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Screening for a gene deletion mutant whose temperature sensitivity is suppressed by FK506 in budding yeast and its application for a positive screening for drugs inhibiting calcineurin a
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Kazunori Kume , Takayuki Koyano , Junpei Takata , Ko Wakabayashi , Masaki Mizunuma , a
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Tokichi Miyakawa & Dai Hirata a
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 kagamiyama, Higashi-Hiroshima, 739-8530 Japan b
Asahi-Shuzo Sake Brewing Co. Ltd., 880-1 Asahi, Nagaoka, 949-5494 Japan Published online: 23 Jan 2015.
Click for updates To cite this article: Kazunori Kume, Takayuki Koyano, Junpei Takata, Ko Wakabayashi, Masaki Mizunuma, Tokichi Miyakawa & Dai Hirata (2015) Screening for a gene deletion mutant whose temperature sensitivity is suppressed by FK506 in budding yeast and its application for a positive screening for drugs inhibiting calcineurin, Bioscience, Biotechnology, and Biochemistry, 79:5, 790-794, DOI: 10.1080/09168451.2014.1003132 To link to this article: http://dx.doi.org/10.1080/09168451.2014.1003132
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Bioscience, Biotechnology, and Biochemistry, 2015 Vol. 79, No. 5, 790–794
Screening for a gene deletion mutant whose temperature sensitivity is suppressed by FK506 in budding yeast and its application for a positive screening for drugs inhibiting calcineurin Kazunori Kume1,*, Takayuki Koyano1, Junpei Takata1, Ko Wakabayashi1, Masaki Mizunuma1, Tokichi Miyakawa1 and Dai Hirata1,2,* 1
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 kagamiyama, Higashi-Hiroshima, 739-8530 Japan; 2Asahi-Shuzo Sake Brewing Co. Ltd., 880-1 Asahi, Nagaoka, 949-5494 Japan Received November 21, 2014; accepted December 16, 2014
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http://dx.doi.org/10.1080/09168451.2014.1003132
Calcineurin, which is a Ca2+/calmodulindependent protein phosphatase, is a key mediator in calcium signaling in diverse biological processes and of clinical importance as the target of the immunosuppressant FK506. To identify a mutant(s) in which calcineurin is activated, inhibiting cellular growth as a result, we screened for a mutant(s) whose temperature sensitivity would be suppressed by FK506 from the budding yeast non-essential gene deletion library. We found that the temperature sensitivity of cells in which the conserved Verprolin VRP1 gene had been deleted, which gene is required for actin organization and endocytosis, was suppressed by either FK506 or by cnb1 deletion. Indeed, the calcineurin activity increased significantly in the Δvrp1 cells. Finally, we demonstrated that the Δvrp1 strain to be useful as an indicator in a positive screening for bioactive compounds inhibiting calcineurin. Key words:
calcineurin; drug screening; FK506; yeast
Calcineurin, a highly conserved Ca2+/calmodulindependent protein phosphatase, is a key mediator of calcium signaling in eukaryotes. Calcineurin regulates numerous cellular processes, including cell proliferation, T-cell activation, cardiac development, memory, and apoptosis.1) Alterations in calcineurin activity have been associated with multiple human diseases including cancer, cardiac hypertrophy, and neurodegeneration.2–4) Calcineurin activity is potently inhibited by the immunosuppressants FK506 (tacrolimus) and cyclosporine A (CsA), which are widely used to prevent organ transplant rejection.5) These compounds are bound by immunophilins, which complexes inhibit calcineurin phosphatase activity and thereby suppress T-cell activation.5,6) This inhibition mechanism acting through immunophilins is conserved from yeast to humans.
