Phosphorylation of the Saccharomyces cerevisiae Grx4p glutaredoxin by the Bud32p kinase unveils a novel signaling pathway involving Sch9p, a yeast member of the Akt / PKB subfamily Caterina Peggion1,*, Raffaele Lopreiato1,*, Elena Casanova1, Maria Ruzzene1,2, Sonia Facchin1, Lorenzo A Pinna1,2, Giovanna Carignani1 and Geppo Sartori1 ` Dipartimento di Chimica Biologica dell’Universita di Padova, Italy Venetian Institute of Molecular Medicine (VIMM), Padova, Italy Keywords Bud32p kinase; EKC ⁄ KEOPS complex; Grx4p glutaredoxin; Sch9p kinase Correspondence G Sartori, Dipartimento di Chimica Biologica ` dell’Universita di Padova, viale G Colombo, 3-35121 Padova, Italy Fax: +39 049 8073310 Tel: +39 049 8276141 E-mail: geppo.sartori@unipd.it *These authors contributed equally to this work (Received 31 July 2008, revised 26 September 2008, accepted October 2008) doi:10.1111/j.1742-4658.2008.06721.x The Saccharomyces cerevisiae atypical protein kinase Bud32p is a member of the nuclear endopeptidase-like, kinase, chromatin-associated ⁄ kinase, endopeptidase-like and other protein of small size (EKC ⁄ KEOPS) complex, known to be involved in the control of transcription and telomere homeostasis Complex subunits (Pcc1p, Pcc2p, Cgi121p, Kae1p) represent, however, a small subset of the proteins able to interact with Bud32p, suggesting that this protein may be endowed with additional roles unrelated to its participation in the EKC ⁄ KEOPS complex In this context, we investigated the relationships between Bud32p and the nuclear glutaredoxin Grx4p, showing that it is actually a physiological substrate of the kinase and that Bud32p contributes to the full functionality of Grx4p in vivo We also show that this regulatory system is influenced by the phosphorylation of Bud32p at Ser258, which is specifically mediated by the Sch9p kinase [yeast homolog of mammalian protein kinase B (Akt ⁄ PKB)] Notably, Ser258 phosphorylation of Bud32p does not alter the catalytic activity of the protein kinase per se, but positively regulates its ability to interact with Grx4p and thus to phosphorylate it Interestingly, this novel signaling pathway represents a function of Bud32p that is independent from its role in the EKC ⁄ KEOPS complex, as the known functions of the complex in the regulation of transcription and telomere homeostasis are unaffected when the cascade is impaired A similar relationship has already been observed in humans between Akt ⁄ PKB and p53-related protein kinase (Bud32p homolog), and could indicate that this pathway is conserved throughout evolution The Bud32p protein of Saccharomyces cerevisiae belongs to the piD261 family of atypical Ser ⁄ Thr protein kinases, which has representatives in virtually all eukaryotic and archaeal organisms Unlike the majority of eukaryotic protein kinases, the protein preferen- tially recognizes acidic substrates [1–3] Several different approaches have shown that Bud32p is able to interact with many yeast proteins [4–6] Particularly remarkable is its tight association with the still uncharacterized Kae1p, as the two proteins make up a single Abbreviations Akt ⁄ PKB, protein kinase B; EKC ⁄ KEOPS, endopeptidase-like, kinase, chromatin-associated ⁄ kinase, endopeptidase-like and other protein of small size; HA, hemagglutinin; PRPK, p53-related protein kinase; pSer, phosphorylated Ser; Ni-NTA, Ni2+-nitrilotriacetate–agarose FEBS Journal 275 (2008) 5919–5933 ª 2008 The Authors Journal compilation ª 2008 FEBS 5919 Role of Bud32p in a new signaling pathway in yeast C Peggion et al polypeptide in some archaeans, and their human homologs are also able to interact [4] Two recent papers have highlighted the importance of this association, by describing a novel and highly conserved protein complex named endopeptidase-like, kinase, chromatin-associated ⁄ kinase, endopeptidase-like and other protein of small size (EKC ⁄ KEOPS) [7,8], composed of Bud32p, Kae1p and three additional small proteins, lacking a known biochemical signature (Pcc1p, Pcc2p, Cgi121p) The EKC ⁄ KEOPS complex is essential for yeast viability, and is functionally related to telomere homeostasis and transcription control, as its mutations cause transcriptional impairment in the expression of specific gene groups, as well as relevant shortening of telomeres Although the molecular mechanism of EKC ⁄ KEOPS activity remains elusive, it has been proposed that the complex might promote the accessibility to chromatin, at telomeres as well as elsewhere on the genome, and regulate the recruitment of specific factors to their site of action [7,8] Although the kinase activity of Bud32p is relevant for the functions of the EKC ⁄ KEOPS complex in yeast, it is actually unknown whether Bud32p-dependent phosphorylation of other subunits of the complex could directly regulate its activity In addition to the components of the EKC ⁄ KEOPS complex, many other proteins have been identified as Bud32p interactors [4–6], suggesting that this protein kinase may have additional roles by specifically phosphorylating other substrates Among these Bud32p interactors, our attention has been drawn to the glutaredoxin Grx4p, which is an in vitro substrate of the protein kinase, being readily phosphorylated by recombinant, purified Bud32p at Ser134 [4], suggesting that Grx4p may be one of the physiological substrates of Bud32p in yeast cells Grx4p belongs to the subfamily of yeast monothiolic glutaredoxins, together with Grx3p and Grx5p [9,10] Whereas the function of Grx5p in mitochondrial Fe–S cluster assembly has been extensively investigated, the role of the nuclear glutaredoxins Grx3p and Grx4p is less well characterized The single deletion of either GRX3 or GRX4 leads to weak growth defects, but the double deletion strongly affects cellular growth and the response to oxidative stress As the two proteins display relevant sequence similarity, they might have overlapping or redundant functions Accordingly, it has been shown that both Grx3p and Grx4p are involved in the transcriptional modulation of the iron regulon, by controlling the nucleo-cytoplasmic shuffle of the transcriptional activator Aft1p [11–13] In this work, we demonstrate that Grx4p is a physiological substrate of Bud32p in yeast cells, and show 5920 that this relationship is influenced by the phosphorylation state of Bud32p In fact, Bud32, as well as its human homolog p53-related protein kinase (PRPK) [14,15], displays a highly conserved C-terminal sequence, rich in basic amino acids, that fulfils the consensus recognized by protein kinase B (Akt ⁄ PKB) (RxxRxS ⁄ THy) [16] Interestingly, the activity of PRPK on its known substrate (Ser15-p53) mainly (but not exclusively) depends on the phosphorylation of its Ser250 residue by Akt ⁄ PKB [17] This prompted us to investigate whether the activity of Bud32p could also be modulated by phosphorylation of its Ser258 residue, possibly mediated by Sch9p, which is considered to be a yeast homolog of mammalian Akt ⁄ PKB Sch9p is an AGC kinase [18] involved in a number of cellular processes, including the response to nutrient-mediated stimuli and the regulation of replicative and chronological lifespan [19–23] Recently, Sch9p has been identified as a transcriptional activator that is recruited, only in stress conditions, to the chromatin of genes induced by osmotic stress [24] Sch9p is also regulated by TOR complex 1, which