CN A Binds Its Substrates with a Wide Range of Affinities
We compared interactions of Cna1, which is one of two homologous CN A subunits in yeast, with its known interacting partners, using the two-hybrid system. The PJ69-4A yeast strain contains two reporter genes: GAL1
, which display low and high stringency for activation, respectively (James, 2001
). Cna1 physically interacts with its substrates Hph1, Slm1, and Slm2; the Rcn1 regulator; and the B subunit Cnb1, as all of these combinations resulted in growth on medium lacking histidine (). However, only the interactions of Cna1 with Slm2 and Cnb1 are sufficiently strong to activate GAL2
and allow growth on medium lacking adenine. Furthermore, the interaction of Cna1 with the substrate Slm2 promoted considerably stronger growth on media lacking histidine than did its interaction with Slm1 or Hph1 (). These results suggest that Cna1 interacts with its substrates with varying affinities. Crz1 was not tested in these experiments, as expression of the Crz1-Gal4AD (activation domain) fusion is toxic to yeast cells.
Yeast Calcineurin Substrates Have a Wide Range of Affinities for CN A
A PxIxIT motif mediates the association of Crz1, Slm1, Slm2, and Hph1 with calcineurin (). Therefore, we examined binding of yeast calcineurin to each of these motifs and to PVIVIT, the consensus PxIxIT peptide previously identified by in vitro selection for increased binding to calcineurin (Aramburu et al., 1999
). Fluorescently labeled PVIVIT peptide was added to increasing concentrations of purified, active, yeast calcineurin holoenzyme, and their interaction was measured using fluorescence polarization (Li et al., 2004
and Experimental Procedures). PVIVIT bound to yeast and human calcineurin with similar affinities (Kd
of ~2 µM and 0.5 µM, respectively) ( and Li et al., 2004
Next, peptides representing the various yeast PxIxIT sites were used to compete with yeast calcineurin-PVIVIT binding (, ). The peptides vary in sequence; however, each contains a core motif with a proline in the first position, hydrophobic residues in the third and fifth positions, and a hydrophilic residue in the sixth position (). The PVIVIT peptide had the highest binding affinity for yeast calcineurin. Peptides encoding the calcineurin-docking sites from Crz1, Slm2, and Slm1 were of decreasing affinity (Ki of 15 µM, 20 µM, and 40 µM, respectively), whereas a peptide representing the substrate Hph1 was of considerably lower affinity (Ki of 250 µM). These results are in general agreement with the yeast two-hybrid experiments () and suggest that targeting of substrates to yeast Cna1, at least via the PxIxIT motif, occurs with a wide range of affinities.
Changing the Calcineurin-Crz1 Interaction via PxIxIT Motif Mutations
As a tool to investigate the biology of calcineurin-substrate interactions, we substituted altered PxIxIT motifs into Crz1, a calcineurin substrate whose biological activity can be reliably assessed in vivo. We replaced the native Crz1 motif, PIISIQ, either with the high-affinity core sequence PVIVIT to create Crz1PVIVIT or with the low-affinity Hph1 sequence PVIAVN to create Crz1PVIAVN. Purified recombinant GST-Crz1 proteins (GST-Crz1, GST-Crz1PVIVIT, and GST-Crz1PVIAVN) were tested in the fluorescence polarization assay for their ability to compete with PVIVIT peptide for binding to calcineurin. All three proteins competed in the binding assay, with GST-Crz1PVIVIT most effective and GST-Crz1PVIAVN least effective (). This activity was due to CRZ1-encoded residues, as control experiments with GST revealed no appreciable interaction with fluorescent PVIVIT peptide in the concentration range used and no competition with calcineurin-PVIVIT binding (data not shown).
Competition by GST-Crz1 and GST-Crz1PVIAVN proteins differed from competition by the synthetic peptides in not achieving complete displacement of fluorescent PVIVIT. We have not investigated whether this difference at high protein concentrations reflects a technical limitation of the assay or an intrinsic property of the GST-Crz1 proteins. In either case, there are detectable differences in interaction at the PxIxIT-binding site at physiological concentrations. This fact encouraged us to proceed with biological studies.
