From the Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021
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ABSTRACT |
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Several recent studies have shown that
Ca2+/calmodulin-dependent protein kinase I
(CaMKI) is phosphorylated and activated by a protein kinase (CaMKK)
that is itself subject to regulation by Ca2+/calmodulin. In
the present study, we demonstrate that this enzyme cascade is regulated
by cAMP-mediated activation of cAMP-dependent protein
kinase (PKA). In vitro, CaMKK is phosphorylated by PKA and
this is associated with inhibition of enzyme activity. The major site
of phosphorylation is threonine 108, although additional sites are
phosphorylated with lower efficiency. In vitro, CaMKK is
also phosphorylated by CaMKI at the same sites as PKA, suggesting that
this regulatory phosphorylation might play a role as a
negative-feedback mechanism. In intact PC12 cells, activation of PKA
with forskolin resulted in a rapid inhibition of both CaMKK and CaMKI
activity. In hippocampal slices CaMKK was phosphorylated under basal
conditions, and activation of PKA led to an increase in
phosphorylation. Two-dimensional phosphopeptide mapping indicated
that activation of PKA led to increased phosphorylation of multiple
sites including threonine 108. These results indicate that in
vitro and in intact cells the CaMKK/CaMKI cascade is subject to
inhibition by PKA-mediated phosphorylation of CaMKK. The
phosphorylation and inhibition of CaMKK by PKA is likely to be involved
in modulating the balance between cAMP- and
Ca2+-dependent signal transduction pathways.
Many of the intracellular actions of Ca2+ in
eukaryotic cells are mediated by activation of the family of
Ca2+/calmodulin-dependent protein kinases
(CaMKs),1 that include the
specific enzymes: myosin light chain kinase, phosphorylase kinase, and
the multifunctional enzymes, CaMKI, CaMKII, and CaMKIV (1-7).
Elongation factor 2 kinase, originally thought to represent a member of
this family now appears to be related to a distinct class of protein
kinases (8-10). CaMKI was initially identified in brain, but
subsequently the enzyme was found to have widespread tissue and
cellular distribution (11, 12). More recently several cDNAs have
been isolated that appear to be isoforms of CaMKI derived from distinct
genes (13). CaMKI was originally purified from brain based on its
ability to phosphorylate the synaptic vesicle protein synapsin I at
site 1 (14), and physiological substrates for CaMKI include synapsin I
and synapsin II (14, 15). In vitro, CaMKI has also been
found to phosphorylate cAMP response element-binding protein (16), and
CF-2, a portion of the R-domain of the cystic fibrosis transmembrane
conductance regulator (17). A potential role for CaMKI in the
phosphorylation and regulation of the transcription factor, ATF-1, has
also been suggested (18). Notably, for all of the well characterized
substrates, CaMKI and cAMP-dependent protein kinase (PKA)
phosphorylate the same residue, although CaMKI phosphorylates only a
subset of PKA substrates, indicating differences in the recognition of
substrates by the two enzymes.
CaMKI, myosin light chain kinase, CaMKII, and CaMKIV are activated upon
binding of Ca2+/CaM to a COOH-terminal regulatory domain of
the enzyme, which in turn leads to removal of a short
pseudosubstrate-like autoinhibitory region from the active site of the
enzyme (7, 19-26). In addition, several recent studies have indicated
that in a manner analogous to the MAP kinases and
cyclin-dependent protein kinases, CaMKI and CaMKIV
activities are strongly dependent on phosphorylation by a highly
specific CaMKI kinase (CaMKK) at an equivalent threonine (Thr177 in CaMKI) in the activation loops of both kinases
(26-36). Two isoforms of CaMKK have been identified that appear to
recognize both CaMKI and CaMKIV (36, 37). However, unlike
autophosphorylated CaM kinase II (4), phosphorylated CaMKI remains
fully dependent on Ca2+/CaM for activity. In contrast to
CaMKI and CaMKIV, CaMKII, myosin light chain kinase, and phosphorylase
kinase are not regulated by phosphorylation within the activation loop.
The precise physiological consequences of linking two CaMKs as part of
an enzyme cascade remains to be clarified. However, this is likely to
contribute to amplification of the Ca2+ signal since CaMKK
appears to be more sensitive to activation by Ca2+/CaM than
CaMKI (26). In addition, by analogy to kinase cascades upstream of the
MAP kinases, the CaMK cascade may serve to integrate information from
other signal transduction pathways. In the present study, we report
that both in vitro and in intact cells the CaMKK/CaMKI cascade is subject to inhibition by PKA-mediated phosphorylation of
CaMKK. The phosphorylation and inhibition of CaMKK by PKA suggests a
novel mechanism to modulate the balance between cAMP- and
Ca2+-dependent signal transduction pathways in
eukaryotic cells. CaMKK is also phosphorylated by CaMKI, reflecting the
overlapping substrate specificities of CaMKI and PKA, and suggesting
the possibility of a classical negative feedback mechanism.
Materials--
ATP, Nonidet P-40, dithiothreitol,
2-mercaptoethanol, EDTA, EGTA, Tris, Coomassie Brilliant Blue R-250,
SDS, and bovine serum albumin were from Sigma. HEPES and
phenylmethylsulfonyl fluoride were obtained from Calbiochem. Nerve
growth factor (NGF) was from Life Technologies, Inc. Forskolin, H89,
and PDBu were obtained from Alexis Laboratories.