In Saccharomyces cerevisiae, calcineurin is required for ion homeostasis, cell-wall biogenesis, and cell-cycle regulation (calcium mediated G2-arrest and onset of mitosis).7–10) An inappropriate activation of calcineurin in a certain genetic background (Δzds1) leads to a severe growth defect (Swe1-dependent G2-arrest) when cells are grown in a medium containing a high concentration of calcium,10) and this growth defect is suppressed by FK506. However, the mechanism underlying calcineurin-dependent growth control remains elusive. Verprolin (Vrp1), which is conserved from yeast to mammals, is required for actin organization and endocytosis.11) Verprolin regulates actin dynamics in 2 ways: one is by directly binding to actin, the other is by binding to the Wiskott–Aldrich syndrome protein (WASP) family proteins, which are activators of the ARP2/3 complex.11–13) Verprolin and its binding partners have been implicated in human diseases such as cancer and are potential targets for cancer therapy.11,14,15) WASP-family verprolin homologous protein 1 (WAVE1) is abundant in the brain, and its ability to regulate ARP2/3 complex-dependent actin polymerization is inhibited by phosphorylation and is activated by dephosphorylation.14) Cdk5 phosphorylates WAVE1 at its Ser310, Ser397, and Ser441 under basal conditions, and calcineurin has been implicated in the dopamine D1-induced dephosphorylation at Ser441 and in the N-methyl-D-aspartate-induced dephosphorylation at Ser397.14) However, the effect of a verprolin-defect on calcineurin activity has not been reported yet. Large-scale genetic interaction mapping studies were pioneered in S. cerevisiae and focused on the identification of a specific type of interaction termed synthetic lethality or rescue.16) Synthetic lethality, in which a combination of mutations in two different genes prevents growth, has been extensively used to identify genes whose products are functionally redundant. Synthetic rescue, in which a growth defect arising from one mutation is rescued by the additional mutation of a second gene, has been used to find a new genetic
*Corresponding authors. Email: [email protected] (K. Kume); [email protected] (D. Hirata) © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
A yeast temperature sensitive mutant is suppressed by FK506
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compensatory mechanism. These genetic interactions are useful for predicting the cellular targets of bioactive compounds.17) Using measurable phenotypes based on genetic interactions, it is possible to develop a drug screen for gene-specific inhibitors. In this study, we used synthetic genetic interaction analysis to identify genes that functionally interact with calcineurin. For this analysis, using a budding yeast non-essential gene deletion library we performed a screening for a mutant(s) whose temperature sensitivity would be suppressed by FK506. In this screening, we identified the vrp1 deletion. Indeed, this deletion significantly increased calcineurin activity, and the growth defect at high temperature was suppressed by deletion of the calcineurin gene. Furthermore, we demonstrated that the vrp1 deletion would be useful as an indicator in a positive screening for bioactive compounds that inhibit calcineurin.
Materials and methods Strains, media, and growth conditions. The budding yeast S. cerevisiae strains used in this study are listed in Table 1. The non-essential gene deletion library on the BY4741 strain background (Invitrogen) was used for this screening. Double mutants were created by tetrad analysis. Media used were as described previously.11) Assay for calcineurin activity. The pmr2A::lacZ reporter plasmid pKC20118) was used for the assay of calcineurin activity. β-Galactosidase assay of cells transfected with pKC201 was performed as described previously.18) Procedures for drug screening. The drug detection was done on solid YPD medium. The indicator cells, of late log phase and grown at 28 °C in liquid YPD medium, were suspended at a cell concentration of 1 × 105 cells/ml in 10 ml of YPD soft-agar medium (0.7% agar, 50 °C) and overlaid on 20 ml of solid YPD medium (2% agar). A filter-paper disc (8 mm diameter; Toyo Roshi Kaisha, Ltd.) was placed on the surface of the top agar, and the samples were applied to the disc. The plates were incubated at a restrictive temperature for the indicator cells.
Results and discussion Screening for a gene deletion mutant whose temperature sensitivity is suppressed by FK506 Using synthetic genetic interaction analysis to find the genes that may functionally interact with calcineurin, Table 1.
Yeast strains used in this study.
Name
MAT Genotype
WT SCDB-5-B-5 SCDB-70-A-3 SCDB-19-A-5 SCDB-42-F-4 JP-8-6D JP-52-5C
a a a a a a a
his3 leu2 met15 ura3 Δzds1::kanr his3 leu2 met15 ura3 Δvrp1::kanr his3 leu2 met15 ura3 Δcnb1::kanr his3 leu2 met15 ura3 Δfkb1::kanr his3 leu2 met15 ura3 Δfkb1::kanr Δvrp1::kanr his3 leu2 met15 ura3 Δcnb1::kanr Δvrp1::kanr his3 leu2 met15 ura3
