(Received for publication, March 18, 1994; and in revised form, October 20, 1994)
From the
Calcineurin (CaN) contains an autoinhibitory element (residues
457-482) 43 residues COOH-terminal of the calmodulin-binding
domain (Hashimoto, Y., Perrino, B. A., and Soderling, T. R.(1990) J. Biol. Chem.265, 1924-1927) that regulates
the Ca-dependent activation of its phosphatase
activity. Substitution of Arg
and Arg
or
Asp
to Ala in the autoinhibitory peptide 457-482
significantly decreased its inhibitory potency. CaN A subunits with
these residues mutated to Ala were co-expressed with the
Ca
-binding B subunit using the baculovirus/Sf9 cell
system. Kinetic analysis showed that although the purified mutants had
no activity in the absence of calcium, they were less dependent than
the wild-type enzyme on calcium and calmodulin for activity. To
determine if additional autoinhibitory motifs were present in the COOH
terminus of calcineurin, the A subunit was truncated at residues 457 or
420 and co-expressed with B subunit. The V
values of both truncation mutants with or without Ca
were increased relative to wild-type calcineurin. The increased
Ca
-independent activity of CaN
relative
to CaN
indicates the presence of additional
autoinhibitory element(s) within residues 420-457. CaN
had similar high V
values with or without
Ca
, but the K
value for
peptide substrate was increased 5-fold to 125 µM in the
absence of Ca
. The K
values of all the expressed calcineurin species were
increased in the absence of Ca
. The CaN A or CaN
A
subunits alone have low V
and
high K
(115 µM) values even
in the presence of Ca
. These results indicate that 1)
there are several autoinhibitory motifs between the CaM-binding domain
and the COOH terminus that are relieved by Ca
binding
to CaM and the B subunit, 2) Ca
binding to the B
subunit also regulates enzyme activity by lowering the K
of the catalytic subunit for substrate,
3) binding of the B subunit is required for high V
values even after removal of the autoinhibitory domain. These
results are consistent with synergistic activation of calcineurin by
Ca
acting through both CaM and the B subunit.
Calcineurin (CaN) ()is the neuronal form of the
widely dis-tributed Ca
/CaM-dependent Ser/Thr
phosphoprotein phosphatase 2B (PP-2B) (reviewed in (1) ). CaN
is involved in diverse physiological functions such as induction of
long term depression in area CA1 of the hippocampus and mediating the
immunosuppresant functions of cyclosporin and FK506 which inhibit
dephosphorylation of the transcription factor NF-AT
by
CaN(2, 3, 4) . Studies with FK506 also
implicate regulatory roles for CaN in Ca
-dependent
transcription of other genes(5, 6, 7) . There
is also evidence that the activities of Na
channels,
L-type Ca
channels, N-methyl-D-aspartate; receptors, and the heat-stable
inhibitors of PP-1 (inhibitor-1 and DARRP-32) are modulated by
CaN(1, 8, 9, 10) .
Type 2B
phosphatases are heterodimers composed of the catalytic A subunit
(57-61 kDa) and a regulatory B subunit (19 kDa)(11) . The
CaN B subunit is an ``EF-hand'' Ca-binding
protein which remains tightly associated with the A subunit in the
presence or absence of Ca
(1) . Ca
binding to the B subunit stimulates CaN phosphatase activity, but
this activity is low compared with that attained in the presence of
Ca
/CaM. CaN A has very low phosphatase activity by
itself, but addition of Mn
/CaM or
Mn
/B subunit gave 5- or 50-fold activations,
respectively(12) . However, addition of CaM to reconstituted A
and B subunits gave a synergistic 600-fold activation. CaM increased
the V
whereas B subunit primarily decreased the K
with a smaller effect on V
(12) .