[ Synthesis of Peptides--
Peptide YLRRRLSDSNF-amide
(corresponding to residues 3-13 of synapsin I peptide) and PKI
fragment-(5-24) were synthesized by the Keck Foundation Biopolymer
Facility at Yale University. Peptides were purified by preparative
reversed-phase high performance liquid chromatography, were >95% pure
as analyzed by high performance liquid chromatography, and had the
expected amino acid composition and mass spectra.
Immunological Reagents--
Antibody CC76 is a polyclonal
antiserum prepared by injection of expressed rat CaMKI into rabbits.
Antibody CC135 is a polyclonal antiserum prepared in rabbits using a
synthetic peptide corresponding to residues 486-505 of CaMKK Site-directed Mutagenesis of CaMKK and Preparation of Truncation
Mutants--
CaMKK
Site-directed mutagenesis of full-length CaMKK Cell Culture--
PC12 cells were incubated in Dulbecco's
modified Eagle's medium plus 1% horse serum 18-24 h prior to drug or
KCl treatment. Replicate cultures (1 × 106 cells)
were treated with agonists for the indicated times and at the
concentrations indicated in the figure legends. Forskolin (10 mM) and H89 (1 mM) were dissolved in
Me2SO. Depolarization of intact cells was accomplished by
raising extracellular K+ to 40 mM by addition
of an appropriate volume of 150 mM KCl.
Immunoblotting and 125I-CaM Overlay--
Proteins
(100 µg) were separated by SDS-PAGE and transferred to nitrocellulose
filters as described (40). Immune complexes were detected using an
enhanced chemiluminescence kit (Amersham). For the 125I-CaM
overlay, nitrocellulose filters were incubated with a blocking buffer
containing: 50 mM Tris-HCl (pH 7.5), 0.1 M
NaCl, 1 mg/ml bovine serum albumin, 0.05% Tween 20, and 1 mM CaCl2. 125I-CaM (Amersham) was
added at a final concentration of 0.5 µg/ml in the blocking buffer
and the filter incubated for 30 min. The filter was then washed in the
blocking buffer for 1 h, and then analyzed using a PhosphorImager
(Molecular Dynamics).
Phosphorylation Assays--
Phosphorylation assays were carried
out for different amounts time at 30 °C. The standard reaction
mixture (100 µl) contained 50 mM HEPES (pH 7.5), 10 mM magnesium acetate, 1 mM EGTA, 5 mM dithiothreitol, 100 µM
[ Immunoprecipitation--
PC12 cells were rinsed with
phosphate-buffered saline, and lysates were prepared as described
previously (41). CaMKI and CaMKK 32P Labeling of Brain Slices--
A Mcllwain tissue
chopper was used to prepare 400-µm thick slices from freshly
dissected rat hippocampus. Dissection, cutting, and preincubation (for
30 min) were carried out in a modified Krebs-bicarbonate buffer
(phosphate free) of the following composition: 124 mM NaCl,
4 mM KCl, 25 mM NaHCO3, 1.5 mM MgSO4, 15 µM
CaCl2, 10 mM D-glucose, which was
saturated with 95% O2, 5% CO2 and adjusted to
pH 7.4. At the end of the preincubation period, the buffer was removed
and replaced with prelabeling buffer of the following composition: 124 mM NaCl, 4 mM KCl, 25 mM
NaHCO3, 1.5 mM MgSO4, 1.5 mM CaCl2, 10 mM
D-glucose, 1 mCi of [32P]orthophosphate (NEN
Life Science Products Inc.). Slices were prelabeled for 60 min before
application of drugs. All compounds were diluted in prelabeling buffer.
Forskolin was dissolved in Me2SO, then diluted into
prelabeling buffer resulting in a final concentration of 0.5%
Me2SO in the slice medium. Incubations were stopped by
rapidly removing the medium with a Pasteur pipette and immersing the
tubes in liquid nitrogen. Slices were homogenized using IP buffer and
immunoprecipitation was performed as described above. Proteins were
analyzed by SDS-PAGE (10% polyacrylamide), and gel pieces containing
CaMKK were identified by alignment with the autradiogram and excised.
After measuring 32P incorporation by scintillation counting
(Cerenkov), gel pieces from triplicate samples were pooled and
processed for two-dimensional phosphopeptide mapping.
Two-dimensional Phosphopeptide Mapping and Phosphoamino Acid
Analysis--
Gel pieces containing 32P-labeled CaMKK were
excised from dried SDS-polyacrylamide gels, and two-dimensional
phosphopeptide mapping and phosphoamino acid analysis were performed as
described (14).
Phosphorylation of CaMKK by CaMKI and PKA--
When incubated
together, CaMKK-WT phosphorylated CaMKI-WT in a
Ca2+/CaM-dependent manner (Fig.
1A). A low but significant
level of phosphorylation of CaMKK-WT was also observed in the presence of Ca2+/CaM (Fig. 1A). In contrast, CaMKK-WT,
when incubated alone, was not phosphorylated under any condition (Fig.