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we first determined the mutants whose growth defect could be suppressed by FK506 from 222 temperaturesensitive (ts) gene deletion strains previously selected in our laboratory from a haploid non-essential gene deletion library of budding yeast. These ts gene deletion mutants showed a growth defect when grown on a YPD agar plate at a high temperature of 35.5 °C. We screened for a mutant(s), whose ts growth would be suppressed by the addition of FK506, from 222 ts gene deletion mutants. In this screening, we used vma22 deletion mutant as a negative control, because previously it has been reported that double mutant between calcineurin and vma mutants showed the synthetic lethal genetic interaction.17) Through this screening, only 1 gene deletion mutant (VRP1-deletion, Δvrp1) was found (Fig. 1(A)). The Δvrp1 cells showed a severe growth defect at 35.5 °C, but this growth defect was almost completely suppressed by FK506 (Fig. 1(A)). FK506 did not affect cell growth of Δvrp1 cells at 28 °C. These results indicate that calcineurin activity was required for the growth inhibition of Δvrp1 cells at the restrictive temperature of 35.5 °C. Suppression of the temperature sensitivity of the VRP1 deletion mutant by calcineurin deletion To confirm that the growth defect of Δvrp1 at the restrictive temperature was indeed dependent on calcineurin (CNB1; gene encoding a regulatory subunit of calcineurin), we investigated the growth properties of a Δvrp1Δcnb1 double-deletion mutant strain at 35.5 °C. As in the case of FK506, the deletion of CNB1 suppressed the growth defect of Δvrp1 cells at the restrictive temperature (Fig. 1(B)). In budding yeast, Vrp1 is critical for cortical actin patch distribution and is required for cytokinesis.19–21) It has been reported that the deletion of VRP1 causes a defect in cytokinesis and an increase in the percentage of large-budded cells, resulting in loss of viability (Fig. 1(C)).19–21) To investigate whether CNB1 deletion (Δcnb1) could suppress the cytokinesis defect of Δvrp1 cells at the restrictive temperature, we observed the cell morphology of Δvrp1Δcnb1 double-mutant cells at 35.5 °C. Two hours after the shift of the Δvrp1 cells from 28 to 35.5 °C, the percentage of large-budded cells increased and that of unbudded ones decreased (Fig. 1(C)). On the other hand, in Δvrp1Δcnb1 cells, no increase in large-budded cells was observed upon this shift to 35.5 °C, unlike in the case of the Δvrp1 single-mutant cells (Fig. 1(C)). These results suggest that the cytokinesis defect in Δvrp1 at high temperature would be mediated directly or indirectly by the function of calcineurin.
Activation of calcineurin in VRP1 deletion mutant Under certain conditions, activated calcineurin dephosphorylates the zinc-finger-type transcription factor Crz1, and this dephosphorylated Crz1 then stimulates the transcription of genes having the promoter of calcineurin-dependent response element (CDRE).18,22,23) To investigate if calcineurin was actually activated in Δvrp1 cells, we performed a calcineurin assay using a reporter plasmid with a lacZ gene under the Crz1-driven PMR2A/ENA1 promoter.18)
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(A)
(B)
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(C)
Fig. 1. Suppression of the growth defect of Δvrp1 mutant by FK506 or deletion of CNB1. Notes: (A) Growth properties of indicated strains on YPD containing FK506. WT, Δvma22, or Δvrp1 cells were spotted on YPD or YPD plates containing FK506 (2.0 µg/ml) and incubated at 28 or 35.5 °C for 2 days. The Δvma22 strain was used as a negative control for FK506.17) (B) Growth properties of the indicated strains on YPD. WT, Δcnb1, Δvrp1, or Δvrp1Δcnb1 cells were spotted on YPD medium and incubated at 28 or 35.5 °C for 2 days. (C) Cell morphology of WT, Δvrp1, and Δvrp1Δcnb1 cells. These cells were grown in YPD liquid medium at 28 °C and then shifted up to 36 °C. At the times indicated, the cells were fixed and stained with Calcofluor-white for monitoring cell morphology, which was categorized into large-budded cell, small-budded cell, and unbudded cell (n > 200).
This reporter plasmid was introduced into the Δvrp1 cells, and the calcineurin activity in these cells was measured by performing a β-galactosidase assay (Fig. 2). As previously reported,24) we observed an increase in calcineurin activity in ZDS1-deleted cells (Δzds1), in which calcineurin is activated, and confirmed the basal expression level of the reporter plasmid in Δcnb1 cells. Under the normal growth condition at both permissive and restrictive temperatures, calcineurin activity in the Δvrp1 cells was more than 2 times higher than that in WT cells, as in the case of Δzds1 cells (Fig. 2). This result indicates that calcineurin had been actually activated in the VRP1-deletion background. Application of the mutant for a positive screening for drugs inhibiting calcineurin Previously, based on the results obtained from the usage of a Ca2+-sensitive Δzds1 strain whose Ca2+dependent growth defect was suppressed by the inhibition of calcineurin, we established a positive screening method for drugs inhibiting calcineurin.25) To investigate whether the Δvrp1 strain would be suitable for a positive screening for drugs inhibiting calcineurin, we examined the suppression of the temperature sensitivity of the Δvrp1 cells by FK506 in a growth assay on YPD solid medium. Under the assay conditions (incubation at the restrictive temperature of 35.5 °C), cell
Fig. 2. Activation of calcineurin by the VRP1 deletion. Notes: The CDRE-dependent reporter gene (pKC201) was expressed in WT, Δcnb1, Δzds1, and Δvrp1 cells. Cells were grown at 36 °C for 2 h, and then, the β-galactosidase assay activity was determined. *p < 0.05, **p < 0.01.