phosphorylates several amino acids situated in its C-terminal sequence [25] Recent data have implicated the PAS kinase Rim15p and the Rps6p protein as substrates of yeast Sch9p [25–28] Here, we identify a novel phosphorylation cascade implicating Sch9p, Bud32p and Grx4p, which apparently does not affect the telomeric and transcriptional activities of the EKC ⁄ KEOPS complex, suggesting an additional function for Bud32p in yeast cells Results and Discussion Grx4p is an in vivo substrate of Bud32p The characterization of Bud32p as a protein kinase was achieved by using a recombinant form of the enzyme, purified from Escherichia coli [1] Bud32p was shown to be able to autophosphorylate and to phosphorylate in vitro the Ser ⁄ Thr residues of acidic substrates, such as casein As is the case with many other protein kinases, autophosphorylation of Bud32p on its activation loop correlated with an increased activity on substrates, whereas all the mutant forms unable to autophosphorylate were also inactive on substrates [1– 3] A subsequent search for Bud32p-associated proteins in yeast identified Grx4p as a Bud32p interactor, and the observation that recombinant Bud32p was able to phosphorylate recombinant Grx4p in vitro [4] suggested that Grx4p might be an in vivo substrate of the protein kinase To verify this assumption, we constructed several yeast strains in which Bud32p (wild-type or mutant) is FEBS Journal 275 (2008) 5919–5933 ª 2008 The Authors Journal compilation ª 2008 FEBS C Peggion et al expressed from its chromosomal location in fusion with the hemagglutinin (HA) epitope at the C-terminus The two mutations analyzed here substitute respectively Asp161 and Lys52, two amino acids that are essential for the catalytic activity of the recombinant protein [2] The two bud32 mutants are characterized by a slow growth phenotype that is, however, less stringent than that exhibited by cells in which BUD32 is deleted (Fig 1A) First, we checked the activity of Bud32p upon immunoprecipitation in an in vitro assay on recombinant, purified Grx4p Preliminary experiments, performed in the conditions used in the biochemical characterization of recombinant Bud32 (i.e in the presence of Mn2+ as bivalent ion) [1–4], showed that the kinase activity of immunoprecipitated Bud32p was extremely low Conversely, the substitution of Mn2+ with Mg2+ significantly improved the enzymatic activ- A B Fig (A) The kinase activity of Bud32p is relevant for its in vivo functionality The wild-type [W303 and BUD32-HA (WT)], the bud32 mutants K52A-HA (K52A), D161A-HA (D161A) and the BUD32 deleted strain (bud32D) were grown until stationary phase in YPD medium, and diluted to · 107 cellsỈmL)1; 10-fold serial dilutions were then spotted onto solid YPD medium Growth was observed after days at 28 °C (B) Immunoprecipitated yeast endogenous Bud32p autophosphorylates and phosphorylates recombinant Grx4p in vitro HA-tagged Bud32p, wild-type or catalytically inactive, was immunoprecipitated from lysates (500 lg of total protein) of strains BUD32-HA (WT), D161A-HA (D161A) and K52A-HA (K52A) grown in complete glucose medium (YPD) until exponential phase Immunocomplexes were subjected to an in vitro phosphorylation reaction in the presence of [33P]ATP[cP] and 500 ng of recombinant, purified Grx4p After SDS ⁄ PAGE, proteins were blotted onto a filter that was autoradiographed (left panel) and then revealed with specific antibodies for Bud32p or Grx4p (right panel) Role of Bud32p in a new signaling pathway in yeast ity of native Bud32p This behavior could be related to subtle differences between the structures of the recombinant and the native protein kinase (e.g protein folding and ⁄ or post-translational modifications) Even in the presence of Mg2+, the catalytic efficiency detected for the native kinase remains low, the results, however, being consistent with those already reported for the recombinant protein [4] It is worth noting that very recent data [29] have indicated that the activity of Bud32p is inhibited by its functional partner Kae1p, suggesting that the protein(s) coprecipitating with Bud32p might reduce its activity also in our in vitro assays Altogether, native Bud32p displays specific kinase activity on recombinant Grx4p: in fact, the wild-type protein, which undergoes autophosphorylation, is able to phosphorylate Grx4p, whereas the mutants D161A and K52A almost completely fail to autophosphorylate and exhibit a significantly lower activity on Grx4p (Fig 1B) The partial phosphorylation of Grx4p still observed in the case of the two mutants (corresponding to 30–40% of that of the wild-type) could be explained either by the intervention of another (contaminant) kinase, copurified with Bud32p, or, alternatively, by residual activity of the mutant proteins, which would still be able to catalyze the kinase reaction in the presence of an excess of exogenous Grx4p substrate Together, these results demonstrate that native Bud32p is able to phosphorylate Grx4p, further suggesting that the same relationship may exist in yeast cells In a further approach, we investigated whether endogenous Grx4p was phosphorylated by Bud32p Native, myc-tagged Grx4p was immunoprecipitated from yeast, and the precipitate was analyzed for the presence of coprecipitated Bud32p The ability of Bud32p to interact in vivo with Grx4p has already been reported, although coimmunoprecipitation of the two proteins depends on the experimental conditions [4,7] In our present experiments, a weak but specific signal was detected when the Grx4p immunoprecipitates were analyzed for the presence of wild-type Bud32p Interestingly, this signal was clearly stabilized in the presence of a catalytic mutant of Bud32p, such as D161A (Fig 2A) This observation is consistent with Grx4p being an in vivo substrate of Bud32p, based on the assumption that the transient interaction between the enzyme and its substrate is strengthened if the course of the catalytic reaction is hampered In accordance with this, the low amount of wild-type Bud32p present in the precipitate is still active on native Grx4p when subjected to in vitro phosphorylation, whereas the reaction is impaired in the case of coimmunoprecipitation of Grx4p with D161A mutant FEBS Journal 275 (2008) 5919–5933 ª 2008 The Authors Journal compilation ª 2008 FEBS 5921 Role of Bud32p in a new signaling pathway in yeast C Peggion et al A C B D Fig Bud32p coimmunoprecipitates with and phosphorylates native Grx4p Myc-tagged Grx4p was immunoprecipitated from 500 lg of total protein from the following strains: GRX4-myc ⁄ BUD32-HA (WT), GRX4-myc ⁄ bud32-D161A-HA (D161A), BUD32-HA (no tag) and GRX4-myc ⁄ bud32D (bud32D), grown as in Fig Immunocomplexes were either directly subjected to SDS ⁄ PAGE and immunoblotting (A, D) or subjected to an in vitro phosphorylation reaction in the presence of [33P]ATP[cP] and then to SDS ⁄ PAGE and western blotting (B, C) The band corresponding to Grx4p in (C) is indicated (*) In (A), the starting amounts of Bud32p present in the cell lysates were equivalent, as revealed by antibody against HA (Input) In (B), the amounts of Bud32p and Grx4p present in the kinase reaction after immunoprecipitation are revealed by antibody against HA or Myc, respectively (Fig 2B) To be sure that the observed Grx4p phosphorylation was catalyzed by coimmunoprecipitated Bud32p, we performed a similar experiment