(Received for publication, September 6, 1994)
From the
Calcineurin (CaN) is the serine/threonine protein phosphatase
(phosphatase 2B) that is activated by binding of Ca to its B subunit and to calmodulin (CaM). This paper identifies
residues between the catalytic region and the CaM-binding domain of the
A subunit as the domain that binds the regulatory B subunit.
A
purified fusion protein containing residues 328-390 of the A
subunit 1) binds CaN B subunit, and 2) inhibits (IC = 0.1 µM) the in vitro stimulation
of CaN A phosphatase activity by purified CaN B subunit. A synthetic
peptide corresponding to residues 341-360 blocked the binding of
CaN B to residues 328-390 in the fusion protein, so 4 hydrophobic
residues within this region (Val
-Phe
and
Phe
-Val
) were mutated to either Glu (E
mutant) or Gln (Q mutant). The wild-type and mutant A subunits were
expressed individually or co-expressed with B subunit in Sf9 cells,
purified and characterized. The mutant A subunits were similar to
wild-type A subunit in terms of basal phosphatase activity (1-3
nmol/min/mg) and activation by Mn
/CaM. Addition of
purified B subunit to purified wild-type A subunit at a 1:1 molar ratio
gave a 40-fold increase in phosphatase activity whereas addition of B
subunit to either of the mutant A subunits had no effect on phosphatase
activity, even at a 3:1 molar excess of B subunit. Furthermore, when
wild-type or mutant A subunits were co-expressed with B subunit and
purified on CaM-Sepharose, the B subunit co-eluted with the wild-type A
subunit but not with either mutant A subunit. These results demonstrate
that residues 328-390 in the A subunit bind B subunit and that
the mutated hydrophobic residues are essential.
Calcineurin (CaN), ()a
Ca
/calmodulin (CaM)-dependent protein Ser/Thr
phosphatase (protein phosphatase 2B), is a major brain protein (1% of
total brain protein as opposed to 0.1-0.05% in other
tissues)(1, 2) . Although the substrate specificity of
CaN is limited compared to protein phosphatases 1 and 2A, CaN is
involved in the regulation of diverse cellular functions such as ion
channels(3) , protein phosphatase 1 activity(4) , gene
transcription(5, 6) , and neuronal long term
depression(7) .
CaN is a heterodimer of a catalytic A
subunit (58-61 kDa) and a regulatory B subunit (19 kDa). In
addition to the NH-terminal catalytic domain (residues
71-325, numbering based on the rat brain
isoform(8) ), the A subunit contains a central CaM-binding
domain (residues 390-414) (9) and a COOH-terminal
autoinhibitory region (residues 420-482) (10) . (
)The myristylated B subunit has four ``EF'' hand
high affinity Ca
-binding sites(2) . The
isolated CaN A subunit has very low phosphatase activity, which is
synergistically activated 200-fold by CaM plus B subunit(11) .
Kinetic studies have shown that Ca
/CaM activates CaN
by increasing the V
, whereas
Ca
-binding to the B subunit decreases the K
and increases the V
.
Thus, binding of Ca
to the B subunit is essential for enzymatic activity and for the
cellular regulation of CaN.
Studies on limited proteolysis of CaN in
the absence of Ca/CaM demonstrated cleavage of the
CaN A subunit NH
-terminal of the CaM-binding domain with
retention of phosphatase activity and B subunit binding(12) .
This result demonstrated that the binding domain for the B subunit was
located between the catalytic domain and the CaM-binding domain.
Dissociation of the CaN A and B subunits requires buffer containing 6 M urea(13) , indicating that hydrophobic interactions
are important in the subunit association. Two regions (residues
347-357 and 363-372) whose hydrophobicity are highly
conserved in CaN A isoforms from human to Drosophila are
likely candidates for binding the B subunit. Using peptide chemistry,
site-specific mutagenesis and enzymology, this paper identifies this
region as important for B subunit interaction.
Figure 1:
Schematic of rat brain CaN A
subunit. A, the localization of the domains for catalytic
phosphatase activity (residues 71-325), CaM binding
(390-414), and one autoinhibitory element (457-482) are
depicted. The sequence 328-390 is also shown with the residues
mutated in this study underlined. B, the hydropathy
index of residues 328-390 for wild-type (upper panel)
and Q mutant (lower panel) were analyzed by the method of Kyte
and Doolittle (22) with a window average of 6 residues. The asterisks indicate the residues mutated in this
study.
Figure 2:
Binding of CaN B to residues 328-390
of CaN A. The CaN B subunit (1 µg) was incubated for 1 h at 4
°C in 0.5 ml containing 40 mM Tris-HCl (pH 7.5), 0.5
mM CaCl, 150 mM 2-mercaptoethanol, 0.2
mg/ml BSA, glutathione-Sepharose 4B (40 µl of 50% (v/v) slurry),
and either 8 µg of fusion protein GST/A328-390 plus 8 µg
of GST (lane 1) or 16 µg of GST (lane 2). The
Sepharose beads were collected by centrifugation, washed three times
with 40 mM Tris-HCl (pH 7.5), 0.5 mM CaCl
, 300 mM NaCl, resuspended in 50 µl
of 2
SDS-PAGE sample buffer, and boiled for 3 min. Eluted
proteins were analyzed by SDS-PAGE (15%), stained by Coomassie Blue (panel A), or transferred to PVDF membrane and immunoblotted
with anti-CaN B antibody (panel
B).
