(Received for publication, April 7, 1995; and in revised form, August 9, 1995)
From the
Calcineurin is a heterodimeric
Ca/calmodulin-dependent protein phosphatase that
regulates signal transduction and is the target of
immunophilin-immunosuppressive drug complexes in T-lymphocytes and in
yeast. Calcineurin is composed of a catalytic A subunit and a
regulatory B subunit that is myristoylated at its amino terminus. We
employed genetic and biochemical approaches to investigate the
functional roles of myristoylation of calcineurin B (CNB1) in Saccharomyces cerevisiae. A calcineurin B mutant in which
glycine 2 was substituted by alanine (CNB1-G2A) did not incorporate
[
H]myristate when expressed in yeast. Both
wild-type calcineurin B and the CNB1-G2A mutant protein are partially
associated with membranes and cytoskeletal structures; hence,
myristoylation is not required for these associations. In several
independent genetic assays of calcineurin functions (recovery from
-factor arrest, survival during cation stress, and viability of a
calcineurin-dependent strain), the nonmyristoylated CNB1-G2A mutant
protein exhibited full biological activity. In vitro, both
wild-type and CNB1-G2A mutant proteins formed complexes with both
cyclophilin A-cyclosporin A (CsA) and FKBP12-FK506 that contained
calcineurin A. Interestingly, expression of the nonmyristoylated
CNB1-G2A mutant protein rendered yeast cells partially resistant to the
immunosuppressant CsA, but not to FK506. This study demonstrates that
calcineurin B myristoylation is not required for function, but may
participate in inhibition by the cyclophilin A-CsA complex.
Calcineurin is a Ca/calmodulin-regulated
serine/threonine-specific protein phosphatase. The fully active enzyme
is a heterotrimer composed of a 60-kDa catalytic A subunit, a 19-kDa
regulatory B subunit, and calmodulin(1) . The regulatory B
subunit is a calcium-binding protein with four EF hands and marked
similarity to calmodulin. Recent NMR studies confirm that calcineurin B
and calmodulin share similar secondary structures(2) . However,
these two calcium-binding proteins cannot replace each other, and both
are required for full activation of calcineurin(1) . In
addition, Ca
binding to calcineurin B and calmodulin
has different effects on calcineurin activity(3) .
Calcineurin plays important roles in mediating different biological
responses including regulation of the vacuolar
Ca-ATPase PMC1 in yeast(4) , the renal
Na
,K
-ATPase in mammals (5) ,
a potassium channel in plants(6) , and the N-methyl D-aspartate channel in vertebrates(7) . Several
independent studies have demonstrated that calcineurin is required for
adaptation of the yeast Saccharomyces cerevisiae to high salt
stress(8, 9, 10) . Calcineurin has also been
shown to participate in neuronal signaling pathways for long-term
depression(11) , neurotoxicity(12) , and neurite
outgrowth(13) .
In complex with the immunophilins
cyclophilin A and FKBP12, the immunosuppressants cyclosporin A (CsA) ()and FK506 bind to and inhibit calcineurin ((14) ;
reviewed in (15, 16, 17) ). The cytoplasmic
subunit of a transcription factor required for T-cell activation
(NF-ATc) is a substrate for calcineurin in vitro(18) . Dephosphorylation of NF-ATc by calcineurin may
promote nuclear translocation and transcription of T-cell activation
genes(19) . Physical interactions between calcineurin and
immunophilin-drug complexes have been previously examined by chemical
cross-linking and photoaffinity
labeling(20, 21, 22) . Recently, a combined
molecular modeling and mutagenic approach has implicated a latch region
of calcineurin B in both immunophilin-drug complex binding and
calcineurin phosphatase activity(23) .
Both immunophilins
and calcineurin are highly conserved. The yeast S. cerevisiae expresses homologs of mammalian cyclophilin A (24) ,
FKBP12(25) , calcineurin A (CMP1 and CMP2 isoforms), and
calcineurin B
(CNB1)(26, 27, 28, 29) . Genetic
studies demonstrated that calcineurin is required for recovery from
-factor mating pheromone in
yeast(26, 29, 30) . Recent studies have
provided direct evidence that yeast cyclophilin A and FKBP12 are
required for CsA and FK506 cytotoxicity, respectively, and that
calcineurin is essential in CsA- and FK506-sensitive
strains(9, 31) . These results demonstrate that, as
previously established in T-cells, the yeast target of the cyclophilin
A-CsA and FKBP12-FK506 complexes is also calcineurin. In addition,
Cardenas et al.(32) have recently demonstrated that
immunophilins and calcineurin functionally interact in the absence of
exogenous immunosuppressants.
