From the
Previous studies with synthetic peptides indicate that residues
290-309, corresponding to the calmodulin (CaM)-binding domain of
Ca
Ca
Like many other protein kinases, CaM kinase II
is regulated by an autoinhibitory mechanism. In the absence of
Ca
For several protein kinases, the autoinhibitory
mechanism has been suggested to be due to interaction of a
pseudosubstrate sequence in the regulatory domain with the active site
of the enzyme
(5, 6, 7, 8) . A
pseudosubstrate type of inhibition has also been proposed for CaM
kinase II
(3, 9) , and in our molecular model of CaM
kinase II
(4) , Arg
Mutagenesis was carried out by
using Sculptor
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
Arg
When we subjected the mutant kinases to
Ca
CaM binding motifs of several
Ca
The unaltered
Ca
Changing a
pseudosubstrate autoinhibitory sequence to a substrate consensus
sequence would be expected to enhance intrasubunit autophosphorylation
in the absence of Ca
The mutations
that we studied were also within the CaM-binding domain, and many of
the mutants (Fig. 1C, Fig. 2, and )
were altered in their abilities to bind to and be activated by
Ca
Hydrophobic residues
Leu
Although several of the mutants in this study had altered CaM
binding properties, none of the mutants demonstrated enhanced
Ca
In summary, the observation
that Ser
Specific activities
were measured for total kinase activities in the presence of 12
µM CaM, 0.5 mM CaCl
Wild-type and K300S mutant enzymes (1 µM) were
incubated for 15 min at 5 °C in 50 mM HEPES buffer, pH
7.5, 10 mM magnesium acetate, 0.5 mM CaCl
/CaM-dependent protein kinase II interact with the
catalytic core of the enzyme as a pseudosubstrate (Colbran, R. J.,
Smith, M. K., Schworer, C. M., Fong, Y. L., and Soderling, T. R.(1989)
J. Biol. Chem. 264, 4800-4804). In the present study, we
attempted to locate the pseudosubstrate motif by generation or removal
of potential substrate recognition sequences (RXXS/T) at
selected positions using site-directed mutagenesis. Based on previous
results, Arg
, Thr
, and Ser
were selected as key residues. Single mutations such as N294S,
K300S, A302R, A309R, and R311A were expressed, purified, and
characterized. Several of the mutants exhibited decreased binding of
and activation by CaM, not surprising since the mutations were within
the CaM-binding domain. None of the mutants exhibited enhanced
Ca
-independent kinase activity toward exogenous
substrate, but the K300S and N294S mutants showed a significant
enhancement in the rate and stoichiometry of
P
incorporation during Ca
-independent
autophosphorylation. Using two-dimensional peptide mapping and
phosphoamino acid analyses, enhanced phosphorylation of the introduced
Ser residue was demonstrated in the K300S mutant but not in the N294S
mutant. This specific Ca
-independent
autophosphorylation of Ser
is consistent with the
hypothesis that Arg
may occupy the P(-3) position
in a pseudosubstrate autoinhibitory interaction with the catalytic core
in the nonactivated state of the kinase.
/CaM-dependent protein kinase II (CaM kinase
II)
(
)
is a multifunctional serine/threonine
kinase known to be involved in a variety of cellular functions
(reviewed in Refs. 1 and 2). The enzyme is present in most tissues but
is particularly abundant in brain. The adult rat brain kinase is an
oligomeric enzyme composed of 10-12 subunits (50-60 kDa
each) of several isoforms in varying molar ratios. All isoforms share
at their NH
termini a highly conserved catalytic domain
characteristic of serine/threonine kinases in general. The central
portion of each subunit contains the regulatory domain comprised of an
autoinhibitory sequence overlapping a CaM-binding region, and the COOH
terminus contains an association domain, which is important in
holoenzyme formation.
