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Mechanisms of protein kinase Sch9 regulating Bcy1 in Saccharomyces cerevisiae

Aili Zhang , Wenxuan Gao
DOI: http://dx.doi.org/10.1111/j.1574-6968.2012.02552.x 10-16 First published online: 1 June 2012

Abstract

In this study, we investigated the mechanisms of Sch9 regulating the localization and phosphorylation of Bcy1. Our research indicated that Sch9 regulated localization of Bcy1 via Zds1 for the following reasons: (1) deletions of SCH9 or ZDS1 both caused nuclear localization of Bcy1; (2) Sch9 and Zds1 interacted physically; (3) overexpression of ZDS1 led to a significantly increased cytoplasmic localization of Bcy1 in sch9Δ cells, whereas overexpression of SCH9 had no visible effect on cytoplasmic localization of Bcy1 in zds1Δ cells. Our study also suggested that Sch9 regulated phosphorylation of Bcy1 via Yak1.

Keywords
  • yeast
  • Sch9
  • Bcy1
  • Zds1
  • Yak1

Introduction

In Saccharomyces cerevisiae, glucose signals activate the production of cellular cAMP. This signaling pathway is called the cAMP-PKA pathway and plays a major role in the regulation of cell growth, metabolism and stress resistance, in particular in connection with the available nutrient conditions (Broach, 1991; Thevelein, 1994). PKA is a heterotetramer consisting of a homodimer of two regulatory subunits (encoded by the gene BCY1) and two catalytic subunits (encoded by the genes TPK1, TPK2 and TPK3) (Toda et al., 1987a, b). The binding of two cAMP molecules to each regulatory subunit in the holoenzyme induced the release of the catalytic subunits and their activation.

In glucose-grown yeast cells, Bcy1 was found to be almost exclusively nuclear with little or no cytoplasmic localization. However, Bcy1 were distributed over nucleus and cytoplasm in cells growing on a nonfermentable carbon source or in stationary phase cells (Griffioen et al., 2000, 2001). The N-terminal domain of Bcy1 served to target it properly during logarithmic and stationary phase (Griffioen et al., 2000). Phosphorylation of its N-terminal domain directed Bcy1 to cytoplasm. Bcy1 modification was found to be dependent on Yak1 kinase (Griffioen et al., 2001). Zds1-mediated cytoplasmic localization of Bcy1 was regulated by carbon source-dependent phosphorylation of cluster II serines (Griffioen et al., 2001; Griffioen & Thevelein, 2002). Recently, we reported that Sch9 was involved in regulating phosphorylation and localization of Bcy1 (Zhang et al., 2011). But the mechanisms of Sch9 regulating Bcy1 are still unknown.

The serine/threonine protein kinase, Yak1, functioned as a negative regulator of the cell cycle in S. cerevisiae, acting downstream of the cAMP-dependent protein kinase (Garrett & Broach, 1989). Yak1 is a dual specificity protein kinase which autophosphorylates on Tyr-530 and phosphorylates exogenous substrates on Ser/Thr residues (Kassis et al., 2000). When glucose is limited, Yak1 accumulates in the nucleus where it phosphorylates Pop2, which is required for proper cell cycle arrest. In the presence of glucose, Yak1 was phosphorylated by an as yet unknown protein kinase at its serine residue(s) and associates with Bmh1 and Bmh2, and was then exported from the nucleus to the cytoplasm (Moriya et al., 2001).

ZDS1 and ZDS2 of S. cerevisiae were reported to be involved in transcriptional silencing, longevity, optimal mRNA export and mitotic exit through regulation of Cdc14 (Roy & Runge, 2000; Estruch et al., 2005; Queralt & Uhlmann, 2008). Zds1 was also reported to control sexual differentiation, cell wall integrity and cell morphology in fission yeast (Yakura et al., 2006). Recently, it was reported that that Zds1/Zds2 primarily control localization of Cdc55, a regulatory B subunit of the PP2A, which plays important roles in mitotic entry and mitotic exit (Rossio & Yoshida, 2011).

Here we report that Sch9 regulates the localization of Bcy1 via Zds1 by showing that: (1) deletion of SCH9 or ZDS1 both caused nuclear localization of Bcy1; (2) Sch9 and Zds1 interacted physically; (3) overexpression of ZDS1 led to a significant increased cytoplasmic localization of Bcy1 in sch9Δ cells, whereas overexpression of SCH9 had no visible effect on cytoplasmic localization of Bcy1 in zds1Δ cells. Additionally, our study suggests that Sch9 regulated the phosphorylation of Bcy1 via Yak1.