1B). This raised the possibility that the phosphorylation of
CaMKK-WT was catalyzed by activated CaMKI-WT. To test this idea
further, CaMKI-293, a constitutively active form of CaMKI, and
CaMKI-299, an autoinhibited form of CaMKI (23, 30), were incubated with
CaMKK-WT (Fig. 1A). With CaMKI-293, phosphorylation of
CaMKK-WT was observed in both the absence and presence of
Ca2+/CaM. However, with CaMKI-299 no phosphorylation of
CaMKK-WT or CaMKI-299 was detected. The failure to observe
phosphorylation of CaMKI-299 was expected since CaMKI has to be in an
active form to act as a substrate for CaMKK (26, 30). Moreover, the
failure to observe phosphorylation of CaMKK when incubated alone or in the presence of CaMKI-299 indicated that active forms of CaMKI catalyzed phosphorylation of CaMKK.
Notably, all substrates for CaMKI identified to date are also
phosphorylated by PKA, and studies of phosphorylation of peptide analogs have indicated that the two enzymes display closely overlapping specificities (42).2 We
reasoned therefore that PKA might also phosphorylate CaMKK and that the
functional effects of any phosphorylation would be easier to analyze.
To investigate this, CaMKK-WT, PKA, and Ca2+/CaM were
incubated togther in various combinations. PKA phosphorylated CaMKK-WT
in either the absence or presence of Ca2+/CaM (Fig.
1B). At short incubation times (<20-30 min)
phosphorylation of CaMKK by either CaMKI or PKA was found to be
predominantly on threonine although phosphorylation on serine was also
observed (Fig. 2). At longer incubation
times (>60 min), phosphorylation of serine increased (data not shown).
In addition, two-dimensional tryptic phosphopeptide mapping indicated
that both CaMKI and PKA largely phosphorylated the same site(s) in
CaMKK (Fig. 2).
Phosphorylation of CaMKK by PKA Inhibits Enzyme Activity--
PKA
was incubated with CaMKK for various times using either
[32P]ATP (Fig.
3A), or non-radioactive ATP.
In the latter case, at each time point PKA activity was terminated by
addition of PKI fragment-(5-24), and the activity of CaMKK was
measured in a second assay using CaMKI as substrate (Fig.
3B). As measured by 32P incorporation,
phosphorylation of CaMKK proceeded in a time-dependent manner reaching a maximal stoichiometry of ~1.8 mol/mol after 60 min
(Fig. 3A and data not shown). The phosphorylation was
accompanied by inhibition of CaMKK activity (Fig. 3B). No
phosphorylation (data not shown) or inhibition of CaMKK was observed in
the absence of PKA (Fig. 3B).
Thr108 Is the Major Site in CaMKK Phosphorylated by PKA
in Vitro--
Analysis of the amino acid sequence of CaMKK indicated
the presence of three good consensus phosphorylation sites for PKA, at
Ser74 (RKFSL), Thr108 (RRPTI), and
Ser458 (RKRSF). Removal of the COOH terminus of CaMKK at
amino acid 433 (CaMKK-433) did not affect significantly phosphorylation
by PKA, or the inhibition of enzyme activity as measured by
phosphorylation of CaMKI-WT (Fig.
4A). This suggested that
Ser458 was not a major site of phosphorylation, and that
phosphorylation did not contribute to inhibition of CaMKK activity,
under the conditions used in this assay. Mutation of Thr108
to alanine (T108A) resulted in a large decrease in phosphorylation by
PKA (Fig. 4B). Additional mutation of Ser74 to
alanine (S74A) resulted in a further small reduction in
phosphorylation. These results indicated that Thr108 was
the major site of CaMKK phosphorylated by PKA in vitro.
However, a low level of phosphorylation of Ser74 was
likely. Moreover, a low level of phosphate was incorporated into
CaMKK-108M-505, indicating that phosphorylation of residues in the
catalytic or regulatory domain also occurred.
Ser458 is present within the CaM-binding domain of CaMKK
(26, 44). The results illustrated in Figs. 1 and 4B
indicated that under our assay conditions little phosphorylation of
Ser458 occurs, and that the presence of
Ca2+/CaM has no significant effect on phosphorylation of
CaMKK by PKA. However, phosphorylation of CaMKK with a concentration of PKA 50-fold higher than that used in the normal assay reduced binding
of Ca2+/CaM to CaMKK as measured by 125I-CaM
overlay (Fig. 4C).
Regulation of CaMKK and CaMKI by PKA in Intact Cells--
As a
first step in the analysis of the regulation of CaMKK and CaMKI in
intact cells, we characterized the tissue and cell distribution of
CaMKK
As shown in part in a previous study in PC12 cells (41), membrane
depolarization, but not treatment with NGF or PDBu, led to activation
of CaMKI (Fig. 6). Membrane depolarization,
NGF, and PDBu did not affect CaMKK activity. However, treatment with forskolin resulted in a decrease in both CaMKK and CaMKI activities to
approximately the same magnitude. The forskolin-dependent
inhibition of both CaMKK and CaMKI activities occurred rapidly, but
transiently, reaching a peak within 5 min, after which both activities
returned to an intermediate level (Fig.