/CaM, low basal kinase activity is due to
interaction of amino acid residues within the regulatory domain with
the catalytic region, thereby blocking access of exogenous protein
substrates and ATP. Binding of Ca
/CaM to the
CaM-binding motif (residues 293-310 in the
subunit) removes
the regulatory domain from the catalytic domain, which converts the
kinase to its fully active form. Several recent studies
(3, 4) have addressed the mechanisms of autoinhibition and
regulation of CaM kinase II. For example, studies with synthetic
peptides corresponding to the regulatory domain of
CaM kinase II
suggest that residues between 281 and 289 interact with the ATP-binding
motif, whereas residues between 290 and 309 interact with the
protein/peptide-binding elements of the catalytic domain
(3) .
Thus, a bisubstrate-directed autoinhibitory mechanism was indicated,
and we have recently proposed a molecular model for
CaM kinase
II, illustrating a possible interaction of the bisubstrate
autoinhibitory domain with the bilobal catalytic domain of the
enzyme
(4) .
was proposed to occupy the
P(-3) position of a putative pseudosubstrate sequence. In the
present study, we have investigated the pseudosubstrate mechanism by
generating or removing substrate consensus sequences (RXXS/T)
at several potential sites in
CaM kinase II through site-specific
mutagenesis. Since most of the mutations were introduced into the
CaM-binding region of the regulatory domain, this study also revealed
some important information about the structural basis of regulation by
CaM in the context of the holoenzyme structure of CaM kinase II.
Oligonucleotide-directed
Mutagenesis
The cDNA encoding the 50-kDa mouse brain CaM
kinase II subunit was inserted into the EcoRI site of
pVL1393 baculovirus transfer vector and digested with XbaI and
PstI. A 680-base pair SphI-PstI fragment
from the 3`-end of the mouse CaM kinase II cDNA (corresponding to the
regulatory and subunit association domains of the kinase) was inserted
into M13 mp19 to generate single-stranded DNA for mutagenesis. The
following oligonucleotides were designed for carrying out the mutations
mentioned (mutated bases underlined): N294S,
5`-TTCCTCCTGGCACTGAACTTCTTCAG-3`; K300S,
5`-GAGGATGGCTCCACTCAGTTTCCTCCT-3`; A302R,
5`-TGGTGAGGATTCTTCCCTTCAGTTTCC-3`; A309R,
5`-GAGAAGTTCCTGGTTCTCAGCATAGTGGT-3`; R311A,
5`-TCCGGAGAAGTTCGCGGTGGCCAGCATAGT-3`.
in vitro mutagenesis kit (Amersham
Corp.). The mutations were confirmed by sequencing the entire 650-base
pair fragment using the Sequenase kit from U. S. Biochemical Corp.
After mutation, the SphI-PstI fragments were ligated
to XbaI-SphI-digested 5`-fragment (corresponding to
catalytic region) of mouse CaM kinase-II
, and the total cDNA
molecule corresponding to the 50-kDa subunit was inserted into
XbaI- and PstI-digested pVL1393 for subsequent
expression in baculovirus/Sf9 expression system.
Expression and Purification of Mutant CaM Kinase II
Enzymes
The wild-type and mutant CaM kinase II enzymes were
expressed in the baculovirus/Sf9 cell expression system as previously
described
(10) with slight modifications. Recombinant viruses
were isolated via homologous recombination and plaque purification.
Isolated recombinant viruses were titered by plaque assay
method
(11) . About 150 ml of Sf9 cell culture at log phase
(1.2-1.6 10
cells/ml) were infected with
recombinant viruses at a multiplicity of infection of 3 in Corning
spinner flasks. Infected cells were harvested between 48-72 h,
frozen in liquid N
, and stored at -70 °C until
purification. The wild-type and mutant CaM kinase II enzymes were
purified by CaM-Sepharose affinity chromatography as described
before
(10) . Because of the poor affinity for CaM, mutants K300S
and A302R were eluted in the salt wash step. All the enzyme
preparations were dialyzed against 100 mM HEPES, pH 7.5, 10%
ethylene glycol, 0.5 mM EDTA, 5 mM dithiothreitol,
50% glycerol and stored at -20 °C.