Materials and methods

Yeast strains, media, growth conditions of plasmids

Yeast cells were grown in YPD [1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) glucose] or in synthetic complete (SC) medium [0.17% (w/v) nitrogen base, with adenine, uracil, histidine, leucine, tryptophan and amino acids as appropriate] but lacking essential components to select for plasmids. Yeast cells were grown into mid-exponential phase (OD600 nm = 1.5) at 30 °C. Carbon source-derepressed cells used for the experiments described in this study were grown on YPG [1% (w/v) yeast extract, 2% (w/v) peptone and 3% (w/v) glycerol)].

The following yeast strains were used in this study: W303-1A (MATa leu2-3, 112 ura3-1 trp1-92 his3-11, 15 ade2-1 can1-100) (Thomas & Rothstein, 1989), sch9Δ (W303-1A sch9Δ::URA3) and zds1Δ (W303-1A zds1Δ::repeat). Oligonucleotides used in this study for construction of strains and plasmids are described in Supporting Information, Tables S1 and S2. The sch9Δ::URA3 deletion cassette was obtained by PCR amplification of genomic DNA of strain YBY1 containing the sch9Δ::URA3 cassette with primers KSCH9-U and KSCH9-D. The deletion cassette was used to delete SCH9 gene in strain W303-1A by one-step gene replacement procedure. Strains zds1Δ::RYUR was generated using a one-step gene replacement procedure transforming W303-1A with a zds1Δ::RYUR deletion fragment, which was obtained by PCR amplification of repeat-URA3-repeat fragment on the plasmid pUC18-RYUR (Kong et al., 2007) using primers KZDS1-U and KZDS1-D. Strains zds1Δ::RYUR were spotted on plates containing 5-fluoroorotic acid to pop-out URA3 marker using the recombination of two repeat fragments and the obtained strain zds1Δ::repeat. CZDS1 and CZDS1-D were used to confirm the deletion of ZDS1 on the genome.

Plasmids constructions

Plasmid YCplac22-3xHA was generated by inserting a triple copy of the HA sequence between the PstI and SphI sites and the 249-bp CYC1 terminator sequences between the SphI and HindШ sites of plasmid YCplac22. Plasmid YEplac181-ZDS1-3xHA was generated as follows: 3317 bp of ZDS1 sequence was amplified using primers ZDS1GFP-U and ZDS1GFP-D from yeast genomic DNA. The PCR fragments were digested with SalI and NotI and inserted in the same enzyme pair-digested plasmid YCplac22-3xHA, creating an in-frame fusion between the ZDS1 ORF and 3xHA. Primers CZDS1-U and CZDS1-D were used to amplify 3861 bp of the ZDS1 sequence from yeast genomic DNA. The PCR fragments were digested with BamHI and SalI and inserted in the same enzyme pair-digested plasmid YEplac195, creating YEplac195-ZDS1.

Plasmid YCplac22-SCH9-13xMYC was generated by inserting 3052 bp of SCH9 sequence amplified from yeast genomic DNA using primers SCH9GFP-U and SCH9GFP-D between the BamHI and SphI sites and 862 bp of 13xMYC-CYC1 sequence between the SphI and HindШ sites of plasmid YCplac22. Plasmid YCplac22-CYC1 was generated by inserting 249 bp CYC1 terminator sequences between the SphI and HindШ sites of plasmid YCplac22. To create YEplac181-SCH9, primers SCH9GFP-U and SCH9GFP-D were used to amplify 3052 bp of SCH9 sequence from yeast genomic DNA. The PCR fragments were digested with BamHI and SphI and inserted in the same enzyme pair-digested plasmid YCplac22-CYC1.

Plasmid YCplac22-YAK1-3xHA was generated as follows: 3100 bp of ZDS1 sequence were amplified using primers YAK1-U and yak1gfp-D from yeast genomic DNA. The PCR fragments were digested with PstI and SphI and inserted in the same enzyme pair-digested plasmid YCplac22-3xHA, creating an in-frame fusion between the YAK1 ORF and 3xHA.

Green fluorescence protein (GFP) fluorescence microscopy

GFP tags were integrated on the N terminus of Bcy1 on the plasmid YCplac22. Cells were used for fluorescence microscopy directly without fixation. Cells were viewed with an Olympus BX51 fluorescence microscope. Images were taken with an Olympus U-LH100HGAPO camera using spot (Version 4.0.2) software and then processed in adobe photoshop CS4.