Since CaN A residues 328-390 bind CaN B
subunit, GST/A328-390 should block the in vitro interaction between the CaN A and B subunits. In agreement with
our previous study(11) , the low phosphatase activity of
baculovirus/Sf9-expressed CaN A subunit (8 nmol/min/mg, Fig. 3A) was greatly enhanced (300-350
nmol/min/mg, Fig. 3A) after in vitro reconstitution with the B subunit (Fig. 3A). When
the CaN A and B subunits were co-expressed in Sf9 cells, the purified
CaN had an activity of about 700 nmol/min/mg CaN A, and purified brain
CaN had an activity of 410 nmol/min/mg CaN A under the same assay
conditions(11) . Thus, reconstitution with the B subunit
resulted in a 40-fold increase in the phosphatase activity of the A
subunit to a value equivalent to purified brain CaN and about half that
of co-expressed CaN. However, increasing concentrations of
GST/A328-390 inhibited this in vitro reconstitution with
an IC of 50-100 nM (Fig. 3B). This represents potent inhibition since
the concentrations of CaN A and B subunits were each 30 nM.
Inhibition by GST/A328-390 was specific since 1) GST alone was
without effect (Fig. 3B), and 2) addition of 600 nM fusion protein to co-expressed CaN A/B gave a small stimulation of
phosphatase activity (not shown).
Figure 3:
Reconstitution of CaN A and B subunits in
the absence and presence of fusion protein GST/A328-390. Panel A, time course of subunit reconstitution. CaN A (50
nM) was incubated with CaN B (50 nM) at 30 °C in
40 mM Tris-HCl (pH 7.5), 1.5 mM Mn,
150 mM
-mercaptoethanol, and 2 mg/ml BSA. At the
indicated times aliquots were removed and assayed 10 min for
phosphatase activity using 60 µM
P-R
peptide and 500 nM CaM as described under
``Experimental Procedures.'' The phosphatase activity of
co-expressed CaN A/B using the same conditions was 668 ± 33
nmol/min/mg CaN A. Panel B, inhibition of CaN A and B subunit
reconstitution by GST/A328-390. CaN A (30 nM) was
incubated with the indicated concentrations of GST/A328-390 or
GST for 1 h prior to incubation for another hour with CaN B (30
nM). The reconstitution conditions and phosphatase assay were
the same as in panel A. Assays in panels A and B were performed in duplicate, and each point represents the mean
± S.D. from two experiments.
These mutant CaN A
subunits were expressed individually in Sf9 cells and purified on
CaM-Sepharose. Analysis by SDS-PAGE and Western blot indicated highly
purified subunits (Fig. 4). The low basal phosphatase activities
of these expressed mutant CaN A subunits, assayed in the presence of
Mn, were equivalent to the basal activity of the
wild-type A subunit (Fig. 5, left bars). Wild-type and
mutant A subunit phosphatase activities were each stimulated
5-6-fold by Mn
/CaM (Fig. 5, middle
bars). Thus, the mutant CaN A subunits had indistinguishable
properties relative to wild-type A subunit in terms of basal
phosphatase activity and activation by CaM. However, the mutant A
subunits did not exhibit activation by B subunit upon in vitro reconstitution whereas the wild-type A subunit was stimulated
40-fold (Fig. 5, right bars). Fig. 5(inset) shows that wild-type A subunit was
maximally activated by a 1:1 molar ratio of B subunit to A subunit, but
the mutants were not affected by 3.4-fold molar excess of B subunit.
Our reconstitution conditions included 1.5 mM Mn
, and we previously determined that the
presence of Ca
had no effect. Since the B subunit is
a Ca
-binding protein, we also tested for
reconstitution of the mutant A subunits with the B subunit in the
presence of 0.5 mM Ca
, but there was still
no detectable activation of the mutant A subunits by B subunit (data
not shown). Because the reconstitution is relatively slow (Fig. 3A), there may be refolding of either or both
subunits during the incubation. If the mutant A subunits were
improperly folded, they may not be able to properly unfold to interact
with B subunit. We added 6 M urea, which is known to cause
dissociation of the A and B subunits(13) , to a mixture of B
subunit plus either wild-type or mutant A subunits, incubated them
overnight and then dialyzed out the urea prior to assaying phosphatase
activity. The resulting phosphatase activities of wild-type and mutant
A subunit plus B subunit incubations were essentially identical to the far right bars in Fig. 5(data not shown).
Figure 4: SDS-PAGE and Western blot analyses of purified wild-type and mutant CaN A subunits. Lane 1, wild-type CaN A; lane 2, mutant E CaN A; lane 3, mutant Q CaN A. Proteins (2 µg) were separated by 10% SDS-PAGE and stained by Coomassie Blue (panel A) or transferred to PVDF membrane and immunoblotted with anti-CaN A antibody (panel B).