Kinase Assays
CaM kinase II activities
were measured routinely at 30 °C for 1 min using standard assay
conditions containing 40 µM syntide-2, 0.5 mM
[-
P]ATP (600-2000 cpm/pmol), and
either 0.5 mM Ca
, 3 µM CaM
(total activity) or 1 mM EGTA
(Ca
-independent activity) in a final volume of 25
µl
(10) . All assays were initiated by the addition of
kinases diluted appropriately in dilution buffer (50 mM HEPES,
pH 7.5, 2 mg/ml bovine serum albumin, and 10% ethylene glycol).
P incorporation was determined by spotting 15-µl
aliquots onto Whatman P-81 phosphocellulose paper followed by washing
in 75 mM phosphoric acid as described
(12) .
Immunodetection of CaM Kinase II
The purified proteins were separated by 10%
SDS-PAGE and transferred to nitrocellulose membrane (Schleicher &
Schuell). Blocking, washing, and antibody dilution were done with 50
mM Tris-HCl buffer, pH 8.0, 150 mM NaCl, and 0.2%
Tween 20. The stock solution of 4-chloro-1-naphthol (3 mg/ml methanol)
was diluted 6-fold in 50 mM Tris-HCl buffer, pH 8.0,
containing 150 mM NaCl. Goat polyclonal anti-rat brain CaM
kinase II was diluted 500-fold. Anti-CaM kinase II was prepared by
Bethyl Laboratories (Montgomery, TX) and was purified by affinity
chromatography using CaM kinase II linked to Affi-Gel 15 (Bio-Rad). The
peroxidase-conjugated swine anti-goat IgG antibody (Boehringer
Mannheim) was diluted 1000-fold.
Subunits in
Western Blot
CaM Overlay
Wild-type and mutant CaM
kinase II enzymes were resolved on 10% SDS-PAGE and then
electrophoretically transferred onto nitrocellulose membrane. The
membrane was blocked with 150 mM NaCl, 50 mM Tris-HCl
(pH 7.4), 1 mM CaCl 0.2% Tween 20, and 5% nonfat
dry milk for 1 h at room temperature. Biotinylated CaM was added at a
final concentration of 0.4 µg/ml in the above buffer without milk
and incubated for 1 h. After washing with buffer alone, the membranes
were incubated with 1:500-diluted avidin D-conjugated horseradish
peroxidase (Vector Laboratories, Inc.)
The membranes were
washed extensively with 50 mM Tris-HCl, pH 7.4, 150
mM NaCl, and 1 mM CaCl
and developed with
4-chloro-1-naphthol and H
O
.
Two-dimensional Peptide Mapping
Mapping
of the phosphorylation sites was performed as previously described
(13) but with some modifications. The P-labeled
proteins or peptides were transferred to nitrocellulose membranes after
separation by 10% SDS-PAGE and 18% Tricine gel. Subsequent digestions
with CNBr or trypsin were carried out on the corresponding membrane
fragments containing the labeled protein or peptides as described
earlier
(13) .
Phosphoamino Acid Analysis
Aliquots of
the P-labeled tryptic peptides for two-dimensional peptide
mapping were dried by speed vac, resuspended in 100 µl of 6
N HCl at 110 °C for 1 h. Hydrolyzed samples were dried and
resuspended in pH 1.9 electrophoresis buffer (formic acid:glacial
acetic acid:water, 25:78:897, v/v/v). The
P-labeled
phosphoamino acids were separated by one-dimension electrophoresis in
pH 1.9 buffer for 1 h at 1.5 kV and detected by autoradiography. For
phosphoamino acid analysis of peptides from two-dimensional maps, the
corresponding spot was scraped off the cellulose plate and washed with
pH 1.9 buffer to elute the phosphopeptide
(14) . The
P-labeled phosphopeptide was hydrolyzed with 6 N
HCl and subjected to phosphoamino acid analysis as described above.