growth of Δvrp1 was not observed around the disc loaded with a solvent (EtOH); on the other hand, the growth zone appeared around the discs loaded with FK506, which zone expanded in a dose-dependent manner (Fig. 3). The inhibition of calcineurin by FK506 is mediated by the immunophilin Fkb1.5,6) To
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Fig. 3. FK506 promotes the growth of Δvrp1 mutant in an Fkb1-dependent manner. Notes: The suspension of Δvrp1 or Δvrp1Δfkb1 cells (1 × 105 cells/ml) in 10 ml of YPD soft-agar medium (0.7%) was overlaid on YPD agar medium (2%). Paper disc with containing 200 or 400 ng of FK506 were placed on the surface of the top agar. These plates were incubated at 35.5 °C for 2 days. Growth zones were measured using Image J.
examine whether the growth suppression of Δvrp1 by FK506 was dependent on Fkb1, we investigated the effect of the FK506 on the growth of the Δvrp1Δfkb1 double mutant at the restrictive temperature. The deletion of FKB1 in Δvrp1 cells canceled the growth zone around the disks loaded with FK506 (Fig. 3). These results indicate that the promotion of growth in Δvrp1 cells at the restrictive temperature by FK506 occurred in a highly specific FKB1-dependent manner and demonstrate that Δvrp1 strain may be a useful tool for a positive screening for novel bioactive compounds inhibiting calcineurin. Another positive screening system for calcineurin inhibitors, one using an rps5 temperature-sensitive mutant strain, was earlier reported.26) Instead of using Δzds1 with a high concentration of calcium,25) the use of Δvrp1 and rsp5 temperature-sensitive mutant strains and high temperature is much easier and will be effective to find highly specific inhibitors for calcineurin. Here, we carried out a screening for a gene deletion mutant whose temperature sensitivity at the restrictive temperature would be suppressed by the immunosuppressant FK506 and identified only a single candidate, that is, Δvrp1. We showed a genetic interaction between Vrp1 and calcineurin in budding yeast. Loss of viability of Δvrp1 cells at the restrictive temperature associated with a defect in cytokinesis is almost suppressed by the deletion of CNB1 (Fig. 1(B) and (C)), suggesting that activated calcineurin inhibits cytokinesis in Δvrp1 at the restrictive temperature. It has been reported that activated calcineurin negatively regulates actin polarization at the bud site to inhibit bud emergence in budding yeast.27) These results implicate that activated calcineurin has a negative role in actin-based cellular processes in budding yeast. The impaired cytokinesis in Δvrp1 cells at the restrictive temperature is due to defects in both the medial actomyosin ring contraction and relocalization of the F-BAR protein Hof1 from two septin rings to a single medial ring at the bud neck.21) This relocalization is required for proper septum formation and contraction of the actomyosin ring to promote efficient cytokinesis.28,29) In addition, Δvrp1 is synthetic lethal with Δhof1,21) suggesting that the function of Hof1 is essential to the viability of Δvrp1 cells at the restrictive temperature. Recently, it has been shown that relocalization of Hof1 to a single ring is facilitated by phosphorylation of Hof1 by the mitotic exit network kinase complex Dbf2-Mob1 during
cytokinesis.28,29) Intriguingly, Hof1 has 4 potential calcineurin binding motifs (PxIxIT) that are important for Ca2+-dependent dephosphorylation of substrates and calcineurin-mediated signaling.30,31) Taken together, we propose a following possible hypothesis in budding yeast. When Δvrp1 cells are shifted to the restrictive temperature, activated calcineurin dephosphorylates Hof1 to inhibit localization of Hof1 to a single medial ring at the bud neck followed by a severe defect in cytokinesis, resulting in loss of viability. Further analysis with physiological and biochemical studies will help address this possibility and reveal molecular mechanism of calcineurin-dependent growth control in budding yeast. It has also been reported that in fission yeast, calcineurin and the Kin1 kinase cooperate to link actin ring assembly and septum synthesis.32) Further analysis will be necessary to clarify a role of calcineurin in cytokinesis of both yeasts. In conclusion, we identified Δvrp1 as a mutant whose temperature sensitivity was suppressed by inhibition of calcineurin. Further analysis of Δvrp1 revealed that calcineurin activity was significantly increased by the deletion of VRP1 and the activated calcineurin has a negative role in cytokinesis in Δvrp1 at the restrictive temperature. We also showed that Δvrp1 was a useful strain for a positive screening for drugs inhibiting calcineurin.
Acknowledgments We are grateful to all other members of Hirata’s laboratory for their help. T.K. is the recipient of a JSPS Fellowship (DC1/PD).
Funding This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to K.K. and D.H.).