7). Pretreatment with H89, a relatively specific inhibitor of PKA, significantly attenuated the effect of
forskolin on both CaMKK and CaMKI activities.
Phosphorylation of CaMKK in Hippocampal Slices--
The low level
of CaMKK in PC12 cells precluded an analysis of the phosphorylation of
CaMKK by PKA in situ. Therefore, we carried out studies in
rat hippocampus, the brain region that expresses the highest level of
CaMKK (see Fig. 5 above). Hippocampal slices were prelabeled with
32P, treated without or with forskolin, and CaMKK was
immunoprecipited (Fig. 8). A significant
level of phosphorylation of CaMKK was measured under basal conditions
and this was increased 2.0 ± 0.3-fold (average of three
experiments) by forskolin. Under the same conditions used for analysis
of phosphorylation of CaMKK, forskolin treatment of hippocampal slices
resulted in ~30% decrease in CaMKK activity (data not shown).
Two-dimensional peptide mapping was used to compare the site(s)
phosphorylated in CaMKK in vitro and in situ
(Fig. 8). In vitro, CaMKK-WT was phosphorylated by PKA at
multiple sites (see also Fig. 2). As revealed by site-directed
mutagenesis, two major peptides and a minor peptide (numbered 1, 2, and
3) were not detected in the peptide map obtained from CaMKK-T108A. In
addition, following deletion of the NH2-terminal 108 amino
acids, only peptide 1' was phosphorylated by PKA. These results
suggested that peptides 1-3 were derived by alternative tryptic
cleavage of amino acids flanking Thr108 in CaMKK, peptides
2', 4, and 5 were derived from the NH2-terminal 108 amino
acids of CaMKK, and peptide 1' was derived from the COOH-terminal 397 amino acids of CaMKK. In hippocampal slices incubated under basal
conditions, CaMKK was phosphorylated at multiple sites, several of
which appeared to be the same as the minor sites phosphorylated by PKA
in vitro (for example, peptides 1', 4, and 5). Incubation
with forskolin not only increased phosphorylation of sites found in the
control condition, but significantly increased phosphorylation of
peptide 1 and either peptides 2 or 2'. In general, the pattern obtained
from CaMKK-WT phosphorylated in vitro with PKA, and that
obtained from CaMKK phosphorylated in hippocampal slices following
incubation with forskolin, were the same. Based on these results, we
conclude that in situ, stimulation of PKA leads to an
increase in the phosphorylation of several sites in CaMKK, one of which
is Thr108.
In the present study, we have shown that the CaMKK/CaMKI cascade
is subject to regulation via phosphorylation and inactivation of CaMKK
by PKA. In in vitro studies, PKA was found to efficiently phosphorylate Thr108 of CaMKK During the completion of this work, Wayman et al. (46)
published results from studies of the effect of PKA on the regulation of the CaMKK/CaMKIV cascade. Several of the general conclusions were
similar to those obtained in the present study. However, it was
concluded that phosphorylation of both Thr108 and
Ser458 by PKA contributed equally to inhibition of CaMKK,
and that as a consequence of phosphorylation of Ser458,
suppression of Ca2+/CaM binding contributed to inhibition
of enzyme activity. The reasons for the differences in the conclusions
are not immediately clear, but notably Wayman et al. (46)
demonstrated at least in some of their studies that phosphorylation of
Ser458 occurred only following prolonged incubation of
CaMKK with PKA. Thus, while phosphorylation of Ser458 or
other serine(s) by PKA may contribute to the inhibition observed in vitro and in intact cells, our results suggest that
Thr108 is likely to be more efficiently phosphorylated than
Ser458, and that phosphorylation of Thr108
represents the major mechanism of regulation of the enzyme.
There are a number of potential mechanisms by which phosphorylation of
Thr108 may lead to inhibition of CaMKK. Recent studies of
several protein kinases have indicated that the NH2
terminus is important for regulation of kinase activity (47-51), and
phosphorylation of Thr108 may confer an autoinhibitory
function. In the catalytic subunit of PKA, the catalytic core of the
enzyme is preceded by a 39-residue NH2-terminal amphipathic
The results obtained using PC12 cells indicate that activation of CaMKK
is primarily dependent on increases in intracellular Ca2+.
In contrast, and in agreement with previous studies (41), phosphorylation of CaMKI by CaMKK leads to a stable activated form of
the enzyme that can be assayed following immunoprecipitation in the
presence of protein phosphatase inhibitors. Notably, forskolin treatment led to a significant decrease in CaMKI activity, that presumably reflects a decrease in the level of basally activated, phosphorylated CaMKI. Moreover, the effect of forskolin on either CaMKK
or CaMKI activity was somewhat transient, most likely resulting from
dephosphorylation of CaMKK and CaMKI by cellular protein phosphatases.
Our preliminary studies suggest that phospho-CaMKI is a good substrate
in vitro for protein phosphatase
2A,3 but the identity of the
phosphatase(s) that dephosphorylate(s) CaMKK or CaMKI in intact cells
is not known.