Other Materials and Methods
CaM was
purified from bovine brain according to the method of Gopalakrishna and
Anderson (15). Syntide-2 was synthesized and purified as previously
described (16). Protein concentration was determined by the Bio-Rad
protein binding assay using bovine serum albumin (Pierce) as standard.
Mutagenesis in the Pseudosubstrate Region of CaM
Kinase II
Previous studies with synthetic peptides showed
that residues 290-309 in the autoinhibitory domain of CaM kinase
II inhibited a catalytic fragment of CaM kinase II competitively with
substrate. This result suggests that residues 290-309 interact as
a pseudosubstrate with the catalytic core of the enzyme in the absence
of Ca/CaM
(3) . In the present study, we
attempted to locate the pseudosubstrate sequence by generating or
removing potential substrate consensus sequences (RXXS/T)
through site-specific mutagenesis of selected residues as shown below.
, Thr
, and Ser
,
indicated by the asterisks, were selected as key residues for
analyses. On the basis of substitution studies in synthetic
peptides
(9) , Arg
was suggested to be an important
determinant for interaction of the autoinhibitory domain peptide of CaM
kinase II with the protein-binding motif of the catalytic core, and it
has been modeled by our laboratory to occupy the P(-3) position
of a pseudosubstrate
(4) . Thr
and Ser
have been shown to be the major autophosphorylation sites in the
absence of Ca
/CaM
(17, 18) . We made
single mutants by introducing Ser residues in place of Asn
and Lys
, which are at the P-0 positions on the
NH
- and COOH-terminal sides, respectively, of Arg
because the relative orientation of the autoinhibitory region
with respect to the active site of the enzyme is not established yet.
Two other single mutants were made by introducing an Arg in place of
Ala
or Ala
, which would place them three
residues to the NH
- and COOH-terminal sides of
Thr
, respectively. It is known that CaM kinase II
recognizes phosphorylation sites that have an Arg three residues
NH
-terminal of the phosphorylated Ser or Thr. Since the
orientation of the autoinhibitory polypeptide sequence in the catalytic
site (see model in Ref. 4) may be opposite to that of the normal
substrate, we wanted to also place an Arg three residues COOH-terminal
of Thr
(i.e. mutant A309R). The R311A mutant was
constructed to test the effect of disruption of the substrate
recognition sequence around Ser
.
General Characteristics of the
Mutants
The mutants were expressed using the
baculovirus/Sf9 cell system and purified on CaM-Sepharose as described
under ``Experimental Procedures.'' All the mutants were at
least 90% pure and gave a major 50-kDa band on SDS-PAGE by Coomassie
stain (Fig. 1A) and by Western analyses
(Fig. 1B). Four of our mutations are in the CaM-binding
region of the autoinhibitory domain, and these mutants showed lower
affinity for CaM. Fig. 1C shows the CaM overlay assay
for all the mutants. The relative CaM binding affinities were wild type
= R311A > N294S > A309R > K300S > A302R. The
abilities of the mutants to be activated by CaM are shown in
Fig. 2
. Mutants N294S and R311A exhibited similar CaM activation
profiles as the wild-type enzyme with A values
of 0.1-0.15 µM in the presence of 0.5 mM
CaCl
The A309R and K300S mutants showed significant loss
in CaM activation with A
values of 0.6 and 1.6
µM, respectively. The A302R mutant showed a severe loss in
CaM activation, and the A
value could not be
determined for this mutant.
Figure 1:
Coomassie, Western blot, and CaM
overlay analyses of purified CaM Kinase II mutants. Equal amounts (2
µg) of wild-type CaM kinase II and the indicated mutants were run
on 10% polyacrylamide gel in the presence of SDS and analyzed by
Coomassie Blue (A), Western blot with anti CaM kinase II
(B), or reacted with biotinylated CaM in the presence of 1
mM CaCl (C) as described under
``Experimental Procedures.'' STDS, molecular mass standards
ranging from 31 to 106 kDa.