Consequences of CN Binding-Affinity Changes on Crz1 Activity In Vivo
To determine the consequences of mutating the PxIxIT motif on Crz1 activity, Crz1, Crz1PVIVIT
, and Crz1PVIAVN
were expressed from low-copy number (centromeric) plasmids, in a strain lacking genomic Crz1 (crz1 Δ
) that contains a reporter gene comprised of four tandem repeats of the Crz1 DNA binding site (4×-CDRE-lacZ
) (Stathopoulos and Cyert, 1997
). We assessed Crz1-dependent transcription in these cells by measuring β-galactosidase activity before and after exposure to CaCl2
(). In these and subsequent experiments, extracellular addition of 50–200 mM CaCl2
was used to activate calcineurin signaling. This addition results in a rapid, transient rise in intracellular Ca2+
followed by a sustained plateau and has only a modest effect on yeast cell growth (Miseta et al., 1999
). A range of CaCl2
concentrations was used to achieve different levels of calcineurin activation. Similarly, in vivo, exposure of yeast to different environmental stress conditions results in Ca2+
signals of differing amplitude (Viladevall et al., 2004
; Denis and Cyert, 2002
PxIxIT Motif Mutations in Crz1 Alter Transcriptional Activity, In Vivo Phosphorylation States, and Nuclear Localization
Cells lacking Crz1 had no detectable CDRE-lacZ
activity (, “vector”). Cells expressing Crz1 displayed low basal CDRE-lacZ
activity, which reflects the low level of calcineurin activity in cells grown in standard media (Stathopoulos and Cyert, 1997
). Addition of CaCl2
caused an increase in CDRE-lacZ
expression whose magnitude was concentration dependent (e.g., 3.8-fold increase for 50 mM CaCl2
and 8.5-fold increase for 100 mM CaCl2
, ). In cells expressing the low-affinity Crz1PVIAVN
protein, both basal CDRE-lacZ
activity and Ca2+
activity were reduced. When exposed to Ca2+
, these cells exhibited substantially decreased Crz1 activation as compared to wild-type (~3.0-fold less than wild-type Crz1 in 50 mM CaCl2
and 1.5-fold less in 100 mM CaCl2
, ). Cells expressing the high affinity Crz1PVIVIT
protein exhibited a surprisingly high level of basal CDRE-lacZ
activity (~13-fold higher than wild-type, ). Upon treatment with Ca2+
, this level was further increased (1.5- to 2.0-fold in 50 mM CaCl2
and 100 mM CaCl2
, respectively, ). The Ca2+
-induced activity of all three Crz1 proteins was severely reduced by the calcineurin inhibitor, FK506, and was thus calcineurin dependent (). These results show that modest differences in the affinity of the Crz1 PxIxIT motif for calcineurin cause corresponding changes in both basal and Ca2+
-induced Crz1-dependent transcription. Furthermore, the amount of Crz1 activity elicited by a given amount of Ca2+
changed with affinity, showing that, in vivo, the Ca2+
concentration dependence of Crz1 is determined in part by its affinity for calcineurin.
Changes in the PxIxIT Motif Affect Crz1 Phosphorylation and Nuclear Localization
activity should directly reflect the extent of Crz1 dephosphorylation by calcineurin. Therefore, we examined each Crz1 protein by immunoblotting, as phosphorylation decreases Crz1 electrophoretic mobility (Stathopoulos-Gerontides et al., 1999
) (). Wildtype Crz1 was largely dephosphorylated in extracts of cells containing active calcineurin and was fully phosphorylated in extracts of FK506-treated cells (, lanes 2 and 3). Crz1PVIAVN
showed a similar FK506-dependent decrease in its electrophoretic mobility, although some slower-migrating forms were detected even in the absence of FK506 (, lanes 4 and 5). In contrast, Crz1PVIVIT
was predominantly in the hypophosphorylated, faster-migrating form, both in the presence and absence of FK506 (, lanes 6 and 7). Only a small fraction of the protein appeared to be phosphorylated in extracts of FK506-treated cells.