The catalytic A subunit is
composed of several functional domains(13, 14) . The
catalytic domain is presumed to be located between residues
71-325 (all numbering will be based on the rat brain
isoform (15) ) because of its sequence homology to PP-1 and
PP-2A(16) . The CaM-binding domain encompasses residues
391-414(13) . Limited proteolysis of CaN in the presence
of Ca
/CaM removes the residues COOH-terminal of the
CaM-binding domain and generates a 57-kDa A subunit which still binds B
subunit and CaM but which no longer requires Ca
or
CaM for full activity(14) . These results suggested the
presence of a COOH-terminal autoinhibitory domain which was localized
to residues 457-482 by use of overlapping synthetic
peptides(17) . In this report we present the results of our
studies, primarily by use of site-specific and truncation mutagenesis,
to identify which residues are critical for the autoinhibitory
interaction and to determine whether additional autoinhibitory motifs
are present in the COOH terminus. Kinetic analysis of the purified
mutants also allowed us to determine mechanisms by which Ca
binding to its B subunit and to CaM activates CaN.
Since many
substrates of CaN contain basic residues just NH-terminal
of the phosphorylated Ser/Thr, we substituted Arg
with Ala. With the R
synthetic peptide, addition of
residues DLDV to the sequence PIPGRFDRRVS
VAAE dramatically
decreased the K
and increased V
for dephosphorylation by CaN(24) , so we substituted the
analogous Leu
and Asp
in the autoinhibitory
CaN peptide. The 2 Glu
residues were chosen since
R
peptide has similarly positioned acidic residues. In
addition, we synthesized a peptide with a 4-residue
NH
-terminal extension to determine if this would increase
inhibitory potency. Fig. 1shows the abilities of these
substituted peptides to inhibit purified bovine brain CaN. Compared to
the parent peptide, the NH
-terminal extension (peptide F)
and the L466A substitution (peptide C) had little or no effect on
IC
The D467A and R476/477A substitutions (peptides D and
E, respectively) strongly decreased inhibitory potency, and the
E461/462A substitution (peptide B) was intermediate in effect.
Figure 1:
Effects of amino acid substitutions
on the inhibitory potency of the CaN autoinhibitory peptide. Purified
bovine brain CaN (50 nM) was assayed using 30 µM [P]R
pep and 150 nM CaM in the presence of 0.5 mM MnCl
(see
``Experimental Procedures''). The indicated concentrations of
the following peptides were also included: peptide A (residues
457-482 of the
-CaN A subunit) (
), peptide B (
),
peptide C (
), peptide D (
), peptide E (
), and
peptide F (
).
Figure 2:
Domain structure of -CaN A subunit. Catalytic, conserved phosphatase domain; CaM,
calmodulin-binding domain; INH, autoinhibitory element. Amino
acid residues are numbered according to the
-CaN A isoform (15) . The positions of the D467A and R467/477A mutants are
shown as well as the truncation mutants A
and
A
.
Figure 3:
SDS-PAGE and Western blot of wild-type and
mutant CaNs purified from Sf9 cells. A, proteins (4
µg/lane) were separated by SDS-PAGE (15%) and stained with
Coomassie Brilliant Blue. Lane 1, molecular mass standards; lane 2, bovine brain CaN; lane 3, wild-type
CaN; lane 4, CaN D467A; lane 5, CaN
R476/477/A; lane 6, CaN
; lane 7,
CaN
. B, proteins (4 µg/lane) were separated
by SDS-PAGE (15%) and transferred to nitrocellulose. Immunostaining was
carried out as described under ``Experimental Procedures.'' Lane 1, bovine brain CaN; lane 2, CaN
; lane 3, CaN D467A; lane 4, CaN R476/477A; lane
5, CaN
; lane 6,
CaN
.
Figure 4:
SDS-PAGE and autoradiography of wild-type
CaN purified from [H]-myristate-labeled Sf9
cells. A, bovine brain CaN (lane 1, 6 µg) and
wild-type CaN (lane 2, 6 µg) were separated by 12%
SDS-PAGE and stained with Coomassie Brilliant Blue. B, autoradiogram of
gel shown in panel A.
Fig. 5shows the K and V
values in the absence and presence of
Ca
and CaM for the wild-type and mutant CaNs. Similar
to purified bovine brain CaN, Ca
binding to the B
subunit increased the wild-type CaN V
(Fig. 5A). An additional 3-5-fold increase
in the V
with little change in the K
resulted from binding of
Ca
/CaM, consistent with displacement of an
autoinhibitory domain. Since the D467A and RR476/477AA peptides were
poor inhibitors, we expected the corresponding mutants to have
increased phosphatase activity in the absence of Ca
.