Figure 2:
Ca/CaM activation of
wild-type and mutant CaM kinase II. Kinase assays were performed in the
presence of 0.5 mM Ca
and the indicated
concentrations of CaM. Equivalent amounts (15 nM) of wild-type
enzyme (
), N294S (
), K300S (
), A302R (
), A309R
(⊞), and R311A (
) mutants were used. Maximal activation
(100%) was the activity at 12 µM CaM (see Table I), where
activation had reached a plateau for all the mutants except
A302R.
Catalytic Properties of the Mutants
All
of the mutants had activities in the absence of
Ca/CaM similar to wild-type kinase (),
and this indicates that the mutations, which are not in the catalytic
domain, had no significant effect on the basic catalytic properties of
the kinase. When assayed in the presence of saturating CaM (12
µM), most of the mutants had specific activities
() between 14-22 µmol/min/mg, comparable to the
wild-type enzyme. Mutant K300S displayed a 10-fold lower specific
activity, probably a consequence of its altered interaction with CaM.
It is known that mutations within CaM can not only affect the apparent
K
for activation of its target proteins but also
the extent of activation at saturating CaM
(19) . It is not
surprising that mutation within the CaM-binding domain of a target
protein may have a similar effect.
Ca
Truncation of CaM kinase
II at residue 316 produces a monomeric species of CaM kinase II that
requires Ca-independent
Autophosphorylation of the Mutants
/CaM for activation
(20) . This
indicates that interaction of the autoinhibitory domain with the
catalytic domain is intrasubunit in the holoenzyme. Furthermore, CaM
kinase II undergoes slow, intrasubunit autophosphorylation at 23 °C
in the absence of Ca
/CaM (i.e. 1 mM
EGTA) (13). It has been demonstrated that this
Ca
-independent autophosphorylation occurs
predominantly at Thr
in the CaM-binding domain, and this
blocks subsequent binding of CaM and prevents activation of the
enzyme
(17, 18) . Therefore, if any of our autoinhibitory
domain mutations converted a pseudosubstrate motif into a substrate
site, we would expect enhanced intrasubunit,
Ca
-independent autophosphorylation of that mutant.
-independent autophosphorylation conditions, the
K300S and N294S mutants showed enhanced rates of
P
incorporation compared with wild-type kinase and the other mutants
(Fig. 3). The stoichiometry of
P incorporation per
mole of enzyme subunit was also increased. For the K300S mutant, about
0.4 mol of
P was incorporated per mol of enzyme subunit in
15 min at room temperature compared with about 0.1 mol for wild-type
kinase (Fig. 3). The N294S mutant incorporated 0.2 mol of
P under identical conditions. The A302R, A309R, and R311A
mutants did not show any significant differences in either rate or
stoichiometry of
P incorporation compared with wild-type
enzyme, and all showed a similar loss (50-70%) in
Ca
/CaM-stimulated activity after 15 min of incubation
in the presence of 1 mM EGTA and 0.5 mM ATP at room
temperature (data not shown). This loss of activation by
Ca
/CaM is consistent with the known
Ca
-independent autophosphorylation of
Thr
. Since the K300S and N294S mutants demonstrated
enhanced Ca
-independent autophosphorylation, we
carefully examined the effects of this autophosphorylation on their
kinase activities. The K300S mutant did exhibit a slightly higher rate
of inactivation, whereas the N294S mutant showed a rate of inactivation
similar to the wild-type kinase (Fig. 4).
Figure 3:
Kinetics and stoichiometry of
P incorporation for Ca
-independent
autophosphorylation of wild-type and mutant CaM kinase II enzymes.
Wild-type and mutant enzymes (1 µM) were incubated for 15
min at 23 °C in 50 mM HEPES, pH 7.5, 10 mM
magnesium acetate, 1 mM EGTA, and 0.5 mM
[
-
P]ATP (12000-17000 cpm/pmol). At
the indicated times, aliquots were removed, and EDTA was added to a
final concentration of 28 mM to stop the autophosphorylation
reaction. Aliquots from these solutions were spotted directly on P-81
paper for measurement of
P incorporation. Symbols are the same as in Fig. 2.