in a bud32D ⁄ GRX4-myc mutant strain; the result, shown in Fig 2C, clearly indicates that Bud32p is responsible for the radioactivity incorporated into Grx4p In this assay, the phosphorylation reaction occurs by addition of the [33P]ATP[cP] directly to the resin containing the native, immunoprecipitated Grx4p and proteins associated with it Any phosphotransferase activity on Grx4p therefore requires the presence in the immunoprecipitate of (at least) one protein kinase Our results confirmed the presence of Bud32p in the Grx4p immunoprecipitate, supporting the idea that Bud32p is responsible (or coresponsible) for the kinase activity observed on Grx4p Accordingly, in the case of the bud32D ⁄ GRX4-myc mutant strain, in which Bud32p is lacking (Fig 2C), the phosphorylation of Grx4p completely disappeared However, the possibility cannot be excluded that another, unidentified kinase may act on Grx4p, but in this case it should be associated with Bud32p rather than with Grx4p, as no activity was detected when the immunoprecipitation was performed in the absence of Bud32p Finally, we showed (Fig 2D) that the phosphorylation state of Grx4p, as revealed by antibody against phosphorylated serines (pSer), was much lower when Grx4p was immuno5922 precipitated from the bud32D mutant strain, as compared to the wild-type However, the detectable presence of pSer on Grx4p, even in the absence of Bud32p, indicates that the glutaredoxin is phosphorylated also by other kinases in yeast cells Taken together, these results demonstrate that Bud32p participates in the in vivo phosphorylation status of Grx4p Phosphorylation of Ser134 of Grx4p by Bud32p contributes to the functionality of the glutaredoxin in yeast cells As indicated by previous in vitro data, phosphorylation of Grx4p by wild-type Bud32p occurs mainly at Ser134 and, more weakly, at Ser133, two residues embedded in a highly acidic stretch of the protein [4] This sequence is situated in the linker region between the thioredoxin-like and the glutaredoxin domains of Grx4p [10], and its modification would be likely to influence, directly or indirectly, the activity of the enzyme To evaluate the contribution of either Ser134 or Ser133 phosphorylation to the biological competence of Grx4p in yeast cells, we created a series of unphosphorylatable mutants (S134A, S133A, S133A ⁄ S134A), as well as the phospho-mimic S134D, in order to compare their behavior with that of the wild-type sequence S cerevisiae, however, possesses another FEBS Journal 275 (2008) 5919–5933 ª 2008 The Authors Journal compilation ª 2008 FEBS C Peggion et al Role of Bud32p in a new signaling pathway in yeast nuclear monothiolic glutaredoxin, Grx3p, which is very similar to Grx4p; the two proteins cooperate and show interchangeable roles, e.g in the transcriptional regulation of iron-dependent genes [11–13] Therefore, to specifically investigate in vivo the effect of mutating Grx4p, we created the double null strain grx3D ⁄ grx4D Surprisingly, we noticed that, unlike what was observed with other commonly used yeast strains (such as BY4742 and CML128), cells containing the double mutations are nonviable in the W303 genetic background (Fig S1), indicating that in the W303 strain the functions of nuclear monothiolic glutaredoxins are essential This may reflect the subtle differences existing between yeast laboratory strains, in particular with regard to the responses to environmental changes or stresses involving these oxidoreductases The different GRX4 coding sequences were inserted in galactose-inducible yeast plasmids (see Experimental procedures), which were used to transform heterozygous diploid grx3D ⁄ grx4D cells After sporulation and tetrad dissection, we isolated a complete set of haploid grx3D ⁄ grx4D strains containing wild-type or mutant GRX4 plasmids We then compared their growth in glucose medium, where the plasmidic alleles are weakly expressed, and observed (Fig 3A, left panel) that, in these conditions, wild-type GRX4 was able to fully restore yeast growth, similarly to the bona fide positive control (wild-type W303 cells carrying the empty plasmid) Although the substitution of Ser133 by Ala (S133A) did not affect the in vivo functionality of Grx4p, the mutation of Ser134 (S134A) slightly affected its function, this impairment, however, being completely restored by the phospho-mimic substitution by Asp (S134D) Accordingly, the double mutation of Ser133 and Ser134 (SS-AA) showed the same effect as the single S134A mutation, confirming that Ser134 of Grx4p has a major role in vivo with respect to Ser133 In addition, we checked the effects of Grx4p overexpression, by growing the yeast strains in galactose medium, in which the expression of plasmid-carried genes is strongly induced (Fig 3A, right panel) We observed that overexpression of wild-type A Fig Ser134 phosphorylation of Grx4p by Bud32p contributes to its functionality in vivo (A) The wild-type W303 strain carrying the empty plasmid and the mutant strain grx4D ⁄ grx3D, carrying the plasmids coding for either wild-type or mutant Grx4p (S134A, S133A, SS-AA, S134D) were grown until stationary phase in SD selective medium and diluted at · 107 cellsỈmL)1 Tenfold serial dilutions were spotted either onto solid SD (Glucose) or SG (Galactose) plates Growth was observed after days at 28 °C See text for details (B) Total protein lysates (500 lg) of yeast cells expressing wild-type, HA-tagged Bud32p (Bud32–HA) were used to immunoprecipitate Bud32p as in Fig Immune complexes were subjected to an in vitro phosphorylation reaction in the presence of [33P]ATP[cP] and 25 ng of recombinant wild-type Grx4p (WT) or 50 ng of the Grx4p double mutant S133A ⁄ S134A (SS-AA) After SDS ⁄ PAGE and blotting, filters were autoradiographed (upper panels), and then visualized with specific antibodies against Bud32p or Grx4p (lower panels) The radiolabeled bands (*) are produced by an unidentified contaminant of the purified Grx4p proteins The strong signals in western blots (**) are from the IgG light chains released by the resin used for Bud32–HA precipitation B FEBS Journal 275 (2008) 5919–5933 ª 2008 The Authors Journal compilation ª 2008 FEBS 5923 Role of Bud32p in a new signaling pathway in yeast C Peggion et al Grx4p was toxic to yeast cells, whereas overexpression of either the single S134A mutant or the double S133A ⁄ S134A mutant was less detrimental, indicating that these substitutions somehow impaired the activity of the glutaredoxin, rendering it less toxic to the cell Remarkably, the phospho-mimic substitution S134D was able to restore the toxicity of the glutaredoxin, supporting the relevance of Ser134 phosphorylation for the biological properties of Grx4p Taken together, our data indicate that under normal growth conditions, Ser134 phosphorylation may be almost dispensable for Grx4p functionality, although it could be relevant in the regulation of specific pathway(s) upon environmental changes, allowing yeast cells to respond appropriately to these stimuli Finally, to evaluate the specific contribution of Bud32p to Grx4p phosphorylation at Ser134-Ser133, we used a mutant version of recombinant, His-tagged Grx4p (S133A ⁄ S134A) as substrate for an in vitro phosphorylation reaction by native Bud32p immunoprecitated from yeast cells Despite several purification attempts, the recovery of wild-type and mutant Grx4p was