However, the D467A and R476A/R477A mutants were inactive in the absence
of Ca
, but the V
values in the
presence of Ca
alone were increased 2- and 3.5-fold,
respectively, compared to wild-type CaN. The mutants also exhibited
significantly higher total phosphatase activities in the presence of
Ca
/CaM compared to wild-type enzyme. The K
values of the mutants were similar to the K
values obtained for the wild-type phosphatase (Fig. 5B). The increases in V
in
the presence of Ca
or Ca
/CaM
indicate that Asp
and Arg
may be
involved in the interaction of the autoinhibitory domain with the
catalytic domain, but the autoinhibitory interaction most likely
involves additional residues since mutation of Asp
and
Arg
by themselves had little or no effect on
Ca
-independent activity.
Figure 5:
Kinetic analysis of wild-type and mutant
CaNs for dephosphorylation of [P]R
pep. CaN was assayed (see ``Experimental Procedures'')
at the following nM concentrations in the presence of 1 mM EGTA (20 min) or 0.1 mM Ca
(10 min):
EGTA: 500, wild-type; 300, D467A; 300, R476/477A; 30,
CaN
; 30, CaN
; N.D. A
N.D.
A
; Ca
: 30, wild-type; 30,
D467A; 30, R476/477A; 30, CaN
30, CaN
; 100
A
100 A
. CaM was present at a 3-fold molar
excess of CaN. Five concentrations of substrate were used in the
presence of EGTA (30-300 µM [
P]R
pep) or Ca
(4-80 µM [
P]R
pep), The solid black bars represent the kinetic
analyses of the wild-type A subunit and A
subunits alone
(20-250 µM [
P]R
pep). The K
and V
values were determined by linear regression analysis of
Lineweaver-Burk data plots. The assays were performed in triplicate,
and the activities shown are the mean ± S.D. (n = 3). A, V
. B, K
.
To examine the effects of step-wise COOH-terminal truncations of the
A subunit on phosphatase activity, the kinetic parameters of mutants
CaN and CaN
were compared to
CaN
. As seen in Fig. 5A, CaN
has an extremely low specific activity of 3-10 nmol/min/mg
in the absence of Ca
. The concentration of enzyme was
increased 10-17-fold (300 nM or 500 nMversus 30 nM) and the assay time doubled in
order to detect significant phosphatase activity in the presence of
EGTA. Compared to CaN
, truncation of the A subunit at
residue 457 increased the V
in the absence of
Ca
10-fold, while truncation at residue 420 resulted
in an additional 6-fold increase in the V
value (Fig. 5A). In contrast to CaN
, the
Ca
-independent activity of CaN
was
greater than the Ca
/CaM-stimulated activity of
CaN
, indicating that the phosphatase activity of
CaN
was totally Ca
/CaM-independent. The
elevated Ca
- and Ca
/CaM-stimulated
activity of CaN
relative to CaN
is similar
to previous findings showing that proteolysis of CaN results in levels
of activity slightly higher than the
Ca
/CaM-stimulated activities of the non-proteolyzed
enzymes(14) . Thus, removal of the autoinhibitory element
located within residues 457-482 by truncation at residue 457 gave
partial Ca
-independence, while truncation at residue
420 generated completely Ca
-independent activity.
These results indicate that the A subunit contains an additional
autoinhibitory element(s) within residues 420-457. Relative to
the wild-type enzyme, the activities of CaN
as well as
the point mutants were elevated by Ca
or
Ca
/CaM. Although the Ca
-stimulated
activities of the two point mutants and CaN
were elevated
to similar levels, an additional 2-fold increase in activity which was
also Ca
-independent was seen with CaN
.
These results provide further evidence for the presence of additional
autoinhibitory elements within residues 420-457.