In both fungi and vertebrates, the
calcineurin B regulatory subunit is modified by amino-terminal
myristoylation(26, 33) . A variety of observations
suggest that myristoylation functions in protein-protein interactions,
membrane association, and protein folding(34) . Several N-myristoylproteins are kinases or phosphatases that play
crucial roles in signal transduction. For instance, myristoylation of
the yeast GTP-binding protein -subunit is essential for membrane
association (35) and inhibition of
G
(36) . Myristoylation is required for
transactivation by the FBR v-Fos protein, a retrovirally transduced
c-fos proto-oncogene homolog(37) . Myristoylation of
pp60
is required for membrane association and
oncogenic transformation(38) . Finally, only myristoylated
forms of the abl tyrosine kinase recruit phosphatidylinositol
3-kinase to the plasma membrane(39) .
In this report, we present genetic and biochemical evidence that myristoylation of calcineurin B is not required for biological activity or for association with the calcineurin A catalytic subunit, with membranes, with a Triton-insoluble cytoskeletal fraction, or with the immunosuppressive FKBP12-FK506 and cyclophilin A-CsA complexes. Thus, we conclude that myristoylation is not required for several calcineurin functions in yeast.
For subcellular localization,
yeast cultures were grown as described above. Cells were broken with a
bead beater, and unbroken cells were pelleted at 5000 rpm for 5 min at
4 °C. Clarified cell lysate was centrifuged at 100,000 g for 30 min at 4 °C, and supernatant was collected. The
pellet was resuspended in 500 µl of lysis buffer and is referred to
as the particulate fraction. Particulate fractions (100 µl) were
further extracted with 1% Triton X-100 or 1% sodium cholate for 1 h at
0 °C. The extracted mixture was centrifuged at 100,000
g for 30 min at 4 °C, and the clear supernatant is
referred to as the soluble fraction. The pellet was resuspended in 100
µl of lysis buffer and is referred to as the resistant pellet
fraction. Yeast cell fractions were subjected to Western blot analysis
as described above.
To test for functional roles of amino-terminal myristoylation of
calcineurin B, a G2A mutation was generated by site-directed
mutagenesis of the cloned CNB1 gene. Plasmids expressing the
CNB1-G2A mutant protein were introduced into a host strain deleted for
the calcineurin B gene, and expression and myristoylation were analyzed
by Western blotting with rabbit anti-CNB1 antisera and in vivo labeling with [H]myristic acid performed in
parallel with the same samples. As shown in Fig. 1A,
both wild-type CNB1 (lanes 1 and 3) and the CNB1-G2A
mutant protein (lane 4) were expressed at equivalent levels,
whereas no CNB1 protein was detectable in the
cnb1 mutant
host strain transformed with the control vector alone (lane
2). To demonstrate that the yeast CNB1-G2A mutant protein was not
myristoylated, equal amounts of protein extracts from cells
metabolically labeled with [
H]myristic acid were
immunoprecipitated with antisera against yeast CNB1, and the
immunoprecipitates were analyzed by SDS-PAGE and autoradiography.
Whereas the wild-type CNB1 protein was labeled with
[
H]myristic acid (Fig. 1B, lanes 1 and 3), the CNB1-G2A mutant protein was not (lane 4). Thus, the G2A mutation abolishes myristoylation of
the yeast calcineurin B protein.
Figure 1:
Wild-type yeast calcineurin B is
myristoylated; the CNB1-G2A mutant protein is not. Isogenic yeast
strains with the indicated genotypes were metabolically labeled with
[H]myristate. A, proteins were extracted
and subjected to 15% SDS-PAGE followed by Western blotting with rabbit
anti-yeast CNB1 antiserum. B, calcineurin B was
immunoprecipitated from equal amounts of protein extracts prepared from
cells metabolically labeled with [
H]myristic
acid. Immunoprecipitates were fractionated by 15% SDS-PAGE and analyzed
by autoradiography with fluorography (see ``Materials and
Methods''). Exposure was for 30 days at -70 °C with an
enhancing screen. Isogenic yeast strains were JK9-3da (wild-type (WT)), TB85 (
cnb1), TB85/pYDZ3 (
cnb1/CNB1), and TB85/pYDZ10 (
cnb1/CNB1-G2A) (see Table 1and ``Materials
and Methods'').