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Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Screening for a gene deletion mutant whose temperature sensitivity is suppressed by FK506 in budding yeast and its application for a positive screening for drugs inhibiting calcineurin a
a
a
a
a
Kazunori Kume , Takayuki Koyano , Junpei Takata , Ko Wakabayashi , Masaki Mizunuma , a
ab
Tokichi Miyakawa & Dai Hirata a
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 kagamiyama, Higashi-Hiroshima, 739-8530 Japan b
Asahi-Shuzo Sake Brewing Co. Ltd., 880-1 Asahi, Nagaoka, 949-5494 Japan Published online: 23 Jan 2015.
Click for updates To cite this article: Kazunori Kume, Takayuki Koyano, Junpei Takata, Ko Wakabayashi, Masaki Mizunuma, Tokichi Miyakawa & Dai Hirata (2015) Screening for a gene deletion mutant whose temperature sensitivity is suppressed by FK506 in budding yeast and its application for a positive screening for drugs inhibiting calcineurin, Bioscience, Biotechnology, and Biochemistry, 79:5, 790-794, DOI: 10.1080/09168451.2014.1003132 To link to this article: http://dx.doi.org/10.1080/09168451.2014.1003132
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Bioscience, Biotechnology, and Biochemistry, 2015 Vol. 79, No. 5, 790–794
Screening for a gene deletion mutant whose temperature sensitivity is suppressed by FK506 in budding yeast and its application for a positive screening for drugs inhibiting calcineurin Kazunori Kume1,*, Takayuki Koyano1, Junpei Takata1, Ko Wakabayashi1, Masaki Mizunuma1, Tokichi Miyakawa1 and Dai Hirata1,2,* 1
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 kagamiyama, Higashi-Hiroshima, 739-8530 Japan; 2Asahi-Shuzo Sake Brewing Co. Ltd., 880-1 Asahi, Nagaoka, 949-5494 Japan Received November 21, 2014; accepted December 16, 2014
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http://dx.doi.org/10.1080/09168451.2014.1003132
Calcineurin, which is a Ca2+/calmodulindependent protein phosphatase, is a key mediator in calcium signaling in diverse biological processes and of clinical importance as the target of the immunosuppressant FK506. To identify a mutant(s) in which calcineurin is activated, inhibiting cellular growth as a result, we screened for a mutant(s) whose temperature sensitivity would be suppressed by FK506 from the budding yeast non-essential gene deletion library. We found that the temperature sensitivity of cells in which the conserved Verprolin VRP1 gene had been deleted, which gene is required for actin organization and endocytosis, was suppressed by either FK506 or by cnb1 deletion. Indeed, the calcineurin activity increased significantly in the Δvrp1 cells. Finally, we demonstrated that the Δvrp1 strain to be useful as an indicator in a positive screening for bioactive compounds inhibiting calcineurin. Key words:
calcineurin; drug screening; FK506; yeast
Calcineurin, a highly conserved Ca2+/calmodulindependent protein phosphatase, is a key mediator of calcium signaling in eukaryotes. Calcineurin regulates numerous cellular processes, including cell proliferation, T-cell activation, cardiac development, memory, and apoptosis.1) Alterations in calcineurin activity have been associated with multiple human diseases including cancer, cardiac hypertrophy, and neurodegeneration.2–4) Calcineurin activity is potently inhibited by the immunosuppressants FK506 (tacrolimus) and cyclosporine A (CsA), which are widely used to prevent organ transplant rejection.5) These compounds are bound by immunophilins, which complexes inhibit calcineurin phosphatase activity and thereby suppress T-cell activation.5,6) This inhibition mechanism acting through immunophilins is conserved from yeast to humans.