Dephosphorylation of Crz1 by calcineurin causes its translocation to the nucleus (Stathopoulos-Gerontides et al., 1999
; Boustany and Cyert, 2002
; Polizotto and Cyert, 2001
). Therefore, we examined the localization of GFP-tagged Crz1, Crz1PVIAVN
, and Crz1PVIVIT
(). As previously observed, Crz1 was predominantly cytoplasmic under standard growth conditions and rapidly translocated to the nucleus after addition of CaCl2
. Nuclear localization peaked 5 min after treatment with 200 mM CaCl2
, when 92% of cells displayed complete or partial nuclear localization. Fifteen minutes after Ca2+
addition, rephosphorylation of Crz1 resulted in its relocalization to the cytoplasm, and the population of cells that displayed full or partial nuclear localization was decreased to 18%. Exposure to 50 mM CaCl2
resulted in a significantly weaker response; 5 min after Ca2+
addition, 65% of cells still displayed cytosolic localization of Crz1.
When cells expressing Crz1PVIAVN were treated with CaCl2, nuclear localization was observed in fewer cells. For example, in response to 50 mM CaCl2, 0% of cells expressing Crz1PVIAVN displayed complete nuclear localization, compared to 12% of cells expressing wild-type Crz1 (). Thus, decreased Crz1PVIAVN activity correlated with decreased nuclear localization. Conversely, Crz1PVIVIT displayed nuclear localization even in cells grown without Ca2+ supplementation. Almost 70% of the cells showed complete or partial nuclear localization of Crz1PVIVIT when grown in standard media (). Upon addition of CaCl2, the percentage of cells in which Crz1PVIVIT was localized to the nucleus increased and then decreased again to steady-state levels ().
Thus, the different transcriptional activities of the Crz1 variants reflect their dephosphorylation by calcineurin and subsequent nuclear localization. We conclude that, compared to wild-type Crz1, calcineurin-dependent dephosphorylation and nuclear localization of Crz1PVIAVN is decreased and that of Crz1PVIVIT is greatly increased.
Calcineurin A Residues NIR (366–368) Mediate Interaction with the PxIxIT Motif
To explore further the contribution of calcineurin targeting to the distinct activities displayed by the Crz1 variants, we constructed an allele of CN A predicted to be defective in substrate interaction. In human CN A, M290 can be cross-linked to the PVIVIT peptide. Replacement of three residues that are adjacent on the protein surface to M290 (residues 330–332 in human CN Aα) with alanine (NIR-AAA), severely compromises binding to the PVIVIT peptide in vitro and dephosphorylation of NFAT in cell extracts (Li et al., 2004
). We mutated the corresponding, conserved residues in CNA1
, amino acids N366, I367, and R368 changed to alanine). These mutations had little effect on catalytic activity, as the bacterially expressed mutant enzyme displayed 80% of wild-type activity in vitro (see Figure S1 in the Supplemental Data
available with this article online). In contrast, Cna1NIR-AAA
was nonfunctional in vivo (), despite being expressed at levels comparable to Cna1 (data not shown). Yeast cells expressing Cna1NIR-AAA
as the only CN A sub-unit displayed sensitivities to salt stress, high pH, and increased concentrations of MnCl2
that were almost as severe as those of cells lacking CN A (). In yeast two-hybrid assays, the interaction of Cna1NIR-AAA
with CN B (Cnb1) was equivalent to that of Cna1 ( and data not shown), but Cna1NIR-AAA
failed to interact with calcineurin substrates Hph1, Slm1, or Slm2 or with the regulator Rcn1 (). These findings underscore the evolutionary and structural conservation of CN A-substrate interaction and highlight its importance for in vivo function.