Western blot analysis

Yeast cell cultures were grown at 30 °C. Cells were harvested by centrifugation at 4 °C and washed in ice-cold sterile water, and the pellets then stored at −80 °C until use. All subsequent steps were carried out at 4 °C. Cells were resuspended in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.1 % NP-40/Igepal CA-630, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM NaF, 1 mM sodium orthovanadate, 10 mM glycerol-2-phosphate, and a mixture of protease inhibitors (Roche). Cells were then disrupted by vortexing them for 30 s in the presence of glass beads using Fastprep FP120 (Bio101 Thermo Savant). The resulting suspension was spun down in a centrifuge at 18 000 g for 5 min. After addition of an equal volume of 2× sample buffer to the supernatant, samples were heated to 95 °C for 5 min before an equal amount of total protein was separated by SDS-PAGE. Immunodetection of proteins was carried out using anti-hemagglutinin (HA) monoclonal antibody [mouse immunoglobulin G (IgG3); Tiangen] or anti-myc antibody (mouse monoclonal antibody; Tiangen). The secondary antibody was anti-mouse IgG conjugated with horseradish peroxidase purchased from Tiangen. Proteins were visualized using LumiGlo (KPL) according to the manufacturer's instructions.

Co-immunoprecipitation

Cells expressing 3xHA-tagged Zds1 and 13xMYC-tagged Sch9 were grown in SC medium lacking essential components to select for plasmids. Total extracts were obtained by glass bead disruption in lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.1% NP-40/Igepal CA-630, 1 mM PMSF, 10 mM NaF, 1 mM sodium orthovanadate, 10 mM glycerol-2-phosphate, supplemented with protease inhibitors (Roche)]. Samples were incubated with 1 µg of the anti-myc antibody (Tiangen) by shaking overnight at 4 °C. Then 20 µL of 50% ImmunoPure Protein G beads slurry (Amersham) were added to it with rocking for 1 h at 4 °C. After that, the beads were washed extensively in lysis buffer. The beads were resuspended in 2× sample buffer. Samples were heated to 95 °C for 5 min before being separated by SDS-PAGE. Immunodetection of proteins was carried out using HA or anti-MYC monoclonal antibody (IgG3; Tiangen). The secondary antibody used was anti-mouse IgG conjugated with horseradish peroxidase purchased from Amersham Biosciences. Proteins were visualized using LumiGlo (KPL) according to the manufacturer's instructions.

Results

As shown in Fig. 1, Bcy1 was predominantly localized in nucleus in rapidly glucose-grown wild-type cells and sch9Δ cells. In glycerol-grown wild-type cells, a large part of Bcy1 transferred from nucleus to cytoplasm, whereas Bcy1 remained in the nucleus in glycerol-grown sch9Δ cells. These results indicated that Sch9 regulated, either directly or indirectly, the localization of Bcy1. Sch9 was predominantly localized in the vacuolar membrane (Jorgensen et al., 2004). How sch9 regulated nucleus or cytoplasm localized Bcy1 is still unknown.

Subcellular localization of Bcy1 in wild-type cells and sch9Δ cells grown on glucose and glycerol. Fluorescence microcopy was carried out in strains containing YCplac22-GFP-BCY1 directly without fixation.

In S. cerevisiae, it was suggested that Zds1 could be a functional homolog of the mammalian A-kinase anchor protein (AKAP; Griffioen et al., 2001). It was also reported that nucleocytoplasmic distribution of Bcy1 required Zds1 (Griffioen et al., 2001). The results of those authors demonstrated that in ethanol-grown zds1Δ cells, cytoplasmic localization of Bcy1 was largely absent, whereas overexpression of ZDS1 led to increased cytoplasmic Bcy1 localization. As shown in Fig. 2, Bcy1 was predominantly localized in nucleus in rapidly glucose-grown wild-type and zds1Δ cells. A large part of Bcy1 transferred from nucleus to cytoplasm in glycerol-grown wild-type cells, whereas Bcy1 remained in the nucleus in glycerol-grown zds1Δ cells. These data were consistent with the research of Griffioen et al. (2001).

Subcellular localization of Bcy1 in wild-type cells and zds1Δ cells grown on glucose and glycerol. Fluorescence microcopy was carried out in strains containing YCplac22-GFP-BCY1 directly without fixation.