The very low
phosphatase activity of the A subunit is synergistically stimulated by
the B subunit and CaM(12) . However, since the high V of the CaN
mutant does not
require Ca
(Fig. 5A), does this
mutant also require the presence of the B subunit? As shown by the solid black bars in Fig. 5A, the wild-type A
subunit and A
mutant subunit without co-expressed B
subunit exhibited very low V
values of less than
10 nmol/min/mg. These low V
values are not an
artifact of improper folding of the A subunits in the absence of B
subunit since in vitro reconstitution with B subunit gave 25-
and 60-fold increases in phosphatase activities for the wild-type A and
A
subunits, respectively (data not shown). These results
indicate that CaN
still requires the B subunit to attain
high V
values in the absence of
Ca
.
Similar to purified brain enzyme, CaM
increased the V of Sf9-expressed CaN
without affecting the K
(27.7 ± 4.2 versus 29.7 ± 2.4 nmol/min/mg) (Fig. 5B). The K
values of the CaN
point mutants and truncation mutants in the presence of Ca
were similar to the values of wild-type CaN and were also
unaffected by CaM (Fig. 5B). These results indicate
that the K
was unaffected by COOH-terminal
alterations and deletions of the A subunit. However, in the absence of
Ca
the K
values of the
wild-type, site-specific, and truncation CaN mutants were
4-5-fold higher (Fig. 5B). This suggested that
Ca
decreased the K
by binding to
the B subunit, and this hypothesis was confirmed by kinetic analysis of
A subunits expressed without the B subunit. Both the wild-type and
A
truncated A subunits alone had K
values of 100-120 µM which were not decreased
by Ca
(Fig. 5B, solid black
bars). These results demonstrate that the ability of
Ca
to lower the K
required the B
subunit.
Since the Ca concentration used (100
µM) in these assays was saturating, we tested whether
these mutants would also exhibit an increased sensitivity to activation
by lower concentrations of Ca
. Fig. 6shows
their Ca
-dependent activation in the absence of CaM.
Wild-type CaN was half-maximally activated at 0.35 µM which is similar to a previous report(31) . All of the
mutants were significantly more sensitive to activation by lower
concentrations of Ca
. The CaN
mutant
was the most sensitive, and in this experiment it was only about 50%
active in the presence of excess EGTA because a K
concentration of substrate (i.e. not a V
value as in Fig. 5A) was used.
Figure 6:
Ca activation of
wild-type and mutant CaNs. The phosphatases were assayed without or
with the indicated Ca
concentrations at 30 °C for
10 min with 70 µM [
P]R
pep. Free [Ca
] was calculated for
Ca
/EGTA buffers. The phosphatase activities are
plotted as the ratio of the activity at 100 µM Ca
to the activity at the indicated
Ca
concentrations. Each point represents the mean of
two experiments performed in triplicate. The phosphatase activity
(nmol/min/mg) at 100 µM Ca
is indicated
prior to the appropriate symbol. Wild-type CaN, (19 ± 0.1,
); CaN D467A, (17 ± 0.4,
); CaN R476/477A, (59
± 1.0,
); CaN
, (61 ± 1.6,
);
CaN
, (90 ± 0.7,
).
Both the A and B subunits undergo conformational changes in the
presence of Ca(32) . Our finding that the K
of the A subunit for substrate was regulated by
Ca
-binding to the B subunit suggests that a
Ca
-induced conformational change in the B subunit
caused a conformational change in the catalytic domain that increased
its affinity for substrate (i.e. decreased the K
). Could such a conformational change in the
catalytic domain also weaken the interaction between the autoinhibitory
and catalytic domains? If so, one would predict that the IC
of the autoinhibitory peptide 457-482 would be higher in
the presence of Ca
. As seen in Fig. 7, similar
inhibition curves of CaN
were obtained in the absence and
presence of Ca
. This suggests that the region of the
catalytic domain that interacted with CaN inhibitor peptide
457-482 was unaffected by the Ca
/B
subunit-induced decrease in K
. This result is
consistent with our previous demonstration that peptide 457-482
had similar IC
values for proteolyzed CaN in the presence
of EGTA and for native CaN in the presence of
Ca
/CaM(17) . However, that experiment used a
single substrate concentration ([
P]myosin light
chain, 2.8 µM) that would be limiting in EGTA but near K
in the presence of Ca
/CaM. In
the present experiment the substrate concentrations were K
under both conditions.