Cna1NIR-AAA Is Nonfunctional Due to a Defect in Substrate Interaction, and Cells Expressing cna1NIR-AAA Show Reduced Activity of Crz1-PxIxIT Proteins
In cells containing Cna1NIR-AAA, the CDRE-lacZ activity promoted by Crz1, Crz1PVIAVN, or Crz1PVIVIT was greatly reduced (~8- to 10-fold) relative to that of Cna1-expressing cells, confirming that the PxIxIT-CN A interaction is critical for their activity (). However, we also noted that the relative activities of Crz1, Crz1PVIAVN, and Crz1PVIVIT were the same in both strains, with Crz1PVIAVN showing the lowest activity and Crz1PVIVIT showing the highest activity (). Thus, residues in the PxIxIT sequence apparently still influence the weak interaction of Cna1NIR-AAA with Crz1.
Hyperactivation of Crz1PVIVIT Requires Ca2+ and CN A
The consistent hyperactivation of Crz1PVIVIT
observed, especially in cells grown in the absence of added Ca2+
, led us to investigate the Crz1PVIVIT
-CN A interaction in more detail. First, we examined the Ca2+
dependence of Crz1PVIVIT
activation by measuring CDRE-lacZ
expression in cells whose cytosolic Ca2+
concentration was lowered by overexpression of Pmc1, a vacuolar Ca2+
-ATPase (Cunningham and Fink, 1994
). Both the basal and Ca2+
-stimulated activity of Crz1 and Crz1PVIVIT
decreased under these conditions (), establishing that activation of each protein was Ca2+
dependent. Similar results were also observed for Crz1PVIAVN
(data not shown).
Hyperactivation of Crz1PVIVIT Requires Ca2+ and Calcineurin Activity
Next, we examined the calcineurin dependence of Crz1PVIVIT
basal activity. We reasoned that increased affinity of Crz1PVIVIT
for calcineurin could cause its hyperactivation in a calcineurin-dependent manner (i.e., through a decrease in the Km
for the dephosphorylation reaction) and in a calcineurin-independent manner (i.e., by preventing association of Crz1PVIVIT
with protein kinases). First, we tested the effect of FK506 on basal CDRE-lacZ
activity and saw a 40% reduction in activity in cells expressing either Crz1 or Crz1PVIVIT
(). This is consistent with the basal activity of these proteins being calcineurin dependent but also suggests that FK506-treated cells retain residual calcineurin activity. By comparison, in cells completely devoid of calcineurin activity and CN A polypeptide (cna1Δ cna2Δ
) both Crz1 and Crz1PVIVIT
are fully phosphorylated and CDRE-lacZ
activity is barely detectable (≤1% of basal activity in WT cells) (). These findings demonstrate that in vivo, both Crz1 and Crz1PVIVIT
can be phosphorylated and fully inactivated by endogenous protein kinases and that the basal activity of Crz1PVIVIT
is dependent on CN A. Finally, we measured the activity of Crz1 and Crz1PVIVIT
in cells lacking CN B (cnb1Δ
). In these cells, CN A is present but should have little or no activity due to the absence of the regulatory subunit (Perrino et al., 1995
; Cyert and Thorner, 1992
). As expected, the basal CDRE-lacZ
activity in cnb1Δ
cells expressing Crz1 was undetectable and the protein was hyperphosphorylated in extracts of these cells (). Likewise, basal CDRE-lacZ
activity was substantially reduced in cnb1Δ
cells expressing Crz1PVIVIT
(), confirming that the high basal activity of Crz1PVIVIT
requires calcineurin activity.