Figure 2:
Myristoylated CNB1 and nonmyristoylated
CNB1-G2A are recovered in both cytosolic and membrane fractions. Total
cell-free lysates (T) or cytosolic (supernatant (S))
and particulate (P) fractions (see ``Materials and
Methods'') were analyzed by SDS-PAGE and Western blotting with
antisera against calcineurin B CNB1 (A), calcineurin A CMP1 (B), PMA1 (C), and yeast cyclophilin A CPR1 (D) as described for Fig. 1A. Strains were
JK9-3da (wild-type (WT)) and TB85/pYDZ10 (cnb1/CNB1-G2A).
The particulate fraction includes both
membranes and components of the cytoskeletal matrix. Densitometric
quantification of the Western blot shown in Fig. 2A revealed that 65% of CNB1 was associated with the particulate
fraction. To test whether CNB1 in the particulate fraction is
associated with the cytoskeletal matrix, the particulate fractions from
strains expressing wild-type CNB1 or nonmyristoylated CNB1-G2A were
further treated with either 1% Triton X-100 or 1% sodium cholate, and
the soluble and resistant fractions were recovered by centrifugation
and analyzed by Western blotting. As shown in Fig. 3and
quantified by densitometry, 40% of CNB1 and CNB1-G2A was extracted by a
mock extraction with buffer alone (Fig. 3, A and B, lanes 1 and 2). In contrast, 80% of
wild-type CNB1 and the CNB1-G2A mutant protein was extracted by Triton
X-100, and similar amounts of both wild-type CNB1 and the CNB1-G2A
mutant protein were detected in the Triton-soluble and Triton-resistant
fractions (Fig. 3, A and B, lanes 3 and 4). Based on previous reports, Triton-insoluble
proteins are associated with cytoskeletal structures in yeast and other
organisms(49, 50, 51, 52, 53) .
As a control for cytoskeletal association, control Western blotting was
performed, revealing that a similar proportion of the known yeast
cytoskeletal protein actin was resistant to Triton extraction (Fig. 3, C and D, lanes 3 and 4). The cytoskeleton association of yeast CNB1 was further
tested by treatment of the particulate fractions with 1% sodium
cholate, which removes proteins associated with the cytoskeleton. The
majority (80-90%) of both wild-type CNB1 and the CNB1-G2A mutant
protein was extracted by sodium cholate (Fig. 3, A and B, lanes 5 and 6). The small fraction not
extractable by sodium cholate may represent insoluble protein. Taken
together, these findings demonstrate that, in yeast, CNB1 associates
with both membranes and cytoskeletal structures and that myristoylation
is not required for either association.
Figure 3:
Membrane and cytoskeletal association of
wild-type CNB1 and the CNB1-G2A mutant protein. Equal amounts of the
particulate fraction (P) were extracted with buffer alone or
with buffer containing 1% Triton X-100 or 1% sodium cholate (see
``Materials and Methods''). Samples analyzed were
buffer-soluble (S; lane 1), buffer-resistant (P; lane 2), Triton-soluble (S; lane
3), Triton-resistant (P; lane 4), sodium
cholate-soluble (S; lane 5), and sodium
cholate-resistant (P; lane 6). Fractions were
analyzed by Western blotting with antisera against the calcineurin B
subunit CNB1 (A and B) or yeast actin (C and D) as described for Fig. 1A. Proteins were
from isogenic wild-type CNB1 strain JK9-3da (wild-type (WT)) (A and C) and CNB1-G2A mutant strain TB85/pYDZ10 (cnb1/CNB1-G2A) (B and D).
Figure 4:
Ca-dependent mobility
shift of wild-type CNB1 and the CNB1-G2A mutant protein. EGTA (5
mM) or Ca
(5 mM) was added to
protein extracts in SDS sample loading buffer, and proteins were
separated by 15% SDS-PAGE and analyzed by Western blotting with
anti-CNB1 antisera as described for Fig. 1A. Protein
extracts were from isogenic wild-type CNB1 strain JK9-3da (wild-type (WT)) and CNB1-G2A mutant strain TB85/pYDZ10 (
cnb1/CNB1-G2A).