low, and the phosphorylation assays were run with quantities of substrate consistently lower than in other experiments (such as the one shown in Fig 1) However, as shown in Fig 3B, the results demonstrated that Bud32p was able to phosphorylate the minimal amount of wild-type Grx4p present in the reaction (left, upper panel), whereas the phosphorylation of mutant Grx4p completely failed to so (right, upper panel), despite the presence of a higher amount of recombinant protein, as revealed by the western blot (lower panels) Accordingly, a parallel experiment, performed with the same Grx4p substrates and an aliquot of recombinant Bud32p, showed that the recombinant protein was also unable to phosphorylate the mutant Grx4p (not shown), thus confirming that the phosphorylation of Grx4p by Bud32p specifically involves Ser134 Activity of Bud32p on Grx4p is regulated through phosphorylation by Sch9p As recently demonstrated [17], the kinase activity of PRPK (the human homolog of Bud32p) on its known substrate Ser15-p53 is positively regulated in vitro and in vivo by phosphorylation of Ser250, which is specifically mediated by Akt ⁄ PKB The observation that Bud32p at the homologous residue (Ser258) also displays the consensus sequence for Akt ⁄ PKB (RxxRxS ⁄ THy), and the existence in yeast of a functional homolog of Akt ⁄ PKB, the protein kinase Sch9p, prompted us to look for the presence in yeast of a 5924 similar enzyme–substrate relationship and to determine whether the activity of Bud32p could be modulated by Sch9p Synthetic genetic interaction between BUD32 and SCH9 In order to find whether a functional relationship existed between Sch9p and Bud32p, we took advantage of a genetic approach, easily carried out in yeast, looking for a possible genetic interaction between sch9 and bud32 mutants Figure 4A shows that the combination of the SCH9 and BUD32 deletions is nonviable, and that deletion of SCH9, when combined with a bud32 catalytically inactive mutant (D161A), affects the growth of yeast cells more severely than each of the two single mutations (Fig 4B) These results supported the hypothesis that Sch9p and Bud32p are functionally related, and prompted us to examine their relationship in depth In vivo phosphorylation of Ser258 of Bud32p is strongly reduced in an sch9D mutant strain Phosphorylation of Bud32p at Ser258 can be identified by the use of antibodies (anti-pSer258) that recognize the phosphorylated target site for Akt ⁄ PKB present at the C-terminus of the protein [17] We first established that wild-type, His-tagged Bud32p, when overexpressed in yeast and purified by Ni2+–nitrilotriacetic acid agarose (NiNTA), is recognized by the antibodies, indicating that the protein is phosphorylated in vivo at Ser258 The antibodies are specific, as a similarly overexpressed and purified Bud32p mutant, in which the Ser258 residue is replaced with Ala (S258A), is recognized very weakly (Fig 5A) Next, to ascertain whether phosphorylation of Ser258 of Bud32p is due to Sch9p, we immunoprecipitated endogenous HA-tagged Bud32p (expressed from its chromosomal location) from a wild-type strain (BUD32-HA) and from a mutant strain in which SCH9 had been deleted (sch9D ⁄ BUD32-HA) Figure 5B shows that Bud32p was recognized by the antibodies against pSer258 when immunoprecipitated from the wild-type strain, but not (or very poorly) when immunoprecipitated from the sch9D mutant, supporting the idea that Sch9p is implicated in the phosphorylation of Bud32p at Ser258 In order to investigate the impact of Ser258 substitution in vivo, we compared the growth of the S258A mutant with that of the wild-type strain and of other bud32 mutants (D161A, K52A) and deletion mutants (bud32D), and observed that cells were almost FEBS Journal 275 (2008) 5919–5933 ª 2008 The Authors Journal compilation ª 2008 FEBS C Peggion et al Role of Bud32p in a new signaling pathway in yeast A B Fig Genetic interaction between BUD32 and SCH9 (A) The double deletion of BUD32 and SCH9 is lethal for yeast Heterozygotic diploid bud32D ⁄ sch9D cells were transformed with a centromeric plasmid (pFL38) carrying the wild-type BUD32 sequence and the URA3 marker [counterselectable on 5-fluoroorotic acid (5-FOA)-containing medium) After tetrad dissection, haploid spores were recovered and genotypes were determined Yeast cells containing the plasmidic URA3 marker and coming from some complete tetrads were plated on 5-FOA medium Only cells containing the double deletion bud32D ⁄ sch9D cannot lose the plasmid and cannot grow on 5-FOA plates Two representative complete tetrads are shown DB, DS, DD are for bud32D, sch9D, and bud32D ⁄ sch9D, respectively (B) The slow-growth phenotype of the bud32-D161A mutant is exacerbated by the additional deletion of SCH9 Wild-type BUD32-HA (WT) and mutant strains D161A-HA (D161A), sch9D ⁄ BUD32-HA (sch9D) and sch9D ⁄ D161A-HA (sch9D ⁄ D161A) were grown until stationary phase in YPD medium, and diluted to · 107 cellsỈmL)1; 10-fold serial dilutions were then spotted onto solid YPD medium Growth was observed after days at 28 °C A B Fig Bud32p is phosphorylated at Ser258 in the wild-type but not in an sch9D mutant strain (A) Antibodies against pSer258 (anti-pSer258) recognize ectopically expressed wild-type Bud32p and not the S258A mutant The W303 wild-type strain, transformed with galactose-inducible vectors carrying the BUD32 sequence (WT or S258A mutant) fused to a His epitope, was grown in YPGal until exponential phase, when Bud32–His (WT or S258A) was purified with the NiNTA resin from the cell lysate The resin was subjected to SDS ⁄ PAGE and immunoblotting (B) Endogenous HA-tagged Bud32p is phosphorylated at Ser258 when immunoprecipitated from the wild-type but not from an sch9D mutant strain The wild-type BUD32-HA strain (WT) and the sch9D ⁄ BUD32-HA mutant (sch9D), both expressing BUD32-HA from its chromosomal location, were grown in YPD medium (until exponential phase) Native Bud32p was immunoprecipitated from cell lysate with the anti-HA resin, and processed by SDS ⁄ PAGE and immunoblotting unaffected when compared to catalytically inactive or null mutants (not shown) Although we cannot completely rule out the possibility that Sch9p phosphorylates Bud32p also at other Ser ⁄ Thr residues (which would be embedded in sequences different from the consensus recognized by Akt ⁄ PKB), we suppose that Ser258 phosphorylation could affect cell growth only in specific situations, and not in the (normal) conditions FEBS Journal 275 (2008) 5919–5933 ª 2008 The Authors Journal compilation ª 2008 FEBS 5925 Role of Bud32p in a new signaling pathway in yeast C Peggion et al here tested In this case, the S258A substitution should not affect the main properties of the kinase, but may operate as a regulatory site for Bud32p, e.g by allowing ⁄ promoting its association with specific partners (or substrates) However, the absence of a growth phenotype for the S258A mutant does not explain the strong genetic interaction observed between BUD32 and SCH9 (see above and Fig 4); we must, then, infer that the two genes, besides being interrelated via phosphorylation of the Bud32p Ser258 residue by Sch9p, have overlapping functions in a still unidentified pathway Sch9p interacts with Bud32p and phosphorylates it in vitro at Ser258 The results described above are consistent with a role for Sch9p in the phosphorylation of Bud32p