A large number of studies have indicated that there is an intricate
inter-relationship between Ca2+/CaM-dependent
and cAMP-dependent signaling pathways in cells. For
example, CaM-dependent adenylyl cyclases, as well as
CaM-dependent cAMP phosphodiesterases play a critical role
in the generation and degradation of cAMP (55, 56). The
CaM-dependent protein phosphatase, calcineurin (protein
phosphatase 2B), antagonizes the actions of cAMP through the
dephosphorylation of various substrates for PKA (57, 58).
Interestingly, recent studies have shown that PKA and calcineurin can
exist in a multienzyme complex through their interaction with the
PKA-anchoring protein, AKAP-79 (59). PKA is also able to phosphorylate
and activate phosphorylase kinase (60) and to phosphorylate elongation
factor-2 kinase, leading to the generation of a CaM-independent enzyme
activity (61, 62). Therefore, phosphorylation of CaMKK CaMKI phosphorylates a subset of proteins that are also phosphorylated
on a common site by PKA. For example, CaMKI and PKA phosphorylate
Ser9 of the neurotransmitter vesicle-associated proteins,
synapsin I and II, and CaMKIV and PKA phosphorylate the transcription
factor cAMP response element-binding protein at Ser133 (16,
63-65). Given that PKA and either CaMKI or CaMKIV phosphorylate common
sites in these substrates, the precise physiological consequences of
regulation of either CaMKI or CaMKIV by PKA remains to be elucidated. On first examination, this regulatory mechanism appears somewhat paradoxical, however, any changes in the phosphorylation of downstream targets for CaMKI or CaMKIV as a result of phosphorylation of CaMKK by
PKA would reflect the relative subcellular distribution of the enzymes,
and more importantly reflect the integration of the activities of
protein phosphatases toward the various phosphorylated species
involved. Regulation of CaMKK by PKA phosphorylation may also be more
relevant to other downstream targets. For example, recent studies have
indicated that CaMKIV can stimulate various MAP kinase cascades,
although the exact mechanism is not yet established (66).
Phosphorylation and inhibition of CaMKK by PKA could therefore contribute to the down-regulation of MAP kinase observed in several cell types (67).
These studies of the regulation of CaMKK by PKA resulted from an
initial observation that CaMKK was phosphorylated by CaMKI. Peptide
mapping studies and phosphoamino acid analysis indicated that
Thr108 was the major site phosphorylated by both PKA and
CaMKI. Other studies have also suggested that CaMKK is phosphorylated
by CaMKIV in vitro (26). These results raise the possibility
that phosphorylation of CaMKK by CaMKI, CaMKIV, or both, may represent
a classical feedback mechanism to control the CaMKK cascade. In this
respect, the situation would be analogous to that observed for the
analog of MEK, STE7, found in Saccharomyces cerevisiae (43,
68). Our present studies did not address this possibility
experimentally. However, preliminary studies have shown in hippocampal
slices that depolarization, or incubation with the Ca2+
ionophore, ionomycin, increased threonine phosphorylation of CaMKK.3 Future studies will hopefully establish whether
phosphorylation and inhibition of CaMKK subserves such a feedback mechanism.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP was from NEN Life Science Products Inc.
TPCK-trypsin was from Worthington. Phosphocellulose P-81 paper was from
Whatman. Nitrocellulose membranes (0.2 µm pore size) were from
Schleicher & Schuell. X-ray film was from DuPont. Cellulose thin layer
chromatogram sheets were from Eastman Kodak. Leupeptin was from
Chemicon. 125I-CaM was from Amersham. CaM was purified from
rabbit brain as described (38). The catalytic subunit of PKA was
purified from bovine heart as described (39). Recombinant
GST-CaMKI-WT, -CaMKI-293, -CaMKI-299, CaMKK-WT, and
CaMKK-433 were produced in Escherichia coli and
purified as described previously (26). Serum from nonimmunized rabbits
(nonimmune serum) was obtained from Pierce. Protein A-Sepharose was
purchased from Pharmacia Biotech Inc. Rats were purchased from Charles
River Laboratory.
(26).
cDNA was prepared as described (26). The
following oligonucleotides were synthesized by OPERON: KK-108M-S
(5'-CCCGGATCCATGGAGTCCCACCATGTGGCC-3') and KK-433-AS
(5'-CCCGAATTCTCACACCTCCTCCTCAGTCACCTC-3'). Oligo KK-108M-S
corresponded to the coding strand and introduced an BamHI site. Oligo KK-433-AS corresponded to the complement
of the coding strand and introduced an EcoRI site. The
truncation mutants were amplified using appropriate primers from 10 ng
of CaMKK
cDNA using pfu polymerase (Stratagene). The amplified
DNA was purified and digested with BamHI and
EcoRI. The fragments were subcloned into the BaMHI and
EcoRI site of pGEX-2T (Pharmacia) using a ligation kit (Takara).
cDNA was carried
out with a Quick Change Site-directed Mutagenesis Kit (Stratagene) according to the manufacturers protocol using pGEX-2T/CaMKK-WT as a template and the following primers: KK-T108A-S
(5'-TGGCGGAGACCCGCCATCGAGTCCCAC-3'), KK-T108A-AS
(5'-GTGGGACTCGATGGCGGGTCTCCGCCG-3'), or pGEX-KK-T108A as a template and
the following primers: KK-S74-S (5'-GCTAGAAAGTTCGCCCTGCAGGAAAGA-3') and
KK-S74A-AS (5'-TCTTTCCTGCAGGGCGAACTTTCTAGC-3'). Polymerase chain
reactions were performed under the following conditions: 16 cycles at
95 °C for 1 min, 55 °C for 1 min, and 68 °C for 14 min. The
reaction mixtures were digested with DpnI for 1 h, then 1 µl of digested samples were transfected into XL1 blue. All
mutations were confirmed by DNA sequencing. GST fusion proteins were
prepared as described (26).