Figure 5:
The nonmyristoylated CNB1-G2A mutant
protein is functional and renders yeast cells partially resistant to
CsA, but not to FK506. Isogenic yeast strains lacking FKBP12 (fpr1, JHY3-3D), cyclophilin A (
cpr1,
MH250-2C), and calcineurin B (
cnb1, TB85) and the
calcineurin B-deficient strain TB85 (
cnb1) expressing
either wild-type CNB1 (
cnb1/CNB1) or the CNB1-G2A mutant (
cnb1/CNB1-G2A) were grown for 96 h at 30 °C on the
following: A, YPD medium; B, YPD medium containing
200 mM LiCl; C, YPD medium containing 200 mM LiCl plus 100 µg/ml CsA; and D, YPD medium containing
200 mM LiCl plus 1 µg/ml
FK506.
Calcineurin is also
required for yeast cells to recover from -factor pheromone-induced
cell cycle arrest(26, 29, 30, 32) .
The ability of the CNB1-G2A mutant protein to function and promote
adaptation to pheromone was tested by halo assay. Cells lacking
calcineurin B (
cnb1) were unable to recover from
pheromone arrest (Fig. 6). In contrast, expression of either the
wild-type or the G2A mutant calcineurin B protein enabled a calcineurin
B mutant host strain to recover and resume growth following growth
arrest by
-factor (Fig. 6). Thus, by a second measure, the
nonmyristoylated CNB1-G2A mutant protein can functionally replace
wild-type yeast calcineurin B.
Figure 6:
Yeast
cells expressing nonmyristoylated CNB1-G2A recover from
-factor-induced G
arrest. Cells of isogenic MATa yeast
strains were plated in YPD top agar medium, and discs soaked with 10
µg of synthetic
-factor were placed on the surface. Arrest of
cell growth by
-factor results in a zone of growth inhibition
(halo) surrounding the disc. With prolonged incubation, wild-type MATa
cells overcome this initial arrest, resume growth, and form colonies
within the halo. In contrast, mutant strains that cannot recover from
cell cycle arrest induced by
-factor yield halos that, even with
prolonged incubation, remain clear. Portions of halos were photographed
after 96 h of incubation at 30 °C. Isogenic strains were as
follows: wild-type (WT), JK9-3da;
cnb1, TB85;
cnb1/CNB1, TB85 expressing wild-type CNB1; and
cnb1/CNB1-G2A, TB85 expressing the nonmyristoylated
CNB1-G2A mutant protein.
Biological function of the
nonmyristoylated calcineurin B mutant protein was also assessed in a
yeast strain whose viability is calcineurin-dependent. In an
independent study, we isolated a recessive mutation, cev1,
which exhibits CsA-FK506 sensitivity and synthetic lethality with a
calcineurin cnb1 mutation. ()Thus, the cev1 mutation renders calcineurin essential for viability, as in other
CsA-FK506-sensitive
strains(9, 31, 56, 57) . We tested
if the CNB1-G2A calcineurin B mutant subunit supports
calcineurin-dependent growth in a cev1 cnb1 double mutant
strain. The cev1 mutant strain was mated to isogenic
cnb1::LEU2 strains of opposite mating type containing the
control plasmid pRS316 or plasmids expressing wild-type CNB1 (plasmid
pYDZ3) or the CNB1-G2A mutant protein (plasmid pYDZ10). The resulting
diploids were sporulated, and meiotic segregants were analyzed by
tetrad analysis. As previously observed, the cev1 and cnb1 mutations exhibited synthetic lethality. In contrast, with the cev1/CEV1 cnb1/CNB1 (CNB1 plasmid) and the cev1/CEV1
cnb1/CNB1 (CNB1-G2A plasmid) diploids, dissection yielded 11
tetrads with 4 viable:0 inviable, six with 3 viable:1 inviable, and
three with 2 viable:2 inviable segregants and 12 with 4 viable:0
inviable, seven with 3 viable:1 inviable, and one with 2 viable:2
inviable segregants, respectively. More important, many segregants were
CsA
FK506
(cev1), Leu
(
cnb1::LEU2), and Ura
5-FOA
(CNB1 or CNB1-G2A plasmid present and essential). Thus, either
wild-type CNB1 or the CNB1-G2A mutant protein restores viability to an
inviable cev1 cnb1 mutant strain. In summary, as determined by
three independent tests (cation resistance, recovery from
-factor,
and viability in a calcineurin-dependent strain), myristoylation of
calcineurin B appears not to be required for function.