at Ser258, but not prove that Bud32p is a direct substrate of Sch9p To clarify this point, we checked the ability of Sch9p to interact with Bud32p and phosphorylate it in vitro First, a pull-down experiment was performed by incubating recombinant, purified Bud32-His, previously bound to the NiNTA resin, with a yeast cellular lysate in which Sch9p was HA-tagged The results shown in Fig 7A indicate that the two proteins are able to interact In fact, a western blot analysis revealed the presence of Sch9p associated with the NiNTA resin only where recombinant Bud32p had been previously bound to the resin The different bands recognized by the antibodies against HA are noteworthy, as they probably highlight different phosphorylation states of Sch9p, as already demonstrated [21] Next, we investigated whether Bud32p is a substrate of Sch9p The HA-tagged Sch9p was immunoprecipitated by the use of the anti-HA affinity matrix: Fig 7Ba confirms the immunoprecipitation of Sch9p (lane 1); as controls, the anti-HA matrix was incubated either with a cellular lysate of the untagged strain, or with no lysate (lanes and 3) The resin was subjected to an in vitro phosphorylation reaction in the presence of recombinant, purified Bud32-His In the autoradiograph (Fig 7Bb), it can be seen that, when immunoprecipitated Sch9p was present (lane 1), radioactivity incorporation was greatly increased (2.5-fold) with respect to the background levels (lanes and 3), which are due to autophosphorylation of recombinant Bud32-His Subsequent detection of the filter with antibodies against pSer258 (Fig 7Bc) confirmed that this phosphorylation takes place at Ser258 Conditions that regulate Sch9p abundance influence Bud32p phosphorylation at Ser258 The abundance of Sch9p, which is involved in the control of numerous nutrient-sensitive processes, is in fact modulated by nutrients [21] To verify whether the onset of conditions that modify the amount of Sch9p has an effect on the phosphorylation of Bud32p at Ser258, we compared the level of Bud32p phosphorylation in wild-type cells grown on a fermentable carbon source (glucose) to that in cells grown on glycerol To this end, we used a yeast strain expressing Bud32p and Sch9p fused to different epitopes (SCH9-myc ⁄ BUD32HA), and first verified the abundance of Sch9p: Fig 6A shows that the amount of Sch9p in the cell lysates (as revealed by western blot) was higher in the case of cells grown in glucose than in the case of cells grown in glycerol Accordingly, phosphorylation at Ser258 of Bud32p, immunoprecipitated from the same lysates, is more extensive in cells grown in glucose than in cells grown in glycerol (Fig 6B) These results suggest that Bud32p might be a physiological target of Sch9p, representing one of the effectors of this protein kinase known to be involved in multiple cellular processes A B Fig Phosphorylation at Ser258 of Bud32p is related to Sch9p abundance (A) Sch9p levels in cell lysates Equal amounts (20 lg of total protein) of cell lysates obtained from strain SCH9-myc ⁄ BUD32-HA, grown in YPD (glucose) or in YP with glycerol (Glycerol) until exponential phase, were subjected to SDS ⁄ PAGE and immunoblotting to visualize Sch9p (B) Phosphorylation state of immunoprecipitated Bud32p HAtagged Bud32p was immunoprecipitated from the same lysates used in (A) (500 lg of total protein), using the anti-HA resin Bound proteins were eluted with SDS ⁄ PAGE loading buffer, electrophoresed, and immunoblotted with the indicated antibodies 5926 FEBS Journal 275 (2008) 5919–5933 ª 2008 The Authors Journal compilation ª 2008 FEBS C Peggion et al Role of Bud32p in a new signaling pathway in yeast A B Fig Sch9p is able to interact with Bud32p and phosphorylate it in vitro (A) Interaction between Sch9p and Bud32p by pull-down assay Five micrograms of recombinant, purified Bud32–His (lane 1), bound to the NiNTA resin, were incubated with 500 lg of a cellular lysate of strain SCH9-HA The resin was washed with a buffer containing imidazole, and subjected to SDS ⁄ PAGE and immunoblotting to detect Sch9p or Bud32p As a reference, the same amount of resin, with no Bud32–His bound, was incubated with the same lysate, and then treated as described and loaded in lane Western blot analysis revealed that Sch9p was retained by the resin only in lane (B) Immunoprecipitated Sch9p phosphorylates recombinant Bud32p HA-tagged Sch9p was immunoprecipitated with the anti-HA resin from 500 lg (total proteins) of a lysate of strain SCH9-HA (lane 1) As a reference, the anti-HA resin was incubated with a lysate of wild-type strain W303, in which SCH9 has no tag (lane 2), or with no lysate (lane 3) The immunoprecipitation of Sch9p only in lane is revealed in (a) The resins were subjected to a phosphorylation reaction in the presence of [33P]ATP[cP] (b) or cold ATP (c) and 100 ng of recombinant His-tagged Bud32p [quantified by antibodies against His in panel (d)] After SDS ⁄ PAGE and immunoblotting, Bud32p phosphorylation was detected as radioactivity incorporation (b) and by anti-pSer258 (c) Taken together, the in vivo and in vitro results on Bud32p phosphorylation by Sch9p indicate that Bud32p is one of the downstream targets of Sch9p, whose substrates are still largely unknown, with few exceptions, e.g the Rps6p ribosomal protein, indicated in a recent report as a probable Sch9p substrate, at least in vitro [25] Our data confirm that Sch9p recognizes the same target sequence as human Akt ⁄ PKB, further supporting the assumption of the similarity between the two protein kinases This raises the question of whether phosphorylation of Bud32p at Ser258 represents a way to regulate its activity Ser258 phosphorylation of Bud32p promotes recognition and phosphorylation of Grx4p We next compared the two forms of Bud32p (phosphorylated, or not, at Ser258) for their ability to phosphorylate recombinant, purified Grx4p The HAtagged Bud32p was immunoprecipitated from the wildtype strain BUD32-HA (Fig 8A, lane 1) and from the sch9D mutant strain sch9D ⁄ BUD32-HA (lane 3), as well as from a mutant in which wild-type SCH9 is present but Ser258 of Bud32p is replaced by Ala (S258A-HA) (lane 2); the precipitates were then subjected to an in vitro phosphorylation reaction in the presence of recombinant Grx4p Figure 8A shows that, when Bud32p was not Ser258-phosphorylated, i.e in the sch9D strain, or when it carried the S258A mutation, phosphorylation of Grx4p was reduced by up to 40% of the wild-type activity, similarly to what was seen with the K52A and D161A mutants The observation that Grx4p phosphorylation was comparable in both mutant strains (bud32-S258A and sch9D, lanes and 3, respectively) rules out the possibility of Grx4p being a direct substrate of Sch9p Nevertheless, unlike with catalytic-defective mutants, autophosphorylation of immunoprecipitated Bud32p was not affected with respect to the wild-type, strongly suggesting that Ser258 modification of Bud32p does not alter the catalytic activity of the protein kinase per se We further confirmed such evidence by checking in vitro the enzymatic activity of native Bud32p on the model substrate casein, and observed that both forms of Bud32p, phosphorylated or not, had similar catalytic properties, as they were able to phosphorylate casein (data not shown) These results may therefore indicate that Ser258 modification could modulate the ability of Bud32p