Surprisingly, a low level of basal CDRE-lacZ
activity was consistently observed in cnb1Δ
cells expressing Crz1PVIVIT
(~10% of that observed for this protein in wild-type cells), and Crz1PVIVIT
was partially dephosphorylated in these cell extracts (). These findings may reflect the presence of residual calcineurin activity in cnb1Δ
cells. Alternatively, enhanced binding of CN A to Crz1PVIVIT
in these cells may interfere with the ability of kinases to phosphorylate Crz1, because two of three Crz1 kinases identified, Hrr25 and PKA, physically associate with the transcription factor (Kafadar and Cyert, 2004
; Kafadar et al., 2003
). Such an effect would also contribute to the hyperactivity of Crz1PVIVIT
Consequences of PxIxIT Motif Mutations on Resistance to Extracellular Stresses
To assess their physiological functions, cells expressing Crz1, Crz1PVIAVN, or Crz1PVIVIT were assayed for growth under a variety of stress conditions (). Cells lacking Crz1 (crz1Δ) showed a growth defect in the presence of high concentrations of Na+, Li+, and Mn2+ and high pH () that was complemented by expression of Crz1. Cells expressing Crz1PVIAVN grew less well than those expressing Crz1 on media containing high levels of Na+, Li+, and Mn2+, as well as the cell wall disruptant Congo red ( and data not shown). Conversely, the growth of cells expressing Crz1PVIVIT was more robust than that of cells expressing Crz1 under these same conditions. Thus, in the presence of Na+, Li+, Mn2+, and Congo red, the amount of growth promoted by each Crz1 protein mirrored its level of transcriptional activity.
Cells Expressing Crz1, Crz1PVIAVN, or Crz1PVIVIT Display Different Growth Properties under Environmental Stress Conditions
In contrast, in high pH medium, a different pattern of growth was displayed. Under these conditions, the growth of cells expressing Crz1 or Crz1PVIAVN was indistinguishable, whereas cells expressing Crz1PVIVIT exhibited a significant growth defect relative to cells expressing Crz1 (). Thus, in vivo, the consequences of changing Crz1-calcineurin affinity varied depending on the environmental conditions to which cells were exposed.
Mutations in the Zinc Finger Domain of Crz1 Elucidate One Mechanism Underlying High pH Sensitivity of CRZ1PVIVIT Cells
Crz1PVIVIT directed higher levels of transcription, suggesting that the alkaline growth defect of cells expressing this protein might result from increased expression of one or more Crz1-dependent gene products. Alternatively, the increased affinity of Crz1PVIVIT for CN A might preclude calcineurin’s interaction with other substrates or regulators whose activity is required for growth in high pH medium.
Nonfunctional Crz1 proteins were used to separate the effects of Crz1 binding to calcineurin from its effects on gene expression. We introduced mutations into the first zinc finger domain in Crz1 that should abrogate DNA binding and thus interfere with its ability to drive transcription (). Two such mutant alleles were constructed, crz1-znfd (for zinc-finger defective) and crz1PVIVIT-znfd. These mutants exhibited little CDRE-lacZ activity and were defective for growth under a variety of stress conditions ( and data not shown). Both mutant proteins were expressed at levels similar to wildtype Crz1 (data not shown).
Mutations in the Crz1 Zinc Finger Domain Separate Transcriptional Activity from CN Binding
Expression of Crz1PVIVIT-znfd in wild-type cells resulted in reduced CDRE-lacZ activity (). Thus, Crz1PVIVIT-znfd dominantly interferes with Crz1 function. This is likely due to titration of CN A, as expression of Crz1-znfd showed no such effect. In contrast, Crz1PVIVIT expression significantly increased CDRE-lacZ activity.
The effects of these mutant alleles on wild-type cell growth were examined under conditions of environmental stress. In growth medium with elevated Li+, Na+, or Mn2+, expression of Crz1PVIVIT, but not Crz1PVIVIT-znfd or Crz1-znfd, improved growth relative to wild-type cells ( and data not shown), confirming that Crz1PVIVIT promotes growth under these conditions by increasing Crz1-dependent transcription. Expression of either Crz1PVIVIT or Crz1PVIVIT-znfd in wild-type cells, but not expression of Crz1-znfd, resulted in decreased growth on high pH medium (). However, as described above, Crz1PVIVIT and Crz1PVIVIT-znfd have opposing effects on CDRE-lacZ expression in these cells (). Thus, expression of Crz1PVIVIT is not detrimental under these conditions because of increased Crz1-dependent transcription. Rather, we conclude that Crz1PVIVIT disrupts calcineurin interaction with substrates other than Crz1 and that de-phosphorylation of non-Crz1 substrates is the principal means by which calcineurin improves growth during alkaline stress.