As Bcy1 was both predominately localized in nucleus in the glycerol-grown sch9Δ cells and zds1Δ cells, we wanted to investigate whether Sch9 and Zds1 interacted. First, we used the yeast two-hybrid system to test whether Sch9 and Zds1 interacted genetically. We found that PJ96-4A cells carrying plasmids pGBT9-SCH9/pGAD424 or pGBT9/pGAD 424-SCH9 could grow on SC minus leucine (Fig. 3). This indicated that Sch9 could activate transcription when fused to a promoter. This was consistent with a previous report which demonstrated that Sch9 could activate transcription when recruited artificially to a promoter (Pascual-Ahuir & Proft, 2007). Thus the yeast two-hybrid system could not be used to test whether Sch9 and Zds1 interact.

Yeast two-hybrid analysis between Sch9 and Zds1.

We then used co-immunoprecipitation to examine whether Sch9 and Zds1 interact. Proteins extracted from wild-type cells carrying plasmids YEplac181-ZDS1-3xHA and YCplac22-SCH9-13xMYC were used directly for co-immunoprecipitation analysis. As shown in Fig. 4, signals were detected in Sch9 co-immunoprecipitated with Zds1. These results demonstrated that Sch9 and Zds1 interacted physically. As an important AGC kinase, Sch9 was involved in many aspects of cell growth and interacted with many proteins. But how Sch9 interacted with Zds1 remains to be clarified.

Sch9 and Zds1 interact physically. (a) Western signals were detected using anti-HA antibody [the signals showed Zds1 (MW: 103.358 Da)]. Equal amounts of cellular protein were used. Sample 1: Lysate, protein extract from sch9Δ cells carrying 3xHA-tagged Zds1 and 13xMYC-tagged Sch9 were directly used for Western analysis. Sample 2: Co-IP, protein extracted from sch9Δ cells carrying 3xHA-tagged Zds1 and 13xMYC-tagged Sch9 were immunoprecipitated with anti-myc antibody and G protein beads (or only G protein beads) and then used for Western analysis. Sample 3: negative control, protein extracted from sch9Δ cells carrying 13xMYC-tagged Sch9 were immunoprecipitated with anti-myc antibody and G protein beads (or only G protein beads) and then used for Western analysis. Sample 4: blank control (beads only). (b) Western signals were detected using anti-myc antibody (the signals showed Sch9; 91.8 kD). Equal amounts of cellular protein were used. Sample 1: Co-IP, proteins extracted from sch9Δ cells carrying 3xHA-tagged Zds1 and 13xMYC-tagged Sch9 were immunoprecipitated with anti-HA antibody and G protein beads (or only G protein beads) and then used for Western analysis. Sample 2: blank control (beads only).

As our results indicated that Sch9 and Zds1 interacted physically, we speculated that Sch9 might regulate localization of Bcy1 via Zds1. To confirm this speculation, we investigated the effects of overexpression of ZDS1 on Bcy1 localization in sch9Δ cells and overexpression of SCH9 on Bcy1 localization in zds1Δ cells. According to Fig. 5, overexpression of ZDS1 led to a significantly increased cytoplasmic Bcy1 in wild-type cells, which was consistent with a previous report (Griffioen et al., 2001), and in sch9Δ cells. As shown in Fig. 6, overexpression of SCH9 resulted in increased cytoplasmic Bcy1 in wild-type cells, but overexpression of SCH9 had no visible effect on cytoplasmic Bcy1 in zds1Δ cells. All these results led us to the conclusion that Sch9 regulated localization of Bcy1 via Zds1.

Overexpression of ZDS1 led to increased cytoplasmic Bcy1 localization in wild-type cells and sch9Δ cells. Cells carrying multi-copy plasmids YEplac195 as control, whereas cells carrying multi-copy plasmids YEplac195-ZDS1 were used to overexpress Zds1.

Overexpression of SCH9 led to increased cytoplasmic Bcy1 localization in wild-type cells but not in zds1Δ mutant cells. Cells carrying multi-copy plasmids YEplac181 were used as control, whereas cells carrying multi-copy plasmids YEplac181-SCH9 were used to overexpress Sch9.