to recognize the Grx4p substrate To confirm this hypothesis, we performed a pulldown experiment in which His-tagged wild-type Bud32p and mutant S258A were bound to the NiNTA FEBS Journal 275 (2008) 5919–5933 ª 2008 The Authors Journal compilation ª 2008 FEBS 5927 Role of Bud32p in a new signaling pathway in yeast C Peggion et al A B Fig Ser258 phosphorylation of Bud32p influences its interaction with Grx4p (A) Yeast endogenous Bud32p phosphorylates the Grx4p substrate only if activated by Ser258 phosphorylation Native, HA-tagged Bud32p was immunoprecipitated from the wild-type BUD32-HA strain (lane 1), the S258A-HA mutant strain (lane 2) and the sch9 null strain (sch9D ⁄ BUD32-HA, lane 3), and subjected to an in vitro phosphorylation reaction in the presence of recombinant Grx4p (B) Grx4p coprecipitates with ectopically expressed wild-type Bud32p and not with the S258A mutant The GRX4-myc ⁄ BUD32-HA strain was transformed with an empty centromeric plasmid (lane 1) and with the same plasmid carrying the wild-type (lane 2) or the S258A mutant (lane 3) forms of the BUD32 sequence, both fused to the His epitope Cells were grown until exponential phase in galactose medium to induce the ectopic expression of the plasmid-borne genes, and aliquots of the lysates containing equal amounts of Grx4p (as revealed by anti-myc detection, shown in Input) were incubated with the NiNTA resin to bind His-tagged Bud32p The resin was finally subjected to SDS ⁄ PAGE and western blotting resin and analyzed for their ability to coprecipitate Grx4p Yeast strains expressing native, myc-tagged Grx4p were transformed with a plasmid bearing wildtype BUD32, or the bud32-S258A mutant, both fused to a His-tag After growth in galactose medium (to induce the expression of the plasmid inserted genes), cells were lysed and treated with the NiNTA resin in order to isolate His-tagged Bud32p together with the associated proteins In this experiment, we analyzed the ability of ectopic Bud32p-His, phosporylated or not at Ser258, to compete with endogenous Bud32p for binding to native Grx4p Figure 8B shows that wild-type His (lane 2) efficiently substituted for the endogenous kinase in the association with Grx4p, whereas a similar amount of the S258A mutant (lane 3) failed to bind Grx4p, as shown by a signal comparable to the background level (lane 1) The (unexpected) high signal of the Grx4p background indicates that NiNTA resin may be somehow able to aspecifically bind myc-tagged Grx4p Taken together, these results indicate that phosphorylation of Bud32p at Ser258 positively regulates the ability of the protein kinase to associate with Grx4p and therefore to phosphorylate it, whereas this modification does not affect the catalytic activity of the protein kinase per se Phosphorylation of Bud32p at Ser258 is unrelated to its functions within the EKC/KEOPS complex The observed phosphorylation of Bud32p at Ser258, besides having an effect on Grx4p, might also influence in yeast cells the activity of the whole EKC ⁄ KEOPS complex, of which Bud32p is a crucial component 5928 Notably, Bud32p-Ser258 modification might impact on both functions (transcription control and telomere homeostasis), in which the EKC ⁄ KEOPS complex is involved [7,8] We therefore investigated whether phosphorylation of Bud32p-Ser258 is linked to these processes, first by analyzing telomere length in several wild-type and mutant strains As shown in Fig 9A, catalytically inactive or null bud32 mutations (K52A; D161A; bud32D) led to telomeres that were shortened in comparison to the wild-type, whereas the telomere length of the S258A mutant was unaffected, being almost identical to that of the wild-type (W303 or BUD32-HA) Remarkably, deletion of either SCH9 or GRX4 did not impair telomere elongation We have then examined the effects of Ser258 phosphorylation of Bud32p on the transcriptional activity of the EKC ⁄ KEOPS complex by analyzing the activation rate of the galactoseinducible gene GAL1, known to be regulated by the complex By using real-time RT-PCR and northern blot analyses (a representative northern blot is shown in Fig 9B), we compared the levels of GAL1 mRNA in wild-type and bud32 mutant strains upon transcription induction, observing (as expected) a reduction of mRNA levels in kinase-dead or null mutants, but no difference between the wild-type and the S258A mutant strains, in accordance with the effects observed on telomere elongation The results presented here thus indicate that the phosphorylation cascade involving Sch9p, Bud32p and Grx4p is apparently not relevant to the telomeric or to the transcriptional function of the Bud32p-associated EKC ⁄ KEOPS complex Accordingly, Grx4p has never been isolated as a FEBS Journal 275 (2008) 5919–5933 ª 2008 The Authors Journal compilation ª 2008 FEBS C Peggion et al A Role of Bud32p in a new signaling pathway in yeast cellular role of Bud32p (via Grx4p phosphorylation), which would be unrelated to the functions of Bud32p as a component of EKC ⁄ KEOPS, but could be involved in responses to environmental stimuli or endogenous stresses Whether Bud32p could achieve the two tasks while being simultaneously associated with the EKC ⁄ KEOPS complex and the Grx4p substrate or, alternatively, whether only a cellular fraction of Bud32p, not included in EKC ⁄ KEOPS, could associate with the glutaredoxin, will be a matter for future investigation Conclusions B Fig Ser258 phosphorylation of Bud32p does not influence its functions in telomere elongation and transcriptional regulation (A) Telomere length analysis in wild-type and in mutant strains impaired in the Sch9–Bud32–Grx4 cascade Genomic DNA of the indicated strains (grown in rich medium until exponential phase) was purified and digested with XhoI, producing telomeric terminal DNA fragments of about kb, which were separated on 1.2% agarose, tranferred onto a nitrocellulose membrane, and checked with a 33P-labeled probe specific for telomeric TG1–3 repeats (B) Transcriptional activation of GAL1 is unaffected by Ser258 mutation Yeast strains were grown in noninducing raffinose medium to exponential phase, and then incubated for 30 in galactose medium to activate the GAL regulon Total mRNAs were extracted and subjected to standard northern blot analysis GAL1 mRNA, and ACT1 mRNA (considered as a loading control), were detected by the use of specific radiolabeled probes (see Experimental procedures) component of EKC ⁄ KEOPS, indicating that the phosphorylation of Grx4p by Bud32p is independent from the known activities of the complex and must therefore be involved in different functions and unrelated pathways Furthermore, we noticed that growth of bud32 mutants lacking the highly conserved C-terminal tail was not affected, similarly to what has been observed in the case of the single S258A substitution, indicating that these mutations not impair the main biological properties of the kinase Moreover, recent data [29] from the 3D structure of an archeal Bud32 homolog (Mj1130p) indicate that the Bud32p C-terminal tail is located far from the catalytic site, suggesting that its alteration should not be detrimental to the overall structure Finally, our data are consistent with a In this article, we describe a novel S cerevisiae signaling pathway that implicates Bud32p and Sch9p (yeast homologs of mammalian PRPK and Akt ⁄ PKB, respectively) in modulating the