Figure 5:
Effects of mutagenesis of CaN A subunit on
phosphatase activities and reconstitution with B subunit. Purified
wild-type CaN A and mutants Gln (Q) and Glu (E) at 50 nM were
assayed at 30 °C for 10 min without (left bars) or with (middle bars) CaM (500 nM) in the presence of 1.5
mM Mn and 60 µM
P-RII peptide. Each subunit (50 nM) was
reconstituted for 2 h with 170 nM CaN B subunit (see
``Experimental Procedures'') prior to determining phosphatase
activities (right bars). The inset shows the effect
of the molar ratio of B subunit to A subunit on the reconstitution. All
assays were performed in duplicate, and each point represents the mean
± S.D. from two experiments.
Figure 6:
Co-expression of CaN B with wild-type and
mutant CaN A subunits. Panel A, Sf-9 cells were co-infected
with wild-type CaN A (lane 2), mutant E CaN A (lane
3), or mutant Q CaN A (lane 4) and CaN B at a m.o.i. of 4
and 6, respectively. Cells were harvested 72 h post-infection,
homogenized in buffer A and centrifuged at 10,000 g for 60 min, and the supernatants were subjected to SDS-PAGE. Lane 1 is a crude extract from non-infected Sf9 cells.
Proteins were transferred to PVDF membranes and treated with anti-CaN A
and anti-CaN B. Panel B, the supernatants were purified on
CaM-Sepharose chromatography, and the EGTA eluants were analyzed by
SDS-PAGE. Western blot analysis was performed as
above.
This study used multiple approaches to identify the region of
the CaN A subunit that is involved in binding of the regulatory B
subunit. The B subunit binding domain was likely to be located between
the catalytic domain and the CaM-binding domain, since limited
proteolysis in the absence of Ca/CaM cleaves the A
subunit NH
-terminal of the CaM-binding domain but the B
subunit is still associated with the catalytic A subunit(21) .
We focused on the highly conserved (Fig. 1) sequence
328-390 between the catalytic domain and the CaM-binding domain.
The fusion protein GST/A328-390 containing the putative B
subunit-binding domain specifically bound CaN B (Fig. 2) and
potently inhibited in vitro interaction of the A and B
subunits (Fig. 3B). Interaction of the CaN A and B
subunits requires 6 M urea for dissociation(13) ,
suggesting the potential importance of hydrophobic interactions. Since
the synthetic peptide corresponding to CaN A sequence 341-360
blocked binding of CaN B to GST/A328-390 (data not shown), we
focused on hydrophobic residues (Phe and Val) within 341-360 and
mutated two hydrophobic clusters to either Gln or Glu. Glu and Gln were
chosen because they are both very hydrophilic and they have side-chain
bulkiness close to Phe and Val. The mutant A subunits were well
expressed in Sf9 cells and purified on CaM-Sepharose (Fig. 4).
However, the two mutant CaN A subunits did not interact with the B
subunit as determined by both enzymatic assay after in vitro reconstitution (Fig. 5) and physical detection after
co-expression and purification on CaM-Sepharose (Fig. 6). The
inability of the Gln mutant to bind the B subunit was presumably due to
the decreased hydrophobicity of residues 347-357, whereas the
lack of B subunit binding of the Glu mutant could additionally involve
the acidic charges. The CaN B subunit is an acidic protein(2) .
The inability of the mutant A subunits to bind B subunit was probably
not due to major conformational changes induced by the mutations since
1) both mutant A subunits alone had Mn-stimulated
phosphatase activities similar to wild-type A subunit (Fig. 5, far left bars), and 2) both mutant A subunits bound (i.e. were purified on CaM-Sepharose) and were stimulated by (Fig. 5, middle bars) Ca
/CaM. Since
the catalytic (residues 71-325) and CaM-binding (residues
391-414) domains are normal in the mutant A subunits and they
flank the region of mutagenesis (residues 349-357), any putative
structural changes would be confined to the B subunit-binding domain.
Furthermore, in vitro reconstitution after exposure of both
subunits to 6 M urea was normal for wild-type A subunit but
did not occur with either mutant. Finally, the mutant A subunits,
unlike wild-type A subunit, did not interact with B subunit when they
were co-expressed in Sf9 cells (Fig. 6).
Binding of the B
subunit to the catalytic CaN A subunit is essential to its activity and
regulation of its phosphatase activity. We have recently determined
that Ca binding to the B subunit activates CaN by
both decreasing its K
and increasing the V
, whereas Ca
binding to CaM
increases only V
.
It will be
important to determine the mechanism by which binding of the B subunit
to the A subunit alters the phosphatase kinetic properties, and this
identification of the domain in the A subunit which binds the B subunit
will facilitate further study. Our results identifying hydrophobic
residues 349/350 and 356/357 in the A subunit as essential for binding
of the B subunit do not exclude the potential involvement of other
residues (e.g. residues 365-371, LVNVLNI, Fig. 1B) in B subunit binding. We are also initiating a
study to determine residues in the B subunit involved in binding to the
A subunit.