Figure 7:
Myristoylated and nonmyristoylated
calcineurin B proteins interact with FKBP12-FK506 and cyclophilin A-CsA
complexes. Binding assays to His-FKBP12-affigel 10 and
His
-cyclophilin A-affigel 10 of cell extracts prepared from
strain TB85 lacking CNB1 (
cnb1) and from strain TB85
expressing wild-type CNB1 (
cnb1/CNB1) or CNB1-G2A (
cnb1/CNB1-G2A) protein from a CEN plasmid were
carried out in the absence or presence of 20 µM FK506 or
100 µM CsA. Following elution of the affinity matrices,
proteins were analyzed by SDS-PAGE and Western blotting. The upper half
of the blot (A) was probed with an affinity-purified
calcineurin A antibody, and the lower half of the blot (B) was
probed with calcineurin B antiserum. Arrows indicate the
positions of migration of calcineurins A and
B.
We next tested if myristoylation is required for calcineurin inhibition by immunophilin-drug complexes in vivo. A yeast calcineurin B-deficient strain was transformed with plasmid pYDZ3 expressing wild-type CNB1 and with plasmid pYDZ10 expressing the CNB1-G2A mutant. Sensitivity of the transformed yeast cells to CsA and FK506 was tested in the presence of LiCl. Yeast strains lacking cyclophilin A or FKBP12 were resistant to CsA plus LiCl or FK506 plus LiCl, respectively (Fig. 5, C and D). Growth of yeast cells expressing wild-type CNB1 was inhibited by either CsA or FK506 (Fig. 5, C and D). Interestingly, cells expressing the CNB1-G2A mutant protein were partially resistant to CsA plus LiCl (Fig. 5C), but not to FK506 plus LiCl (Fig. 5D). Although myristoylation of CNB1 is not required for cyclophilin A-CsA binding to calcineurin (Fig. 7), this observation suggests that it may be involved in inhibition of calcineurin by the cyclophilin A-CsA complex.
Physiological functions of myristoylation have been studied
for viral and cellular proteins including Ras- and Src-related proteins (58) and G protein -subunits(59) . Myristoylation
can participate in protein-membrane associations and, in some cases, is
required for biological function(59) . However, not all
myristoylated proteins associate with membranes(60) , and for
some proteins, such as the catalytic subunit of protein kinase A,
myristoylation is not required for membrane association or biological
function(58, 61) .
The calcineurin B regulatory subunit from mammals and yeast is covalently myristoylated at its amino-terminal glycine residue(29, 33) . In addition to the hydrophobic myristate, calcineurin is the target for two hydrophobic inhibitors, CsA and FK506, and acidic phospholipids bind to calcineurin B and activate calcineurin(62, 63) . Interactions between these hydrophobic moieties could influence calcineurin folding, assembly, activity, or association with proteins, substrates, or membranes. To test this, we introduced a G2A mutation to prevent myristoylation of yeast calcineurin B in vivo and used genetic and biochemical approaches to examine whether myristoylation of the calcineurin B regulatory subunit is required for cellular localization, biological functions, or association with and inhibition by the cyclophilin A-CsA or FKBP12-FK506 immunophilin-drug complex.
We find that both wild-type calcineurin B and the G2A mutant protein
are localized to both cytosol and particulate fractions (Fig. 2). A portion of calcineurin B in the particulate fraction
is detergent-extractable, indicating that calcineurin B is
membrane-associated and that myristoylation is not required for this
association (Fig. 3). A smaller fraction of the CNB1-G2A mutant
protein was pelletable (Fig. 2), suggesting that the myristoyl
group at least partially contributes to membrane association of yeast
calcineurin B. Recent studies of Src-related proteins suggest how
nonmyristoylated mutants of myristoylated proteins continue to
associate with membranes. Three alternating amino-terminal lysine
residues function, in conjunction with myristate, to bind proteins to
membranes by electrostatic interactions with negatively charged
membrane
phospholipids(58, 64, 65, 66, 67, 68) .
A similar amino-terminal basic amino acid motif in yeast calcineurin B
(NH-GAAPSKIDRDEIERLRKRFMKLDRD-COOH)
may participate in membrane association.