phosphorylation state of Grx4p in yeast cells, with possible implications for the regulation of its activity Notably, we show that Sch9p phosphorylates, both in vitro and in vivo, Ser258 of Bud32p, and that this modification does not affect the catalytic properties of the enzyme, but promotes its ability to associate with its substrate Grx4p and, consequently, to phosphorylate it This event appears to be physiologically regulated by the cellular levels of Sch9p, suggesting that nutrient-mediated stimuli, detected by Sch9p, may also be important in controlling Bud32p phosphorylation state and finely tuning its functions These results highlight for the first time Bud32p as a substrate of Sch9p and probably one of the effectors of this crucial protein kinase Interestingly, this pathway is unrelated to the known functions of the Bud32p-associated EKC ⁄ KEOPS complex in both telomere metabolism and transcription, suggesting that Bud32p participates in multiple pathways in yeast cells An important aspect of these data concerns the evolutionary conservation of this regulatory system It has been demonstrated that in human cells, phosphorylation at Ser250 of PRPK (Bud32p homolog) by Akt ⁄ PKB positively regulates in vivo the activity of PRPK on its physiological substrate Ser15-p53 [17] However, it remains to be established whether Ser250 modification directly influences the PRPK catalytic properties or, alternatively, promotes the association with its substrate p53 On the basis of the results described here, we are tempted to speculate that in eukaryotic cells, phosphorylation by the Akt ⁄ PKB kinases on Bud32-like proteins would create adhesive surfaces, which promote the association with specific targets, without major effects on the catalytic activity of the enzyme FEBS Journal 275 (2008) 5919–5933 ª 2008 The Authors Journal compilation ª 2008 FEBS 5929 Role of Bud32p in a new signaling pathway in yeast C Peggion et al Experimental procedures Yeast and bacterial strains The yeast strains used in this study (Table S1) are derived from the W303 strain [30] E coli InvaF¢ [endA1, recA1, hsdR17 (r)K, m)K), supE44, k), thi-1, gyrA, relA1, F80lacZaDM15D (lacZYA-argF), deoR+, F¢] was used for DNA manipulation, and E coli BL21 (DE3) [F), ompT, hsdSb (rb), mb)) gal dcm (DE3)] was used as the host for expression of heterologous proteins, as described in [1] and [4] Media YP (1% yeast extract, 1% bactopeptone) contained 2% dextrose (YPD) or 2% galactose (YPGal) or 3% glycerol (YPGly) as carbon source; SD (0.67% yeast nitrogen base without amino acids, 2% dextrose or galactose) contained auxotrophic requirements as needed The bacterial medium was LB (1% Bactotryptone, 0.5% yeast extract, 0.5% NaCl and, when requested, 100 lgỈmL)1 ampicillin) Solid media were obtained by the addition of 2% agar Media components were from Difco (Becton Dickinson and Company, Franklin Lakes, NJ, USA), and auxotrophic requirements were from Sigma (St Louis, MO, USA) Overexpression of genes from the pYeDP plasmid [31] was obtained by pregrowing the transformed cells in SD (galactose) Then, the cultures were diluted to D600 nm = 0.2 and grown in YPGal until mid-log phase Cells were routinely incubated at 28 °C Construction of mutant yeast strains Standard DNA manipulation was performed as described in [32] Gene deletions and C-terminal epitope-tagging of wild-type and mutant alleles were performed using the PCR-based one-step in vivo strategies described, respectively, in [33] and [34] The mutagenized strains have been verified by PCR analysis and, when necessary, direct DNA sequencing of the manipulated genomic regions The wild-type, along with the D161A and K52A mutant alleles of BUD32 inserted in the pFL38 plasmid under the control of the BUD32 promoter, were already available [4] The S258A mutant form of BUD32 was obtained using the QuikChangeTM Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA), in combination with the S258A-S and S258A-AS primers (Table S2), on the pFL38 plasmid bearing the wild-type BUD32 allele All the BUD32 mutagenized sequences were isolated from the pFL38 plasmid by BamHI ⁄ PstI digestion and cloned in the YIplac211 integrative plasmid [35], modified by deletion of the unique EcoRI restriction site Substitution at the appropriate genomic locus of the wild-type BUD32 gene with the mutagenized bud32 alleles was performed as described in [36] and verified by DNA sequencing 5930 The pYeDP–BUD32–His recombinant plasmid was obtained as follows The BUD32 coding sequence was amplified by PCR from yeast wild-type genomic DNA using the BUD-S and BUD-AS primers (Table S2) The forward primer (BUD-S) introduces the restriction site BamHI (underlined) two nucleotides upstream of the ATG codon, and the reverse primer (BUD-AS) introduces a sequence coding for six His residues (lower-case) in-frame with the 3¢-end of the gene, immediately followed by a stop codon and (underlined) the restriction site recognized by the KpnI enzyme The resulting amplification product was cloned in the pYeDP-1 ⁄ 8.2 vector at the BamHI ⁄ KpnI sites [31] In this construct, the expression of the cloned gene is under the control of the CYC1–GAL1-10 promoter, allowing galactoseinducible overproduction of the Bud32–His6 fusion protein pYeDP–bud32-S258A–His was obtained using pYeDP– BUD32–His as template DNA and the QuikChangeTM Site-Directed Mutagenesis Kit, as previously described The same strategy was used to construct the pYeDP– GRX4 recombinant plasmid Amplification of the GRX4 coding sequence from genomic DNA was performed by PCR using the GRX4-S and GRX4-AS primers (Table S2), carrying, respectively, the restriction site for BamHI (underlined) two nucleotides upstream of the ATG codon, and the restriction site for KpnI (underlined) two nucleotides downstream of the TAA stop codon After digestion with these two enzymes, the PCR product was ligated in the pYeDP-1 ⁄ 8.2 vector digested with the same enzymes Starting from this recombinant plasmid, we obtained the S134A, S133A, S133A ⁄ S134A and S134D mutagenized forms of GRX4 as described previously, using, respectively, the S134A-S ⁄ S134A-AS, S133A-S ⁄ S133A-AS, S133A-S134AS ⁄ S133A-S134A-AS and S134D-S ⁄ S134D-AS pairs of primers (Table S2) Transformation of yeast cells with recombinant plasmids was performed as described in [37] Purification of wild-type and mutant S133A ⁄ S134A Grx4 proteins from E coli cells The pET–GRX4 plasmid [4] was used to overexpress the His-tagged wild-type form of Grx4 in E coli To produce the S133A ⁄ S134A double mutant form of Grx4 in this expression system, we mutagenized the sequence of the pET–GRX4 plasmid, following the same strategy used to create this mutant in the pYeDP-1 ⁄ 8.2 vector (see above) Purification of both wild-type and mutant, His-tagged forms of Grx4 from E coli was performed as described in [4] Northern blot analysis Northern blot analyses were performed as described in [32] ACT1-specific (as internal control) and GAL1-specific probes were amplified by PCR from genomic yeast DNA using, respectively, the ACT-S ⁄ ACT-AS and GAL-S ⁄ GALAS pairs of primers listed in Table S2 About 25 ng of the FEBS Journal 275 (2008) 5919–5933 ª 2008 The Authors Journal compilation ª 2008 FEBS C Peggion et al PCR products were radiolabeled using DECAprime II (Applied Biosystems ⁄ Ambion, Austin, TX, USA) with [32P]dCTP[aP] and used to probe 