Figure 4:
Inactivation of N294S and K300S mutants
due to Ca-independent autophosphorylation. Wild-type
CaM kinase II, N294S, and K300S mutant enzymes were autophosphorylated
in the absence of Ca
/CaM as described in Fig. 3. At
the indicated times, aliquots were removed, and autophosphorylation
reactions were stopped by adding EDTA. An aliquot from these solutions
was diluted 35-150-fold in dilution buffer, and total kinase
activities (i.e. + Ca
/CaM) were
measured as described under ``Experimental Procedures.''
Assays were done in duplicate, and the experiment is representative of
three similar experiments. Kinase activities of the wild-type
(
), N294S (
), and K300S (
) mutant enzymes are
expressed as a percentage of corresponding controls incubated under
identical conditions except for the absence of
ATP.
When
autophosphorylation was performed in the presence of
Ca/CaM, all of the mutants, except A302R, generated
similar Ca
-independent activity indicative of
autophosphorylation of Thr
(data not shown except for
K300S mutant in ). Also, enhanced
P
incorporation was observed for autophosphorylation of the K300S mutant
only in the absence (Fig. 3) and not in the presence of
Ca
/CaM ().
Two-dimensional Phosphopeptide Mapping and
Phosphoamino Acid Analysis Studies
Finally, we analyzed the
phosphorylation sites for the K300S and N294S mutants by
two-dimensional P peptide mapping. To simplify the
analysis, we restricted our analysis to the 3-kDa CNBr fragment, which
contains the autophosphorylation sites of interest
(13) . The
patterns of the CNBr/tryptic phosphopeptide maps were the same for
wild-type enzyme, N294S and K300S mutants, showing only one major
radioactive
P peptide with comparable mobility
(Fig. 5). Since Ser
and Thr
are
present in the same CNBr/tryptic phosphopeptide (LS*GAILTT*M), only one
spot was expected to be generated due to phosphorylation of either
Ser
or Thr
. The absence of any additional
spots in the phosphopeptide map of the N294S mutant indicated that
Ser
was not phosphorylated. We also carried out
phosphoamino acid analyses on the 3-kDa CNBr/tryptic peptide fragment
from the wild-type enzyme, N294S and K300S mutants as described under
``Experimental Procedures.'' For wild-type enzyme and N294S
mutant, only Thr residues were phosphorylated (Fig. 6A,
lanes1 and 2). However, the K300S mutant
gave phosphorylation of both Thr and Ser (Fig. 6A,
lane3). Phosphoamino acid analysis of the
phosphopeptide 1 (Fig. 5D) also showed the presence of
both phosphoserine and phosphothreonine (Fig. 6B) in the
same spot, thereby confirming the phosphorylation of Ser
in the K300S mutant.
Figure 5:
Two-dimensional P peptide
mapping of N294S and K300S mutants after
Ca
-independent autophosphorylation. Wild-type, N294S,
and K300S mutants (2 µM) were autophosphorylated in the
presence of 50 mM HEPES, pH 7.5, 10 mM magnesium
acetate, 1 mM EGTA, and [
-
P]ATP (9
Ci/mmol) at 23 °C for 15 min. The 50-kDa
P kinase
subunits were isolated by SDS-PAGE and cleaved with CNBr. The 3-kDa
CNBr fragments, corresponding to residues 282-307, were isolated,
digested with trypsin, and subjected to two-dimensional peptide mapping
as described under ``Experimental Procedures.'' In panelA, synthetic peptide 290-309 was specifically
P labeled on Thr
, digested with CNBr and
trypsin, and subjected to two-dimensional peptide mapping as described
(13). PanelsB-D represent
Ca
-independent autophosphorylation of wild-type,
N294S, and K300S mutants, respectively.
Figure 6:
Phosphoamino acid analysis of
P-labeled tryptic fragments from N294S and K300S mutants.
A, aliquots of the
P-labeled CNBr/tryptic
peptides prepared for two-dimensional peptide maps as described in Fig.