Recoverin, a myristoylated
member of the EF-hand superfamily that is a Ca sensor
in the visual system, associates with membranes by a
Ca
-myristoyl switch in which Ca
binding promotes extrusion of the myristoyl group and exposure of
hydrophobic groups in recoverin(69, 70) . A similar
GTP-myristoyl switch mediates GTP-dependent association of the
ADP-ribosylation factor with membranes(71, 72) . A
Ca
-myristoyl switch could also operate in the
calcineurin B regulatory subunit; purified calcineurin B contains
exposed hydrophobic patches that predispose to aggregation(2) .
Because of the association of yeast CNB1 with the particulate
fraction, we examined the possible association of yeast CNB1 with
cytoskeletal matrices by detergent treatment of particulate fractions.
This technique has been used to analyze cytoskeletal association of
p60(49, 51, 73) ,
v-Fgr(74) , p120
(75) , Nef of human
immunodeficiency virus type 1(52, 76) , and the yeast
SEC1 protein(53) . By Triton extraction of the yeast
particulate fraction, a fraction of the yeast calcineurin B subunit is
cytoskeleton-associated. The nonmyristoylated CNB1-G2A calcineurin B
mutant protein was also associated with the cytoskeleton (Fig. 3), indicating that myristoylation is not required for
this association. In mammals, calcineurin has also been found to be
associated with the cytoskeleton in adrenal cells (77, 78) and neurons(79) .
Although we found that myristoylation of the calcineurin B regulatory subunit was not essential for association with cellular membranes or the cytoskeleton, myristoylation could have been required for calcineurin subunit interactions or activity. However, our genetic studies reveal that the CNB1-G2A mutant is fully functional in several independent biological assays, and we conclude that myristoylation of calcineurin B is not required for calcineurin function in yeast. Our findings are in accord with previous biochemical studies that revealed myristoylation of calcineurin B was not required for association of calcineurin A and B subunits (80, 81) or for association of heterologous calcineurin subunits and reconstitution of phosphatase activity (82) and with the recent demonstration that reconstituted nonmyristoylated calcineurin is enzymatically active in vitro(83) .
Calcineurin transduces signals required for
T-cell activation and yeast pheromone response and regulates the renal
Na,K
-ATPase(5) , a potassium
channel in plants(6) , and cation fluxes in
yeast(4, 8, 9) . Myristoylation of the
calcineurin B regulatory subunit could be required for only a subset of
functions or under special conditions not explored in our assays.
Alternatively, as in other examples where myristoylation is not
detectably required for function, such as protein kinase A, the
nonmyristoylated mutant could confer only subtle defects. In the case
of protein kinase A, myristoylation is not required for activity, but
does contribute to stability(84, 85) .
Numerous studies have established that the calcineurin B subunit is required for and plays a central role in FKBP12-FK506 and cyclophilin A-CsA binding and inhibition of calcineurin(20, 21, 23, 32, 86, 87, 88) . However, it had not been tested whether myristoylation of the calcineurin B subunit participated in binding or inhibition of calcineurin by the cyclophilin A-CsA or FKBP12-FK506 complex. We found that both wild-type calcineurin B and the nonmyristoylated G2A mutant protein bound to FKBP12-FK506 and cyclophilin A-CsA affinity matrices (Fig. 7B). In addition, the calcineurin A catalytic subunit CMP1 associated with either immunophilin-drug complex irrespective of whether calcineurin B was myristoylated or not (Fig. 7A). Thus, myristoylation is not required for association of calcineurin B with calcineurin A or with either immunophilin-drug complex.
We next tested whether myristoylation is required for inhibition of calcineurin by CsA or FK506. Expression of the nonmyristoylated G2A calcineurin B mutant protein rendered yeast cells partially resistant to CsA, but not to FK506 (Fig. 5). In vitro, the CNB1-G2A mutant protein bound the cyclophilin A-CsA complex to the same extent as wild-type CNB1 (Fig. 7). One possibility is that our in vitro assay is not sensitive enough to detect a subtle decrease in binding affinity or does not faithfully mimic in vivo binding conditions. Alternatively, the CNB1-G2A mutation may confer partial CsA resistance by inhibiting some step subsequent to binding required for full inhibition of calcineurin. The cyclophilin A-CsA complex is a noncompetitive calcineurin inhibitor(89) , and binding of cyclophilin A-CsA stimulates calcineurin activity toward the substrate p-nitrophenyl phosphate(14, 90) . Thus, binding of cyclophilin A-CsA likely induces conformational changes that inhibit or activate calcineurin. The calcineurin B myristoyl group may participate in these conformational changes.