In Saccharomyces cerevisiae, calcineurin is required for ion homeostasis, cell-wall biogenesis, and cell-cycle regulation (calcium mediated G2-arrest and onset of mitosis).7–10) An inappropriate activation of calcineurin in a certain genetic background (Δzds1) leads to a severe growth defect (Swe1-dependent G2-arrest) when cells are grown in a medium containing a high concentration of calcium,10) and this growth defect is suppressed by FK506. However, the mechanism underlying calcineurin-dependent growth control remains elusive. Verprolin (Vrp1), which is conserved from yeast to mammals, is required for actin organization and endocytosis.11) Verprolin regulates actin dynamics in 2 ways: one is by directly binding to actin, the other is by binding to the Wiskott–Aldrich syndrome protein (WASP) family proteins, which are activators of the ARP2/3 complex.11–13) Verprolin and its binding partners have been implicated in human diseases such as cancer and are potential targets for cancer therapy.11,14,15) WASP-family verprolin homologous protein 1 (WAVE1) is abundant in the brain, and its ability to regulate ARP2/3 complex-dependent actin polymerization is inhibited by phosphorylation and is activated by dephosphorylation.14) Cdk5 phosphorylates WAVE1 at its Ser310, Ser397, and Ser441 under basal conditions, and calcineurin has been implicated in the dopamine D1-induced dephosphorylation at Ser441 and in the N-methyl-D-aspartate-induced dephosphorylation at Ser397.14) However, the effect of a verprolin-defect on calcineurin activity has not been reported yet. Large-scale genetic interaction mapping studies were pioneered in S. cerevisiae and focused on the identification of a specific type of interaction termed synthetic lethality or rescue.16) Synthetic lethality, in which a combination of mutations in two different genes prevents growth, has been extensively used to identify genes whose products are functionally redundant. Synthetic rescue, in which a growth defect arising from one mutation is rescued by the additional mutation of a second gene, has been used to find a new genetic
*Corresponding authors. Email: [email protected] (K. Kume); [email protected] (D. Hirata) © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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compensatory mechanism. These genetic interactions are useful for predicting the cellular targets of bioactive compounds.17) Using measurable phenotypes based on genetic interactions, it is possible to develop a drug screen for gene-specific inhibitors. In this study, we used synthetic genetic interaction analysis to identify genes that functionally interact with calcineurin. For this analysis, using a budding yeast non-essential gene deletion library we performed a screening for a mutant(s) whose temperature sensitivity would be suppressed by FK506. In this screening, we identified the vrp1 deletion. Indeed, this deletion significantly increased calcineurin activity, and the growth defect at high temperature was suppressed by deletion of the calcineurin gene. Furthermore, we demonstrated that the vrp1 deletion would be useful as an indicator in a positive screening for bioactive compounds that inhibit calcineurin.
Materials and methods Strains, media, and growth conditions. The budding yeast S. cerevisiae strains used in this study are listed in Table 1. The non-essential gene deletion library on the BY4741 strain background (Invitrogen) was used for this screening. Double mutants were created by tetrad analysis. Media used were as described previously.11) Assay for calcineurin activity. The pmr2A::lacZ reporter plasmid pKC20118) was used for the assay of calcineurin activity. β-Galactosidase assay of cells transfected with pKC201 was performed as described previously.18) Procedures for drug screening. The drug detection was done on solid YPD medium. The indicator cells, of late log phase and grown at 28 °C in liquid YPD medium, were suspended at a cell concentration of 1 × 105 cells/ml in 10 ml of YPD soft-agar medium (0.7% agar, 50 °C) and overlaid on 20 ml of solid YPD medium (2% agar). A filter-paper disc (8 mm diameter; Toyo Roshi Kaisha, Ltd.) was placed on the surface of the top agar, and the samples were applied to the disc. The plates were incubated at a restrictive temperature for the indicator cells.
Results and discussion Screening for a gene deletion mutant whose temperature sensitivity is suppressed by FK506 Using synthetic genetic interaction analysis to find the genes that may functionally interact with calcineurin, Table 1.
Yeast strains used in this study.
Name
MAT Genotype
WT SCDB-5-B-5 SCDB-70-A-3 SCDB-19-A-5 SCDB-42-F-4 JP-8-6D JP-52-5C
a a a a a a a
his3 leu2 met15 ura3 Δzds1::kanr his3 leu2 met15 ura3 Δvrp1::kanr his3 leu2 met15 ura3 Δcnb1::kanr his3 leu2 met15 ura3 Δfkb1::kanr his3 leu2 met15 ura3 Δfkb1::kanr Δvrp1::kanr his3 leu2 met15 ura3 Δcnb1::kanr Δvrp1::kanr his3 leu2 met15 ura3
791