Figure 7:
Effect of CaN inhibitor peptide
457-482 on CaN phosphatase activity in the presence
or absence of Ca
. CaN
(30 nM)
was assayed with the indicated concentrations of autoinhibitory peptide
for 10 min at 30 °C in the absence (
) or presence (
) of
Ca
. The concentrations of
[
P]R
pep used in the absence or
presence of Ca
were 115 µM and 30
µM, respectively. The 100% activities of CaN
were 58.5 ± 3.2 and 80.5 ± 3.2 nmol/min/mg without
and with Ca
, respectively. The assays were performed
in triplicate, and the data shown are the mean ± S.D. from three
experiments.
We previously utilized overlapping synthetic peptides to
localize an autoinhibitory element within residues 457-482 of the
CaN A subunit (17) . In the present study we made substitutions
in the autoinhibitory peptide 457-482 to identify essential
autoinhibitory residues. The D467A and R476/477A substitutions strongly
decreased the inhibitory potency, indicating their importance for
inhibitory function. However, the corresponding mutations in the A
subunit generated little or no Ca-independent
activity in the expressed CaN mutants, while the
Ca
-dependent activity was significantly higher than
wild-type CaN (Fig. 5A). It is likely that multiple
intrasubunit interactions occur between the autoinhibitory and
catalytic domains such that disruption of one interaction by
site-specific mutagenesis minimally effects inhibitory potency. Since
the intermolecular interaction of the synthetic peptide with the A
subunit catalytic domain is not subject to intrasubunit structural
constraints, single substitutions in the peptide may be more effective
at disrupting inhibitory interactions. These results are similar to
those observed with the autoinhibitory domain of CaM-kinase II where
single substitutions in the autoinhibitory peptide have large effects,
but the corresponding site-specific mutations in the enzyme are less
dramatic(27, 28) .
The CaN mutants were activated
by lower Ca concentrations than wild-type CaN (Fig. 6). Since the B subunit has four EF hand
Ca
-binding domains(1) , activation of
wild-type CaN may require all four sites to be occupied by
Ca
, whereas fewer occupied sites might be required to
activate the mutants. Alternatively, there could be synergistic
interactions between the B subunit and the autoinhibitory domain such
that deletions or disruptions of the autoinhibitory domain increase the
affinity of the B subunit for Ca
. These findings are
relevant to understanding structure-function aspects of CaN, since
several different isoforms of the A subunit have been
described(11) . Notably, the A subunit carboxyl-terminal region
is less conserved than other functional domains, suggesting that these
differences may impart different substrate specificities, distinct
tissue or subcellular distribution, or variable Ca
sensitivity(33) .
The site-specific mutants were not
completely Ca/CaM-independent, so we constructed a
truncation mutant (CaN
) by inserting a stop codon at
residue 457, the NH
-terminal boundary of the previously
identified autoinhibitory element. Since Ile
is located
43 residues COOH-terminal of the CaM-binding domain (residues
391-414), we also made a truncation at residue 420
(CaN
) to ascertain if additional autoinhibitory elements
were present in this region. Limited proteolysis of CaN removes the
region COOH-terminal of the CaM-binding domain and results in levels of
Ca
-independent activity similar to the
Ca
/CaM-stimulated activity of the non-proteolyzed
enzyme(14) . These findings provided strong evidence for the
presence of an autoinhibitory domain COOH-terminal of the CaM-binding
domain. Similarly, stepwise deletion of the A subunit COOH-terminal
region elevated the V
values of both CaN
and CaN
in the absence of Ca
.
However, the observation that CaN
showed partial
Ca
-independence while CaN
was fully
Ca
-independent demonstrated that additional
inhibitory elements were present within the sequence 420-457. The
finding that Ca
/CaM was required to further increase
the activity of CaN
to similar levels of phosphatase
activity as CaN
with Ca
alone is also
consistent with displacement of an autoinhibitory domain.
Kinetic
analysis of wild-type and mutant CaNs also confirmed that
Ca regulates the displacement of the autoinhibitory
domain from the catalytic domain by binding both the B subunit and CaM.