5 were hydrolyzed in 6 N HCl, and the released
P
amino acids were separated by thin layer electrophoresis at pH 1.9 as
described under ``Experimental Procedures.'' Lane1 represents wild-type enzyme; lane2,
N294S mutant; lane3, K300S mutant, B,
peptide 1 from panelD of Fig.
5.
/CaM-dependent enzymes act as autoinhibitory
domains of their respective enzyme functions
(21, 22) .
In some cases, autoinhibitory domains have been proposed to interact
with the catalytic core of the enzyme as a
pseudosubstrate
(6, 7) . In CaM kinase II, residues
281-290 interact with the ATP-binding pocket of the catalytic
domain, whereas residues 290-309, which also comprise the
CaM-binding domain, have been proposed on the basis of synthetic
peptide and mutagenesis studies
(3, 9) to act as a
pseudosubstrate by blocking the protein-substrate motif. The present
study was aimed at exploring the pseudosubstrate sequence in the
autoinhibitory domain by generating or removing possible substrate
consensus sequences around three potential sites: Arg
,
Thr
, or Ser
.
-independent specific activities of the mutants
() indicated there were no major changes in basic catalytic
properties of the enzymes due to mutations. This conclusion was
supported by the examination of other catalytic properties of the
enzymes, such as autophosphorylation properties. After activation by
Ca
/CaM in the presence of Mg
/ATP,
CaM kinase II autophosphorylates itself on Thr
in the
autoinhibitory domain in an intersubunit
manner
(13, 23) , and the enzyme becomes partially
Ca
/CaM independent. All the mutant enzymes, except
A302R, exhibited normal Ca
/CaM-dependent
autophosphorylation and generation of Ca
-independent
activity similar to the wild-type enzyme. Because the specific activity
of the A302R mutant in the presence of Ca
/CaM was so
low, this mutant could not be tested for any
Ca
/CaM-dependent phenomena.
/CaM. The A302R, A309R, and R311A
mutants exhibited the same rates and stoichiometries of
P
incorporation during Ca
-independent
autophosphorylation as the wild-type enzyme, thereby excluding
Thr
and Ser
as possible pseudosubstrate
sites. This is consistent with previous data indicating that the
autoinhibitory domain resides within residues 281-302
(9) .
However, both the K300S and the N294S mutants exhibited enhanced rates
and stoichiometries of Ca
-independent
autophosphorylation (Fig. 3). When we measured the corresponding
rates of inactivation for these two mutants due to
Ca
-independent autophosphorylation, only the K300S
mutant showed a higher rate of inactivation compared with wild-type
enzyme (Fig. 4). As discussed below, Lys
of the CaM
kinase II peptide interacts with glutamate residues in the
NH
-terminal lobe of Ca
/CaM
(24) ,
and the K300S mutation in CaM kinase II reduced CaM binding
(Fig. 1C). It is likely that introduction of negative
charge by phosphorylation of Ser
in the K300S mutant
would further disrupt the interaction with CaM, causing an enhanced
rate of inactivation during Ca
-independent
autophosphorylation. Phosphorylation of Ser
, but not
Ser
, was demonstrated by two-dimensional peptide mapping
and phosphoamino acid analysis ( Fig. 5and 6). The enhanced
autophosphorylation observed with the N294S mutant was due to
phosphophorylation of an unknown site. Multiple sites are subject to
autophosphorylation in the absence and presence of
Ca
/CaM
(1, 2) , and we did not attempt
to identify this site in the N294S mutant since its autophosphorylation
did not produce any detectable changes in the kinase.