we first determined the mutants whose growth defect could be suppressed by FK506 from 222 temperaturesensitive (ts) gene deletion strains previously selected in our laboratory from a haploid non-essential gene deletion library of budding yeast. These ts gene deletion mutants showed a growth defect when grown on a YPD agar plate at a high temperature of 35.5 °C. We screened for a mutant(s), whose ts growth would be suppressed by the addition of FK506, from 222 ts gene deletion mutants. In this screening, we used vma22 deletion mutant as a negative control, because previously it has been reported that double mutant between calcineurin and vma mutants showed the synthetic lethal genetic interaction.17) Through this screening, only 1 gene deletion mutant (VRP1-deletion, Δvrp1) was found (Fig. 1(A)). The Δvrp1 cells showed a severe growth defect at 35.5 °C, but this growth defect was almost completely suppressed by FK506 (Fig. 1(A)). FK506 did not affect cell growth of Δvrp1 cells at 28 °C. These results indicate that calcineurin activity was required for the growth inhibition of Δvrp1 cells at the restrictive temperature of 35.5 °C. Suppression of the temperature sensitivity of the VRP1 deletion mutant by calcineurin deletion To confirm that the growth defect of Δvrp1 at the restrictive temperature was indeed dependent on calcineurin (CNB1; gene encoding a regulatory subunit of calcineurin), we investigated the growth properties of a Δvrp1Δcnb1 double-deletion mutant strain at 35.5 °C. As in the case of FK506, the deletion of CNB1 suppressed the growth defect of Δvrp1 cells at the restrictive temperature (Fig. 1(B)). In budding yeast, Vrp1 is critical for cortical actin patch distribution and is required for cytokinesis.19–21) It has been reported that the deletion of VRP1 causes a defect in cytokinesis and an increase in the percentage of large-budded cells, resulting in loss of viability (Fig. 1(C)).19–21) To investigate whether CNB1 deletion (Δcnb1) could suppress the cytokinesis defect of Δvrp1 cells at the restrictive temperature, we observed the cell morphology of Δvrp1Δcnb1 double-mutant cells at 35.5 °C. Two hours after the shift of the Δvrp1 cells from 28 to 35.5 °C, the percentage of large-budded cells increased and that of unbudded ones decreased (Fig. 1(C)). On the other hand, in Δvrp1Δcnb1 cells, no increase in large-budded cells was observed upon this shift to 35.5 °C, unlike in the case of the Δvrp1 single-mutant cells (Fig. 1(C)). These results suggest that the cytokinesis defect in Δvrp1 at high temperature would be mediated directly or indirectly by the function of calcineurin.
Activation of calcineurin in VRP1 deletion mutant Under certain conditions, activated calcineurin dephosphorylates the zinc-finger-type transcription factor Crz1, and this dephosphorylated Crz1 then stimulates the transcription of genes having the promoter of calcineurin-dependent response element (CDRE).18,22,23) To investigate if calcineurin was actually activated in Δvrp1 cells, we performed a calcineurin assay using a reporter plasmid with a lacZ gene under the Crz1-driven PMR2A/ENA1 promoter.18)
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(A)
(B)
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(C)
Fig. 1. Suppression of the growth defect of Δvrp1 mutant by FK506 or deletion of CNB1. Notes: (A) Growth properties of indicated strains on YPD containing FK506. WT, Δvma22, or Δvrp1 cells were spotted on YPD or YPD plates containing FK506 (2.0 µg/ml) and incubated at 28 or 35.5 °C for 2 days. The Δvma22 strain was used as a negative control for FK506.17) (B) Growth properties of the indicated strains on YPD. WT, Δcnb1, Δvrp1, or Δvrp1Δcnb1 cells were spotted on YPD medium and incubated at 28 or 35.5 °C for 2 days. (C) Cell morphology of WT, Δvrp1, and Δvrp1Δcnb1 cells. These cells were grown in YPD liquid medium at 28 °C and then shifted up to 36 °C. At the times indicated, the cells were fixed and stained with Calcofluor-white for monitoring cell morphology, which was categorized into large-budded cell, small-budded cell, and unbudded cell (n > 200).
This reporter plasmid was introduced into the Δvrp1 cells, and the calcineurin activity in these cells was measured by performing a β-galactosidase assay (Fig. 2). As previously reported,24) we observed an increase in calcineurin activity in ZDS1-deleted cells (Δzds1), in which calcineurin is activated, and confirmed the basal expression level of the reporter plasmid in Δcnb1 cells. Under the normal growth condition at both permissive and restrictive temperatures, calcineurin activity in the Δvrp1 cells was more than 2 times higher than that in WT cells, as in the case of Δzds1 cells (Fig. 2). This result indicates that calcineurin had been actually activated in the VRP1-deletion background. Application of the mutant for a positive screening for drugs inhibiting calcineurin Previously, based on the results obtained from the usage of a Ca2+-sensitive Δzds1 strain whose Ca2+dependent growth defect was suppressed by the inhibition of calcineurin, we established a positive screening method for drugs inhibiting calcineurin.25) To investigate whether the Δvrp1 strain would be suitable for a positive screening for drugs inhibiting calcineurin, we examined the suppression of the temperature sensitivity of the Δvrp1 cells by FK506 in a growth assay on YPD solid medium. Under the assay conditions (incubation at the restrictive temperature of 35.5 °C), cell
Fig. 2. Activation of calcineurin by the VRP1 deletion. Notes: The CDRE-dependent reporter gene (pKC201) was expressed in WT, Δcnb1, Δzds1, and Δvrp1 cells. Cells were grown at 36 °C for 2 h, and then, the β-galactosidase assay activity was determined. *p < 0.05, **p < 0.01.