-32P]ATP (specific activity, 2-5 × 102 cpm/pmol). Reaction mixtures with CaMKK contained 1 µM CaM, 1.5 mM Ca2+, CaMKI wild
type or mutants. The final concentrations of kinases in the reaction
mixtures were as follows: PKA, 0.2-10 µg/ml; CaMKI, 10 µg/ml;
CaMKK, 1-10 µg/ml. After 2 min of preincubation at 30 °C,
reactions were initiated by addition of [
-32P]ATP or
nonradioactive ATP. Reactions were terminated by the addition of 100 µl of SDS sample buffer (final concentrations, 1% SDS, 60 mM Tris-HCl (pH 6.8), 5% (v/v) glycerol, 0.2 M
-mercaptoethanol). Samples were subjected to SDS-PAGE (10%
polyacrylamide), gels were stained with Coomassie Brilliant Blue,
destained, dried, and subjected to autoradiography or analysis using a PhosphorImager.
were immunoprecipitated with CC76
or CC135, respectively. Cultures were rinsed in ice-cold
phosphate-buffered saline and lysed in immunoprecipitation (IP) buffer
(1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl,
pH 8.0, 25 mM sodium fluoride, 5 mM EDTA, 5 mM EGTA, 2 mM phenylmethylsulfonyl fluoride,
aprotinin (60 kallikrein inactivating units/ml), 100 nM
okadaic acid, and 1 mM sodium orthovanadate). Lysates were
allowed to stand on ice for 10 min. After preclearing the lysates with
6 mg of protein A-Sepharose, 1 mg of lysate protein was incubated for
1 h with 1 µl of CC76, 10 µl of CC135 or nonimmune serum.
Immune complexes were precipitated with 3 mg of protein A-Sepharose,
washed twice with 1 ml of IP buffer, and then twice with 1 ml of 50 mM Tris-HCl (pH 7.6). Immune complexes were immediately resuspended in an assay mixture consisting of: 50 mM Tris
(pH 7.6), 0.5 mM dithiothreitol, 0.5 mg/ml bovine serum
albumin, 1 mM CaCl2, 1 µM CaM, 10 mM MgCl2, 200 µM
[32P]ATP (100 cpm/pmol). For CaMKI activity, 50 µM synapsin site 1 peptide was used as substrate. For
CaMKK activity, GST-CaMKI or GST-CaMKI-293 (10 µg/ml) were used as
substrate. For CaMKI, the assay mixtures were incubated at 30 °C for
5 min, at which time 20-µl aliquots were removed and 32P
incorporation into synapsin site 1 peptide was quantified by adsorption
of the peptide to phosphocellulose P81 papers and scintillation counting. For CaMKK, the assay mixtures were incubated at 30 °C for
20 min. Reactions were stopped by adding SDS sample buffer, samples
were analyzed by SDS-PAGE and 32P incorporation into
GST-CaMKI was quantified by PhosphorImager analysis. Assays were linear
with time and protein concentration.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (38K):
[in a new window]
Fig. 1.
Phosphorylation of CaMKK by CaMKI and
PKA. A, CaMKK (1 µg/ml) was incubated with CaMKI-WT,
-CaMKI-293, or -CaMKI-299 (10 µg/ml) in the absence or presence of
Ca2+/CaM for 10 min at 30 °C as described under
"Experimental Procedures." B, CaMKK-WT (1 µg/ml) was
incubated in the absence or presence of PKA (0.2 µg/ml), in the
absence or presence of Ca2+/CaM for 20 min. For both
A and B, samples were analyzed by SDS-PAGE and
autoradiograms of the dried gels are shown.
View larger version (70K):
[in a new window]
Fig. 2.
Peptide mapping and phosphoamino acid
analysis of CaMKK phosphorylated by CaMKI or PKA. CaMKK was
phosphorylated with PKA or CaMKI as described in the legend to Fig. 1.
CaMKK was digested with TPCK-trypsin and digests were separated on thin
layer cellulose plates by electrophoresis in the first dimension and
chromatography in the second dimension. O, origin in
lower left. Upper panel, tryptic digest of CaMKK
phosphorylated by CaMKI. Lower panel, tryptic digest of
CaMKK phosphorylated by PKA. Right panels, aliquots of
tryptic digests were hydrolyzed with HCl and phosphoamino acids were
separated by electrophoresis. [32P]Phosphoamino acids
were visualized by autoradiography. The position of phosphotyrosine
(pY), phosphothreonine (pT), and phosphoserine
(pS) standards are indicated. Analysis of additional peptide
maps of CaMKK phosphorylated by PKA or CaMKI, and of CaMKK mutants (see
Fig. 8) suggested that peptides 1, 2, and 3 may be derived from
alternative tryptic digestion of residues surrounding a single
phosphorylation site.