/CaM. This is to be expected since the crystal
structure of Ca
/CaM bound to peptide 290-314 of
CaM kinase II
(24) has shown that residues 293-310 form
electrostatic and hydrophobic interactions with CaM. The cluster of
three basic residues (Arg
-Arg
-Lys
and Lys
in the NH
-terminal half of CaM
kinase II peptide form salt bridges with glutamate residues on both the
NH
- and COOH-terminal lobes of CaM.
and Ile
on the NH
-terminal
half and Leu
on the COOH-terminal half of the peptide are
involved in hydrophobic interactions with the COOH- and
NH
-terminal lobes, respectively, of CaM. We found that
insertion of charge at position 302 (A302R mutant), which should
disrupt the hydrophobic interaction with the COOH-terminal lobe of CaM,
caused the most severe loss in binding (Fig. 1C) and
activation by CaM (Fig. 2, ). The hydrophobic
interaction with the NH
-terminal lobe of CaM, on the other
hand, appeared to be more involved in CaM binding
(Fig. 1C) than in kinase activation (Fig. 2,
), as observed with the A309R mutant. Similar but less
pronounced effects were observed with the N294S mutant, probably
because the charge was not altered. The K300S mutant showed a strong
effect on binding to and activation by CaM, presumably by disrupting
the electrostatic interaction of Lys
with glutamate
residues in the NH
-terminal lobe of CaM. Although 12
µM CaM was not able to fully activate the K300S mutant
(), autophosphorylation on Thr
, which
normally does not increase total kinase activity assayed in the
presence of Ca
/CaM, caused a 4-fold increase in total
kinase activity of the K300S mutant (). This result is
consistent with impaired binding to and activation by CaM for this
mutant. Thus, our mutagenesis results suggest that although both lobes
of CaM are involved in binding to CaM kinase II through electrostatic
and hydrophobic interactions, the COOH-terminal lobe of CaM probably
plays the major role in the binding and activation mechanism.
-independent activity toward exogenous substrate
(i.e. syntide 2) in the absence of Ca
/CaM
compared with wild-type kinase (). This indicates that none
of the mutations weakened the interaction of the autoinhibitory domain
with the catalytic domain. Similar findings were obtained with another
Ca
/CaM-regulated enzyme, myosin light chain
kinase
(25, 26) . Mutation to acidic residues of the
basic residues RRK on the NH
-terminal side of the
CaM-binding domain of myosin light chain kinase altered the CaM binding
affinity but did not give rise to Ca
-independent
activity
(26) . It appears, therefore, that the mechanisms of CaM
activation and mechanism of autoinhibition involve different residues.
This is contrary to a ``flip-flop model'' proposed
earlier
(27) , which assumed an autoinhibitory mechanism in which
the CaM-binding domain interacted with a ``CaM-like binding
region'' on the enzyme surface.
is autophosphorylated in the absence of
Ca
/CaM is consistent with our previous synthetic
peptide study
(4) and our molecular model
(4) , indicating
that Arg
may occupy the P(-3) position of a
pseudosubstrate inhibitor. Our results also support some of the
conclusions made from the crystal structure of CaM with the synthetic
CaM-binding peptide complex
(24) . In addition, this study shows
in the context of the entire enzyme structure which residues are
obligatory for high affinity CaM binding versus those that may
be necessary for conformational changes and those that participate
directly in modulation of enzyme activity.
Table:
Specific
activities of the wild-type and mutant kinases
, 10 mM magnesium acetate, 0.5 mM [
-
P]ATP, and 40 µM syntide 2 as described under ``Experimental
Procedures.'' Ca
-independent specific activities
were measured in the absence of Ca
/CaM under standard
conditions (see ``Experimental Procedures''). All assays were
done in duplicate. Values obtained from more than two independent
assays are given as the mean ± S.D.
Table:
Effect of
Ca/CaM-dependent autophosphorylation on K300S mutant
, 27 µM CaM, with or without 0.5
mM [
-
P]ATP (6600 cpm/pmol).
Autophosphorylation reactions were stopped by adding EDTA to a final
concentration of 28 mM. Aliquots from these solutions were
spotted directly on P-81 paper for measurement of
P incorporation or assayed for CaM-kinase II activities
(±Ca
/CaM) as described under
``Experimental Procedures'' after appropriate dilution.
/calmodulin-dependent protein kinase II; PAGE,
polyacrylamide gel electrophoresis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.