Bcy1 modification was found to be dependent on Yak1 (Griffioen et al., 2001). It was reported that faster-migrating iso-form of Bcy1-HA was detected in exponential phase wild-type cells, whereas a predominant slower-migrating iso-form of Bcy1-HA was detected in stationary phase wild-type cells. But a predominant faster-migrating iso-form of Bcy1-HA was detected in yak1Δ mutant in either exponential phase or stationary phase. Recently, we reported that the faster-migrating iso-form of Bcy1-HA was detected in sch9Δ mutant cells, either in exponential phase or in stationary phase (Zhang et al., 2011). To investigate whether Sch9 regulated Bcy1 phosphorylation via Yak1, we tested whether Sch9 affected the protein level of Yak1. Figure 7a, shows that the protein level of Yak1 in log-phase glucose-grown sch9Δ cells (Line 2) was markedly lower than in log phase glucose-grown W303-1A (Line 1). The protein level of Yak1 in stationary phase W303-1A (Line 3) was dramatically lower than in the log-phase W303-1A (Line 1), whereas a predominant slower-migrating iso-form of Yak1-HA was detected in stationary phase sch9Δ (Line 4). As shown in Fig. 7b, the protein level of Yak1 in glycerol-grown sch9Δ cells was markedly lower than in glycerol-grown W303-1A (Line 1). Phosphatase treatment of this slower-migrating iso-form of Yak1-HA resulted in the fast-migrating iso-form of Yak1-HA (Fig. 7b). These results suggest that Sch9 is involved in the regulation of Yak1 phosphorylation.

Effect of Sch9 on protein levels of Yak1 (MW: 91.245 Da) in glycerol-grown or glucose-grown W303-1A and sch9Δ cells. Proteins extracted from W303-1A and sch9Δ cells carrying YCplac22-YAK1-3xHA were used directly for Western analysis. Proteins were extracted, equal amounts of cellular protein (50 µg) were separated on a 10% SDS-PAGE gel. Anti-HA monoclonal antibody [mouse immunoglobulin G (IgG3); Tiangen] were used. (a) Protein levels of Yak1 in log phase or stationary phase glucose-grown W303-1A and sch9Δ cells. (b) Phosphatase treatment of Yak1 in stationary phase glucose-grown sch9Δ cells, and protein levels of Yak1 in glycerol-grown W303-1A and sch9Δ cells.

Discussion

In multicellular organisms, AKAPs targeted PKA holoenzyme to specific subcellular locations (Griffioen & Thevelein, 2002). AKAP-mediated targeting of PKA was thought to confer spatio-temporal control of PKA signaling to phosphorylate specific localized substrates. Compartmentalization of signal transduction pathways is an important spatio-temporal control of PKA signaling. In this way, signaling molecules of the same pathway are brought into close vicinity, thereby increasing the probability that they only affect each other appropriately. Recently, we reported that Bcy1 was predominately localized in nucleus in sch9Δ cells, whereas a large part of catalytic subunits of PKA transferred from nucleus into cytoplasm in sch9Δ cells (Zhang et al., 2011). Thus the liberated catalytic subunits were not restricted by the regulatory subunits and consequently able to phosphorylate preferentially substrates located nearby (e.g. fructose-1,6-bisphosphatase, trehalase), all leading to a high PKA activity phenotype of sch9Δ cells. Our research indicated that Sch9 regulated localization of Bcy1 via Zds1. In yeast, Zds1 may act as the anchor protein of PKA. We report for the first time that Sch9 and Zds1 interact physically. However, the mechanisms of Sch9 regulating Zds1 still need to be clarified.

It was reported that Bcy1 phosphorylation was dependent on Yak1 kinase (Griffioen et al., 2001). In this study, we found that the protein level of Yak1 decreased markedly in sch9Δ cells compared with wild-type cells. Thus Bcy1 could not be phosphorylated efficiently by Yak1 in sch9Δ cells. Earlier reports suggested that Yak1 and Sch9 acted in the parallel pathway. However, our results suggest for the first time that Sch9 is involved in regulating phosphorylation of Yak1. Additionally, stabilization of Yak1 in stationary phase sch9∆ was higher than in stationary phase wild type. It was reported that when glucose was limited, Yak1 accumulated in the nucleus, where it phosphorylated Pop2p, which was required for proper cell cycle arrest (Moriya et al., 2001). Higher stabilization of Yak1 in stationary phase sch9∆ was perhaps responsible for the long G1 phase in sch9∆ mutant cells.

Authors' contribution

A.Z. and W.G. contributed equally to this work.

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Table S1. Primers used in this study.

Table S2. Plasmids used in this study.

Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

Acknowledgements

We particularly thank Prof. Pingsheng Ma for constructive advice in this study.

References

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