10 lg of total yeast RNAs blotted on a nitrocellulose membrane The membrane was scanned directly using the Cyclone apparatus (Perkin Elmer, Waltham, MA, USA) Preparation of cell extracts, immunoprecipitation and immunoblotting Immunoblots, as well all the assays performed with immunoprecipitated proteins, are representative of at least three independent experiments Approximately 109 yeast cells, grown until mid-log phase, were harvested by centrifugation at 3068 g for and washed with ice-cold water Cells were resuspended in IP buffer [50 mm Tris ⁄ HCl, pH 8.0, 150 mm NaCl, 0.1% NP-40, 10 mm NaF, mm Na3VO4, mm phenylmethanesulfonyl fluoride, 50 mm b-glycerophosphate, 0.2% complete protease inhibitor cocktail (Roche Diagnostics Ltd, Burgess Hill, UK), mm EDTA, mm EGTA], at a concentration of · 107 cells per 10 lL, in the presence of an identical volume of glass beads (diameter 0.5 mm), and lysed by vortexing for 30 s at 6000 r.p.m in a MagnaLyser apparatus (Roche Diagnostics) For the interaction between endogenous Bud32-HA (or ectopically expressed Bud32–His6) and Grx4–myc, cells were lysed in a modified IP buffer (20% glycerol instead of 0.1% NP-40) The soluble fraction was obtained by centrifugation (20 at 10 000 g at °C), and the protein concentration was determined by the Bradford method Lysate proteins (0.5 mg) were incubated with the specific affinity matrix at °C for h, or overnight Immunocomplexes were washed with 50 mm Tris ⁄ HCl (pH 8.0), 150 mm NaCl (500 mm in the case of phosphorylation assays), and 200 lm phenylmethanesulfonyl fluoride Bound proteins were eluted by heating the beads for at 95 °C in SDS ⁄ PAGE loading buffer Samples were subjected to 11% SDS ⁄ PAGE, blotted on a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA), and processed with the indicated antibodies; detection was obtained by enhanced chemiluminescence (Amersham ⁄ GE Healthcare Ltd, Chalfont St Giles, UK), and the signal was quantified on a Kodak Image Station 440CF and analyzed with Kodak 1d image software Monoclonal antibodies against phosphoserine (cat no P3430) (anti-pSer), polyhistidine (cat no H1029) (anti-His) and HA (cat no H9658) were from Sigma, and monoclonal antibody against c-myc (anti-c-myc) (clone 9E10) was from Roche For immunoprecipitation of HA or c-myc-tagged proteins from yeast extracts, we used, respectively, the anti-HA and the anti-c-myc affinity matrix (Covance, Richmond, CA, USA) The phosphospecific antibody that recognizes the pSer258 residue of Bud32 (anti-pSer258) is described elsewhere [17] Role of Bud32p in a new signaling pathway in yeast Bud32–His6 pull-down assay To identify the interaction between Bud32 and Sch9, a pull-down analysis was carried out using the NiNTA affinity matrix (Ni2+–nitrilotriacetic acid agarose) (Qiagen, Valencia, CA, USA) preloaded with lg of recombinant, purified Bud32–His6, prepared as described elsewhere [1] Subsequently, 20 mL of NiNTA ⁄ Bud32–His6 resin were incubated for h at °C with approximately 500 lg of cell lysate obtained from the SCH9-HA3 strain (see Table S1) In this protocol, yeast cells were lysed (as previously described) with IPI buffer (IP buffer, 20 mm imidazole) The resin was washed twice with IPI buffer, and bound proteins were eluted with SDS ⁄ PAGE loading buffer and analyzed by immunoblotting, using antibodies as indicated As a negative control, the same cellular lysate was incubated with the NiNTA matrix without any bound protein, and the sample was subjected to the same protocol Immune complex protein kinase assays The protein kinase activity of endogenous wild-type Bud32–HA and of its mutant forms, immunoprecipitated from a cellular lysate (as described in a previous section), was routinely assayed by incubating about 50 ng of Bud32– HA, bound to anti-HA sepharose beads (Covance), at 37 °C for 30 in 20 lL of a medium containing 50 mm Tris ⁄ HCl (pH 7.5), 10 mm MgCl2, and 25 lm [33P]ATP[cP] (Amersham ⁄ GE Healthcare; specific radioactivity 2000– 3000 c.p.m.Ỉpmol)1) and 500 ng of purified, recombinant Grx4–His (prepared as described in [4]) as phosphorylatable substrate All experiments were conducted in the presence of lm K25, a specific inhibitor of the casein kinase protein Phosphorylation assay on the S133A ⁄ S134A mutant form of recombinant, His-tagged Grx4 (see Supporting information) was performed in the same way, but with a lower ( 25 ng) amount of recombinant substrate Phosphorylation of recombinant Bud32–His6 (100 ng) by endogenous Sch9 was performed by incubation of Sch9– HA3, immunoprecipitated from yeast cells as previously described, in 20 lL of a medium containing 50 mm Tris ⁄ HCl (pH 7.5), 12 mm MgCl2, and 10 lm [33P]ATP[cP] ( 1500 c.p.m.Ỉpmol)1) for 20 at 30 °C The reaction was stopped by addition of gel electrophoresis loading buffer, and samples were subjected to 11% SDS ⁄ PAGE Proteins were blotted onto a nitrocellulose membrane (Biorad), and the membranes were directly scanned on the Cyclone apparatus (Packard) and detected by immunoblotting using the appropriate antibodies Telomere length measurement Telomere length was measured by Southern blotting as described in [39] Yeast cells were grown in YPDA medium FEBS Journal 275 (2008) 5919–5933 ª 2008 The Authors Journal compilation ª 2008 FEBS 5931 Role of Bud32p in a new signaling pathway in yeast C Peggion et al until exponential phase, when genomic DNAs were extracted XhoI-digested genomic DNA fragments were separated by gel electrophoresis in 1.2% agarose, transferred to a Hybond-N+ membrane (Amersham Biosciences), and probed with a radiolabeled Y¢-TG1–3 DNA fragment Acknowledgements This work was supported by grants from: Ministero ` Italiano dell’Universita e della Ricerca (MIUR), Progetto PRIN 2005, and University of Padova, Progetto di Ateneo 2005 References Stocchetto S, Marin O, Carignani G & Pinna LA (1997) Biochemical evidence that Saccharomyces cerevisiae YGR262c gene, required for normal growth, encodes a novel Ser ⁄ 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Inactivation of six genes from chromosome VII and XIV of Saccharomyces cerevisiae and basic phenotypic analysis of the mutant strains Yeast 16, 255–265 Marcand S, Gilson E & Shore D (1997) A proteincounting mechanism for telomere length regulation in yeast Science 275, 986–990 Supporting information The following supplementary material is available: Fig S1 Activity of the Grx3 ⁄ Grx4 nuclear monothiolic glutaredoxins is essential in the W303 yeast strain Table S1 Yeast strains [38] Table S2 Oligonucleotides This supplementary material can be found in the online version of this article Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary material supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 275 (2008) 5919–5933 ª 2008 The Authors Journal compilation ª 2008 FEBS 5933 ... for the endogenous kinase in the association with Grx4p, whereas a similar amount of the S25 8A mutant (lane 3) failed to bind Grx4p, as shown by a signal comparable to the background level (lane... phosphorylation states of Sch9p, as already demonstrated [21] Next, we investigated whether Bud32p is a substrate of Sch9p The HA-tagged Sch9p was immunoprecipitated by the use of the anti-HA... Bud32p in yeast cells Results and Discussion Grx4p is an in vivo substrate of Bud32p The characterization of Bud32p as a protein kinase was achieved by using a recombinant form of the enzyme,