
To determine the effect of this proline-directed phosphorylation upon
the ability of PKA to phosphorylate Thr-35, inhibitor-1 was
phosphorylated to a stoichiometry of greater than 0.9 by Cdk1 and
repurified to homogeneity. Phosphorylation of Ser-67 altered the
apparent Km and Vmax for
phosphorylation of inhibitor-1 by PKA from 2.1 ± 0.3 (n = 6) and 0.9 ± 0.04 (n = 4) to
9.5 ± 2.3 (n = 6) and 1.4 ± 0.1 (n = 4) (Fig. 8),
respectfully. Thus, phosphorylation at Ser-67 caused a significant
reduction in catalytic efficiency
(Vmax/Km) from 0.42 ± 0.08 to 0.23 ± 0.03 (n = 4, p = 0.0076, Student's paired t test).
We report here that protein phosphatase inhibitor-1 is efficiently
phosphorylated at Ser-67 by three different proline-directed kinases
in vitro, Cdk1, Cdk5, and MAP kinase. The apparent
Km values for these phosphorylation reactions were
similar to the concentration of inhibitor-1 estimated to occur in
striatal medium spiny neurons (29, 30, 57, 58). Moreover, immunoblot
analysis indicated that Cdk5 and MAP kinase, but not Cdk1, are present in adult striatum. Treatment of striatal slices with selective protein
kinase inhibitors indicated that Cdk5 phosphorylates Ser-67 inhibitor-1
in the striatum but that MAP kinase does not. These studies using
striatal slices were consistent with our biochemical results and
indicate that Cdk5 is the predominant kinase responsible for
phosphorylation of inhibitor-1 at Ser-67 in intact striatal tissue.
Cdk5 functions in neurite outgrowth (59), development of the nervous
system (60), and regulation of dopamine signaling through
phosphorylation of DARPP-32 in the striatum (35). Cdk5 may also play a
role in muscle development (61). A substantial level of basal
phosphorylation of inhibitor-1 at Ser-67 was also detected in adult rat
hippocampus, and in skeletal muscle and kidney
tissue.3 These observations
suggest that Cdk1 and/or MAP kinase could also be responsible for the
phosphorylation of Ser-67 in peripheral tissue.
Phospho-Ser-67 inhibitor-1 was found to be a substrate for protein
phosphatases PP-2A and PP-2B in vitro. In other in
vitro protein phosphatase assays using purified protein
phosphatase catalytic subunits and standard substrates as controls,
PP-2B was found to be more efficient than PP-2A and PP-2C at
dephosphorylating phospho-Ser-67 inhibitor-1. PP-1 could not
dephosphorylate phospho-Ser-67 inhibitor-1 at all (data not shown).
Both phospho-Thr-35 of inhibitor-1 and phospho-Thr-34 of DARPP-32 have
been shown to be very efficient substrates for PP-2B (62), and PP-2B is
highly concentrated in striatal neurons (63). Treatment of slices with
NMDA, which activates PP-2B by increasing intracellular
Ca2+, caused an almost complete loss of phospho-Ser-67
levels. Conversely, cyclosporin A, a PP-2B inhibitor, increased
phospho-Ser-67 levels. Basal phospho-Ser-67 inhibitor-1 levels were
increased in striatal slices from PP-2B Phosphorylation of inhibitor-1 at Ser-67 by Cdk5 had no effect on PP-1
inhibitory activity. These results are also in complete agreement with
numerous previous reports that used inhibitor-1 purified from rabbit
muscle. It has been demonstrated that inhibitor-1, as well as its
homolog, DARPP-32, only become potent inhibitors of PP-1 after
phosphorylation by PKA and that the Thr-35 nonphosphorylated form of
inhibitor-1 is devoid of PP-1 inhibitory activity (1, 50, 58, 64). In
early studies, inhibitor-1 isolated from rabbit skeletal muscle was
found, by direct amino acid sequence analysis, to be phosphorylated at
Ser-67 with a stoichiometry of 0.5-0.7 mol/mol (50). That preparation
was found to inhibit PP-1 with a Ki of 1.6 nM only when phosphorylated at Thr-35 by PKA. Without
phosphorylation by PKA, the endogenous protein could not inhibit PP-1
at detectable levels even at a concentration 1,000-fold above the
Ki. Peptide fragments lacking the sequence
surrounding phospho-Ser-67 were fully active when phosphorylated by
PKA, and a phosphopeptide containing residues 61-71 was inactive (5,
6). All these findings are in contradiction to a recent report by Huang
and Paudel (65) that suggested that phosphorylation of inhibitor-1 at
Ser-67 converted it into a potent inhibitor of recombinant PP-1
purified from bacteria.