growth of Δvrp1 was not observed around the disc loaded with a solvent (EtOH); on the other hand, the growth zone appeared around the discs loaded with FK506, which zone expanded in a dose-dependent manner (Fig. 3). The inhibition of calcineurin by FK506 is mediated by the immunophilin Fkb1.5,6) To
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Fig. 3. FK506 promotes the growth of Δvrp1 mutant in an Fkb1-dependent manner. Notes: The suspension of Δvrp1 or Δvrp1Δfkb1 cells (1 × 105 cells/ml) in 10 ml of YPD soft-agar medium (0.7%) was overlaid on YPD agar medium (2%). Paper disc with containing 200 or 400 ng of FK506 were placed on the surface of the top agar. These plates were incubated at 35.5 °C for 2 days. Growth zones were measured using Image J.
examine whether the growth suppression of Δvrp1 by FK506 was dependent on Fkb1, we investigated the effect of the FK506 on the growth of the Δvrp1Δfkb1 double mutant at the restrictive temperature. The deletion of FKB1 in Δvrp1 cells canceled the growth zone around the disks loaded with FK506 (Fig. 3). These results indicate that the promotion of growth in Δvrp1 cells at the restrictive temperature by FK506 occurred in a highly specific FKB1-dependent manner and demonstrate that Δvrp1 strain may be a useful tool for a positive screening for novel bioactive compounds inhibiting calcineurin. Another positive screening system for calcineurin inhibitors, one using an rps5 temperature-sensitive mutant strain, was earlier reported.26) Instead of using Δzds1 with a high concentration of calcium,25) the use of Δvrp1 and rsp5 temperature-sensitive mutant strains and high temperature is much easier and will be effective to find highly specific inhibitors for calcineurin. Here, we carried out a screening for a gene deletion mutant whose temperature sensitivity at the restrictive temperature would be suppressed by the immunosuppressant FK506 and identified only a single candidate, that is, Δvrp1. We showed a genetic interaction between Vrp1 and calcineurin in budding yeast. Loss of viability of Δvrp1 cells at the restrictive temperature associated with a defect in cytokinesis is almost suppressed by the deletion of CNB1 (Fig. 1(B) and (C)), suggesting that activated calcineurin inhibits cytokinesis in Δvrp1 at the restrictive temperature. It has been reported that activated calcineurin negatively regulates actin polarization at the bud site to inhibit bud emergence in budding yeast.27) These results implicate that activated calcineurin has a negative role in actin-based cellular processes in budding yeast. The impaired cytokinesis in Δvrp1 cells at the restrictive temperature is due to defects in both the medial actomyosin ring contraction and relocalization of the F-BAR protein Hof1 from two septin rings to a single medial ring at the bud neck.21) This relocalization is required for proper septum formation and contraction of the actomyosin ring to promote efficient cytokinesis.28,29) In addition, Δvrp1 is synthetic lethal with Δhof1,21) suggesting that the function of Hof1 is essential to the viability of Δvrp1 cells at the restrictive temperature. Recently, it has been shown that relocalization of Hof1 to a single ring is facilitated by phosphorylation of Hof1 by the mitotic exit network kinase complex Dbf2-Mob1 during
cytokinesis.28,29) Intriguingly, Hof1 has 4 potential calcineurin binding motifs (PxIxIT) that are important for Ca2+-dependent dephosphorylation of substrates and calcineurin-mediated signaling.30,31) Taken together, we propose a following possible hypothesis in budding yeast. When Δvrp1 cells are shifted to the restrictive temperature, activated calcineurin dephosphorylates Hof1 to inhibit localization of Hof1 to a single medial ring at the bud neck followed by a severe defect in cytokinesis, resulting in loss of viability. Further analysis with physiological and biochemical studies will help address this possibility and reveal molecular mechanism of calcineurin-dependent growth control in budding yeast. It has also been reported that in fission yeast, calcineurin and the Kin1 kinase cooperate to link actin ring assembly and septum synthesis.32) Further analysis will be necessary to clarify a role of calcineurin in cytokinesis of both yeasts. In conclusion, we identified Δvrp1 as a mutant whose temperature sensitivity was suppressed by inhibition of calcineurin. Further analysis of Δvrp1 revealed that calcineurin activity was significantly increased by the deletion of VRP1 and the activated calcineurin has a negative role in cytokinesis in Δvrp1 at the restrictive temperature. We also showed that Δvrp1 was a useful strain for a positive screening for drugs inhibiting calcineurin.
Acknowledgments We are grateful to all other members of Hirata’s laboratory for their help. T.K. is the recipient of a JSPS Fellowship (DC1/PD).
Funding This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to K.K. and D.H.).
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