The Ca
-induced increase in V
in the absence of CaM can only be mediated by the B subunit.
Binding of Ca
to CaM further increased the V
of recombinant CaN (Fig. 5A).
The findings that the activities of the D467A, RR476/477AA mutants, and
CaN
in the presence of Ca
alone are
increased relative to wild-type CaN indicate that the autoinhibitory
region is sensitive to Ca
binding to the B subunit.
Similar to Ca
/CaM, the Ca
/B
subunit-induced increase in V
may be mediated by
conformational changes in the COOH-terminal autoinhibitory domain.
Furthermore, the findings that the D467A, RR476/477AA mutants and
CaN457 had elevated Ca
-stimulated activities but
still required CaM for full activation indicate that the effects of
Ca
on the autoinhibitory elements within residues
420-457 and 457-482 are mediated by both the B subunit and
CaM. In addition, the wild-type and truncated A subunits alone had very
little phosphatase activity in the presence of Ca
(Fig. 5A, inset), but their activities
were increased 25- and 60-fold respectively, by in vitro reconstitution with purified B subunit. These results provide
strong evidence that the autoinhibitory domain of CaN is regulated by
Ca
/CaM as well as Ca
/B subunit.
Ca also decreased the K
, but
this effect was mediated only by the B subunit and not through CaM.
Thus, wild-type CaN and all the mutants had K
values of 100-125 µM in the absence of
Ca
which was reduced to 20-30 µM in the presence of Ca
regardless of whether CaM
was present (Fig. 5B). Even the CaN
mutant which did not require Ca
for its maximal V
still required Ca
to
decrease its K
. The fact that the expressed A
subunits alone had K
values of 100-125
µM in the presence of Ca
documents the
need for the B subunit to mediate the decrease in K
(Fig. 5B, inset).
Thus, binding of
Ca to the B subunit appears to change the
conformation of the catalytic pocket to decrease the K
for substrate. One could imagine that a conformational change in
the catalytic domain which alters K
might also
partially disrupt the interaction with the autoinhibitory domain and
thereby account for the increase in V
mediated
by Ca
binding to B subunit. However, the results
shown in Fig. 7are not consistent with this interpretation. The
IC
of autoinhibitory peptide 457-482 for CaN
was the same in the absence or presence of Ca
,
indicating that the affinity of the catalytic domain for the
autoinhibitory peptide was unaffected by Ca
. This
result suggests that CaN substrates and the autoinhibitory region
interact with the catalytic domain at distinct sites, and is consistent
with our previous reports that inhibition of CaN by peptide
457-482 was not competitive with
substrate(17, 29) . This possibility is being tested
with the complete autoinhibitory domain including the CaM-binding
domain (i.e. residues 390-482).
Our previous study on in vitro reconstitution of CaN phosphatase activity using
expressed wild-type A subunit and purified brain B subunit and/or CaM
showed that both Ca-binding proteins were required
for synergistic activation(12) . CaM had a strictly V
effect whereas the B subunit primarily
effected K
with a small effect on V
. The more extensive kinetic analysis presented
in this study clearly demonstrates that Ca
-binding to
the B subunit lowered the K
for substrate and
increased the V
. Ca
/CaM
further increased the V
by abolishing the
interaction between the catalytic domain and the COOH-terminal
autoinhibitory domain. In summary, Ca
binding to both
the intrinsic B subunit and extrinsic CaM is required to displace the
autoinhibitory domain and increase the V
, while
the K
is regulated by Ca
binding
to the B subunit. It is also clear that our previously defined
autoinhibitory element (residues 457-482) represents a minimal
sequence, and additional elements are present between residues
420-457. Ongoing studies of the interactions between the A and B
subunits as well as the interactions between the catalytic and
autoinhibitory domains should further our understanding of the
mechanisms of Ca
regulation of CaN phosphatase
activity.
Addendum-Since
submission of this manuscript, a report has been published
demonstrating that the B subunit of proteolytically activated CaN has a
higher affinity for Ca than the native enzyme
(Stemmer, P. M., and Klee, C. B.(1994) Biochemistry33, 6859-6866).