The present studies are also completely consistent with extensive
studies that have established that PP-1 is inhibited by a common
conserved region at the NH2 terminus of inhibitor-1 and DARPP-32 (5, 66, 67). In addition to the region surrounding the
phosphorylated Thr-35 residue (Thr-34 in DARPP-32), which interacts
with the active site of PP-1, a short docking motif (RKIXF,
residues 8-12) in the two proteins is required for potent inhibition
and interacts with a defined region that is removed from the active
site on PP-1 (5, 54, 66, 67). Phosphorylation of DARPP-32 at other
sites does not directly affect PP-1 inhibitory activity (33, 34).
Our results indicated that phosphorylation of inhibitor-1 at Ser-67
slightly altered its efficiency as a substrate for PKA when
phosphorylated on Ser-67. The stoichiometry of phosphorylation in
striatal tissue under basal conditions was determined to be 0.34 mol/mol, allowing an estimate of the concentration of phospho-Ser-67 in
the striatum of about 1.7 µM. By increasing the
Km for PKA phosphorylation from 1.7 to 9.5 µM, phospho-Ser-67 may serve as a fine control mechanism
for regulating the degree to which the effects of PKA activation are
amplified. Recently, additional PP-1 inhibitor proteins have been
identified, some of which demonstrate selective inhibitory activity
only when PP-1 occurs in association with other regulatory factors
(68-71). Similarly, phospho-Ser-67 inhibitor-1 may serve a regulatory
function only in the context of a protein complex. Phospho-Ser-67
inhibitor-1 could also facilitate subcellular localization or be
involved in the regulation of some unknown function of inhibitor-1. It
will also be interesting to see if phosphorylation of inhibitor-1 at
Ser-67 is involved in the mechanisms underlying synaptic plasticity in
the brain and carbohydrate and lipid metabolism in peripheral tissues.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP was from
PerkinElmer Life Sciences. Glutathione-Sepharose 4B was from Amersham
Pharmacia Biotech. Antibodies to cyclin-dependent protein
kinases were from Santa Cruz Biotechnology, Inc. Phospho-p44/42 MAP
kinase antibodies were from New England Biolabs, Inc. NMDA was from
Research Biochemicals. Okadaic acid and cyclosporin A were from Alexis
Biochemicals. Calmodulin was purified as described previously (36).
Peptides and phosphopeptides were synthesized at the Rockefeller
University Protein/DNA Technology Center.
-32P]ATP.
Activated MAP kinase, p44mpk, isolated from sea star
oocytes was used in some experiments (37). Alternatively, a mixture of
GST-ERK 1 (recombinant glutathione S-transferase fusion
protein containing the 44-kDa isoform of MAP kinase) together with a
constitutively active form of MEK, GST-MEK2E (38), was used. For
kinetic analyses, activated recombinant rat MAP kinase (Calbiochem) was
used. Cdk1 assays were conducted in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, and 1 mM
dithiothreitol, with either Cdk1/cyclin B purified from sea star
oocytes (39-41) or recombinant p34cdc2/cyclin B
(New England Biolabs). Cdk5 assays were conducted using either
partially purified recombinant GST-Cdk5 with GST-p25 from bacteria (42)
or Cdk5 and p35-His6 co-expressed in insect
Sf9 cultures using baculovirus
vectors2 in reaction mixtures
containing 10 µM inhibitor-1, 200 µM ATP, 30 mM MOPS (pH 7.2), and 5 mM
MgCl2. Kinetic analyses for MAP, Cdk1, and Cdk5 were
performed under empirically defined linear steady-state conditions
according to methodology described previously (34).