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Fig. 3.
Phosphorylation and inactivation of CaMKK by
PKA. A, CaMKK (1 µg/ml) was incubated for various
times at 30 °C with PKA (0.2 µg/ml). Reactions were stopped by the
addition of SDS sample buffer, samples were analyzed by SDS-PAGE (10%
acrylamide) and autoradiography was performed. Lane a, CaMKK
alone incubated for 60 min in the presence of Ca2+ and CaM.
B, effect of phosphorylation on CaMKK activity. CaMKK was
incubated in the absence or presence of PKA as described in
A except that non-radioactive ATP was used. 50 µl of each
reaction mixture was diluted 1:2 with ice-cold 50 mM HEPES
(pH 7.5), 10 mM magnesium acetate, 1 mM EGTA, 5 mM dithiothreitol, 100 µM
[ -32P]ATP (specific activity, 2-5 × 102 cpm/pmol), 100 nM PKI, 1 µM
CaM, 1.5 mM CaCl2, and CaMKI-WT (10 µg/ml),
then incubated for 30 min at 30 °C. Reactions were terminated with
SDS sample buffer and samples were separated by SDS-PAGE and analyzed
using a PhosphorImager. Results are plotted as a % of the activity of
CaMKK in the absence of PKA at zero time. The data represent the mean
values of two independent experiments.
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Fig. 4.
Phosphorylation of CaMKK mutants by PKA.
A, CaMKI-WT (10 µg/ml) was incubated with CaMKK (1 µg/ml) or CaMKK-433 (1 µg/ml) in the absence or presence of PKA
(0.2 µg/ml) for 20 min at 30 °C with Ca2+/CaM.
Reactions were terminated by addition of SDS sample buffer and samples
were separated by SDS-PAGE and then analyzed using a PhosphorImager.
B, CaMKK-WT, CaMKK-433, CaMKK-T108A, CaMKK-T108A/S74A, and
CaMKK-108M-505 (all ~1 µg/ml) were incubated with PKA (0.2 µg/ml)
for 60 min. Samples were separated by SDS-PAGE and analyzed using a
PhosphorImager. 32P incorporation into the CaMKK mutants
were 100, 95, 15, 9, and 3% (% CaMKK-WT), respectively. Upper
panel, autoradiogram; lower panel, Coomassie Blue
stain. The results shown are representative of two different
experiments. C, CaMKK-WT was incubated in the absence of PKA
(0 min) or with PKA (60 min). In the result shown in the left
panel, 0.2 µg/ml PKA was used; in the result shown in the
right panel, 10 µg/ml PKA was used. Samples were separated
by SDS-PAGE, transferred to nitrocellulose, and then analyzed by
125I-CaM overlay and PhosphorImager.
using an antibody prepared against a synthetic peptide derived
from the COOH terminus of the enzyme. In all brain regions examined,
this antibody recognized a single band of ~66 kDa (Fig.
5A). CaMKK
expression was
highest in cortex and hippocampus, intermediate in striatum and nucleus
accumbens, and low in cerebellum. Very low levels of CaMKK
were
detected in non-neuronal tissues but only following longer exposure of
the immunoblot (data not shown). In cell lines, CaMKK was also
expressed at much lower levels than that found in brain tissue.
However, measurable amounts were found in PC12 cells, with other cell
lines expressing barely detectable amounts (Fig. 5B). Based
on these results, forebrain regions or PC12 cells appeared good
preparations for further analysis.
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Fig. 5.
Distribution of CaMKK in rat tissues
and cell lines. 100 µg of each sample was subjected to
immunoblotting. Molecular weights (in kDa) are shown on the
left of each panel. A, rat tissues;
Striat, striatum; Nuc Ac, nucleus accumbens;
Panc, pancreas; Salivery G, salivery gland.
B, cell lines. CHO, Chinese hamster ovary. The
autoradiogram shown in panel B was exposed ~5 times longer
than that shown in panel A. Based on these and other
results, we estimate that PC12 cells contained ~20% of the amount of
CaMKK found in hippocampus.
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Fig. 6.
Regulation of CaMKK and CaMKI activity in
PC12 cells. PC12 cells were treated with control solution, NGF (50 ng/ml), PDBu (2 µM), forskolin (5 µM), a
depolarizing concentration of KCl (40 mM), KCl plus NGF, or
KCl plus PDBu for 5 min. CaMKI and CaMKK activities were measured
following immunoprecipitation, using synapsin I peptide or CaMKI-293,
respectively. Upper panel, CaMKI activity results are
representative of three experiments, and standard deviations are shown.
Lower panel, CaMKK assay samples were separated by SDS-PAGE
and analyzed using a PhosphorImager. 32P incorporation into
CaMKI-293 for the samples was 100, 90, 98, 102, 47, 115, and 113% (% control), respectively.
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Fig. 7.
Time course of regulation of CaMKK and CaMKI
by PKA in PC12 cells. PC12 cells were treated with forskolin
(5 µM) for the indicated times in the absence or presence
of H89 (0.5 µM; preincubated for 20 min). CaMKI
(A) or CaMKK (B) activity was measured following
immunoprecipitation, using synapsin I peptide or CaMKI-WT,
respectively. Results are the average of three experiments, and
standard deviations are shown in A and B.