-32P]ATP, as previously described
(34).
-32P]ATP to a stoichiometry of 0.84 mol/mol. The
reaction mixture was then subjected to proteolytic digestion with
trypsin for 18 h at 37 °C. Peptides were separated by C18
reversed phase HPLC using a linear gradient elution in a buffer
containing 0.1% trifluoroacetic acid and increasing concentrations of
acetonitrile with monitoring for absorbance at 214 µm. Eluted
fractions were subjected to Cerenkov counting, phosphopeptide mapping
analysis, phosphoamino acid analysis, and microsequencing.
-32P]ATP. The reaction solution was subjected to
SDS-PAGE using a 15% acrylamide gel, and protein was
electrophoretically transferred to polyvinylidene fluoride (PVDF)
membrane (Millipore). The 32P-labeled phospho-inhibitor-1
was localized by autoradiography, and the membrane was excised and
subjected to proteolytic digestion with clostropain (Arg-C) (Roche
Molecular Biochemicals). Eluted peptides were separated by a C18
reversed phase capillary HPLC apparatus including a PerkinElmer Life
Sciences/Applied Biosystems pump 140D, a 785A detector, and a 112A
oven/injector. Fractions eluting from the capillary column were spotted
directly onto a strip of PVDF membrane using a 153 microblotter
(PerkinElmer Life Sciences). Methylene blue dye was used to align the
A210 absorbance profile of the HPLC chromatogram
and the autoradiogram of the PVDF strip. Membrane pieces containing the
32P-labeled phosphopeptides were excised and subjected to
automated NH2-terminal protein microsequencing and
matrix-assisted laser desorption ionization mass spectrometry (46,
47).
2 position, relative to Ser-67, an alanine
residue occurs in the rabbit sequence whereas a serine occupies that
position in the rat amino acid sequence. Two antibodies were produced
as follows: one used a rat-specific phosphopeptide, and the other used
a rabbit-specific peptide. However, each antibody exhibited equal
cross-reactivity with rabbit and rat phospho-Ser-67 inhibitor-1. The
antibody raised against the rat phospho-peptide was used in all studies
described here.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (48K):
[in a new window]
Fig. 1.
Phosphorylation of inhibitor-1 by
proline-directed protein kinases. Cdk1 (A), Cdk5
(B), and MAP kinase (C) were used to
phosphorylate dephospho-inhibitor-1 in in vitro time course
reactions. For Cdk1 and MAP kinase, rabbit inhibitor-1 was used. For
Cdk5, recombinant rat inhibitor-1 was used. The higher intensity of
signal for the Cdk5 reaction is due to higher specific activity of the
[ -32P]ATP used in the reaction. The radiographic
images shown in A-C were used to derive the quantified and
plotted values (D).
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[in a new window]
Fig. 2.
Identification of Ser-67 as
the site on inhibitor-1 phosphorylated by Cdk1, Cdk5, and MAP
kinase. A, phosphoamino acid analysis and tryptic
phosphopeptide maps of 32P-labeled rabbit
phospho-inhibitor-1 from in vitro phosphorylation reactions
using Cdk1 (left) and Cdk5 (right). For the
phosphoamino acid analyses, the positions of co-migrating
phosphoserine, phosphothreonine, and phosphotyrosine standards and the
origin are indicated. For the phosphopeptide maps, the positions of
comigrating phosphopeptides are indicated by numbers. The
positions of the origins (circles, bottom center) and phenol
red markers (ovals, upper left) are also indicated.
B, determination of the site of inhibitor-1 phosphorylation
by MAP kinase. The reversed phase HPLC chromatogram, PVDF strip with
methylene blue markers, and autoradiogram of the PVDF strip for rat
32P-inhibitor-1 phosphorylated by MAP kinase are shown in
alignment. Peaks corresponding to dye marker and radiolabeled peptides
(filled circles) are indicated (left).
Matrix-assisted laser desorption, mass spectrometry, and Edman amino
acid microsequencing indicated the peptide mass and sequence shown
(right), with the phosphorylated serine preceding a proline
(underlined).
Ala inhibitor-1.
Inhibitor-1 phosphorylation by Cdk1, MAP kinase, and Cdk5 was greatly
attenuated by the mutation of Ser-67 to Ala in comparison to the wild
type isoform in time course in vitro phosphorylation
reactions (Fig. 3A).
View larger version (41K):
[in a new window]
Fig. 3.
Confirmation of Ser-67 as the major site on
inhibitor-1 phosphorylated by Cdk1, MAP kinase, and Cdk5 by
site-directed mutagenesis, and the generation of a phosphorylation
state-specific antibody to that site. A, plots of
quantitated values for in vitro phosphorylation over time of
wild type (WT) versus Ser-67 Ala
(S67A) inhibitor-1 by Cdk1, MAP kinase, and Cdk5.
B, immunoblots of reaction mixtures similar to those in
A using an antibody specific for the detection of either
phospho-Ser-67 (top) or total inhibitor-1
(middle). Quantitation of signal for phospho-Ser-67
inhibitor-1 is also shown (bottom).
Ala mutant was used as a substrate in the reactions.
Equal amounts of protein could be detected at all time points when
blots were reprobed using an antibody specific for total inhibitor-1.
In addition to demonstrating the specificity of the phospho-Ser-67
inhibitor-1 phosphorylation state-specific antibody, these results
confirm that Cdk1, Cdk5, and MAP kinase phosphorylate inhibitor-1 at
Ser-67.
View larger version (64K):
[in a new window]
Fig. 4.
Developmental expression of various
cyclin-dependent protein kinases in the striatum.
Striatal tissue homogenates were prepared at the various time points
during embryogenesis and postnatal development. Samples were subjected
to SDS-PAGE and immunoblotting using antibodies to the various proteins
as indicated at the left. The relative intensity of signals
is not comparable between panels.
View larger version (51K):
[in a new window]
Fig. 5.
Cdk5 phosphorylates inhibitor-1 in the
striatum but MAP kinase does not. Striatal slices were incubated
for 60 min with the MEK inhibitor, PD 98059, or the Cdk5 inhibitor,
roscovitine, at the indicated concentrations. Homogenates were
subjected to SDS-PAGE and immunoblotted for detection of phospho-Ser-67
inhibitor-1 (A), total inhibitor-1 (B),
phospho-Thr-75 DARPP-32 (C), and diphospho-MAP kinase
(D). Quantitation of phospho-Ser-67 inhibitor-1 levels from
multiple experiments are also shown (E). **,
p < 0.01, Student's unpaired t test,
n = 6.
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[in a new window]
Fig. 6.
Phospho-Ser-67 inhibitor-1 is
dephosphorylated by PP-2A and PP-2B in vitro and in
intact neurons. A, in vitro
dephosphorylation of 32P-labeled phospho-Ser-67 inhibitor-1
by phosphatases in striatal homogenate is shown in the absence or
presence of thio-phospho-Thr-34 DARPP-32 (S-P-D32, 100 nM)
(a highly selective inhibitor of PP-1), 2 nM okadaic acid
(OKA) (predominantly an inhibitor of PP-2A), 1 µM okadaic acid (an inhibitor of both PP-1 and PP-2A),
Ca2+/calmodulin
(Ca2+/CaM, an activator of PP-2B),
and Mg2+ (an activator of PP-2C). Data represent means ± S.E. for four experiments. *, p < 0.01 compared
with control; Student's t test. B, upper panel,
phospho-Ser-67 inhibitor-1 levels in striatal slices from wild type and
PP-2B /
mice under basal conditions and
in response to treatment with NMDA were assessed by immunoblotting with
phospho-Ser-67 (upper) and total inhibitor-1
(middle) antibodies. Quantification by densitometry is also
shown (lower). C, effect of PP-2B activation in
the absence and presence of PP-2B or PP-2A inhibitors in intact
neurons. Striatal slices were treated with the PP-2B inhibitor,
cyclosporin A (CyA) or the PP-2A inhibitor okadaic acid
(OKA), and the homogenates were subjected to SDS-PAGE and
immunoblotted with the phospho-Ser-67 antibody. The amount of
phospho-Ser-67 inhibitor-1 was quantified by densitometry, and the data
were normalized to the values obtained with untreated slices. Data
represent means ± S.E. for 6-7 experiments. *, p < 0.01, Student's unpaired t test.