Representative autoradiograms showing CaMKK activities are shown in
C.
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Fig. 8.
Phosphorylation of CaMKK in hippocampal
slices and comparative tryptic phosphopeptide mapping. Lower
left panel, slices were metabolically labeled with
32P for 60 min and treated with control solution or
forskolin (10 µM) for 5 min. CaMKK was immunoprecipitated
and subjected to SDS-PAGE and PhosphorImager analysis. Upper
panels and lower right two panels, CaMKK-WT,
CaMKK-T108A, and CaMKK-108M-505 were phosphorylated by PKA as described
in the legend to Fig. 4 (not shown). Gel pieces containing
32P-labeled CaMKK phosphorylated in vitro or
phosphorylated in hippocampal slices were excised and subjected to
phosphopeptide mapping and autoradiography. Origin is lower
left. The numbering used follows that of Fig. 2.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, a consensus PKA site
(RRPTI) located at the NH2 terminus of the catalytic domain
of the enzyme. Phosphorylation of Thr108 was sufficient to
mediate the inhibition of enzyme activity. Under conditions where
Thr108 is the major site phosphorylated by PKA, CaMKK
activity was significantly inhibited. In addition, CaMKK-433, a
constitutively active form of the enzyme that does not contain the
autoinhibitory or CaM-binding domains, was also phosphorylated by PKA
and inhibited. However, direct assessment of the role of
Thr108 phosphorylation was precluded by the fact that
CaMKK-T108A was inactive when expressed in bacteria (data not shown).
In vitro, phosphorylation on serine was also found within
residues 109-505 of CaMKK, and a consensus PKA site at
Ser458 (RKRSF) may be a potential candidate.
Ser458 is present within the CaM-binding domain of CaMKK
(26, 36, 44), and autophosphorylation of CaMKII and CaMKIV, within or close to the CaM-binding domains, inhibits enzyme activity by influencing the binding of Ca2+/CaM (4, 45). Under our
standard assay conditions, we did not detect any effect of
Ca2+/CaM on the phosphorylation of CaMKK by PKA, or of
phosphorylation of CaMKK on binding of Ca2+/CaM. However,
phosphorylation with high concentrations of PKA resulted in a reduction
in binding of Ca2+/CaM, presumably as a result of a higher
stoichiometry of phosphorylation of Ser458. In intact
cells, CaMKK was basally phosphorylated, and stimulation of PKA
activity was associated with increased phosphorylation. Two-dimensional
peptide mapping suggested that Thr108 was one of several
sites phosphorylated by PKA. Therefore, the present studies support the
conclusion that phosphorylation of Thr108 leads to
inhibition of CaMKK in vitro and that this is likely to
contribute to the inhibition observed in intact cells.
-helical domain (the A helix) that is important for the stability of
the enzyme and for maintaining conformation at the active site (52,
53). Thr108 would be predicted to be at the junction of the
NH2-terminal and catalytic domains of CaMKK.
Phosphorylation by PKA may therefore modulate a stabilizing influence
of the NH2-terminal domain. Studies of the enzymes within
the various MAP kinase cascades have revealed that protein-protein
interactions away from the active site of the upstream kinase, and away
from the activation loop of the downstream kinase, are important for
directing the specificity of a given cascade (54), and for mediating
high affinity interactions (49, 50). These interactions have been found
to be close to the NH2 terminus of the catalytic domain
(54), or within NH2-terminal extensions of the particular
kinases (49, 50). Thus rather than directly inhibiting CaMKK activity,
phosphorylation of Thr108 of CaMKK by PKA may interfere
with its ability to interact with and/or to phosphorylate CaMKI or
CaMKIV.
by PKA
represents a new mechanism that adds to our understanding of the
interaction of Ca2+ and cAMP signaling mechanisms.
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ACKNOWLEDGEMENTS |
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We thank Akinori Nishi and Gretchen Snyder for help with the preparation of hippocampal slices. We also thank Gloria Bertuzzi for excellent technical assistance.
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed: Laboratory of
Molecular and Cellular Neuroscience, The Rockefeller University, 1230 York Ave., New York, NY 10021. Tel.: 212-327-8871; Fax: 212-327-7888; E-mail: nairn{at}rockvax.rockefeller.edu.
2 T. Karasawa, A. C. Nairn, P. Huynh, C. Koenigsberger, C. Wiedemann, J. I. Elliott, A. J. Czernik, and M. R. Picciotto, submitted for publication.
3 M. Matsushita and A. C. Nairn, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: CaM, calmodulin; CaMKI, Ca2+/CaM-dependent protein kinase I; CaMKIV, Ca2+/CaM-dependent protein kinase IV; CaMKK, Ca2+/CaM-dependent protein kinase kinase; PKA, cAMP-dependent protein kinase; GST, glutathione S-transferase; MAP kinase, mitogen-activated protein kinase; NGF, nerve growth factor; PDBu, phorbol 12-dibutyrate 13-acetate; IP, immunoprecipitation; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone.
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REFERENCES |
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