/
) mice. Phospho-Ser-67 levels
were assessed under basal conditions and in response to activation of
NMDA-type glutamate channels (Fig. 6B). In slices from wild
type mice the basal level of phospho-Ser-67 inhibitor-1 was
dramatically reduced by NMDA treatment for 5 min. The basal level was
higher in the PP-2B
/
mice. NMDA caused a
reduction in the level of phosphorylation, possibly due to a
Ca2+-dependent activation of the
-isoform of
PP-2B, which is expressed in these mice (56). However, detectable
levels of phospho-Ser-67 inhibitor-1 remained after NMDA treatment.
Total levels of inhibitor-1 were the same in control and treated slices
from both wild type and PP-2B
/
mice.
View larger version (26K):
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Fig. 7.
Comparison of PP-1 inhibition by dephospho-,
phospho-Thr-35, and phospho-Ser-67 inhibitor-1. Protein
phosphatase-1 inhibition was analyzed using dephospho- (open
circles), phospho-Thr-35 (open squares), phospho-Ser-67
(filled squares), or phospho-Thr-35/phospho-Ser-67
(open triangles) inhibitor-1. Quantified values represent
the average of 4-6 different experiments performed in duplicate.
View larger version (15K):
[in a new window]
Fig. 8.
Effect of phosphorylation of inhibitor-1 at
Ser-67 on the ability of PKA to phosphorylate inhibitor-1.
Lineweaver-Burk analysis of PKA phosphorylation of dephospho-
(open squares) and phospho-Ser-67 (filled
circles) inhibitor-1. Values represent the average of four
experiments using duplicate samples.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice. The residual effects of NMDA on Ser-67 in
PP-2B
/
may be attributed to the activity
of the
-isoform of PP-2B (56). Thus, a variety of data suggest that
both PP-2A and PP-2B may dephosphorylate phospho-Ser-67 inhibitor-1
under basal conditions, but PP-2B may function as the predominant
phosphatase in the presence of elevated Ca2+ levels.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Shirish Shenolikar, Duke University
Medical Center, for the pUC120 inhibitor-1 subclone; Alan R. Saltiel
and David T. Dudely, Parke-Davis Pharmaceutical Research Division, for
the GST-ERK1 and GST-MEK bacterial expression vectors and PD 98059; J. G. Seidman, Harvard Medical School for the
PP-2B/
mice; Laurent Meijer, Center
National de la Recherché Scientifique for roscovitine and sea
star Cdk1; and Steven L. Pelech, University of British Columbia for sea
star Cdk1 and activated MAPK. We acknowledge the technical assistance
of Jean Whitesell at Cocalico Biologicals, Inc., and Joseph Fernandez
of the Rockefeller University Protein/DNA Technology Center.
![]() |
FOOTNOTES |
---|
* This work was supported by a National Research Service award (to J. B.) and by the National Institute of Mental Health and the National Institute of Drug Abuse (to G. L. S., A. C. N., and P. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This paper is dedicated to the memory of George Kuzmycs (1916-1996) whose contribution to the generation of polyclonal antibodies used in our laboratory has been and will continue to be an invaluable resource.
§ To whom correspondence should be addressed. E-mail: bibbj@rockvax.rockefeller.edu.
Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M007197200
2 T. Saito, R. Onuki, G. Kusakawa, K. Ishiguro, T. Kishimoto, and S. Hisanaga, manuscript in preparation.
3 J. Bibb, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PKA, cAMP-dependent protein kinase; MOPS, 4-morpholinepropanesulfonic acid; PP, protein phosphatase; MAP, mitogen-activated protein; MAPK, MAP kinase; PCR, polymerase chain reaction; NMDA, N-methyl-D-aspartic acid; GST, glutathione S-transferase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride; HPLC, high pressure liquid chromatography.
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