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Yale University School of Medicine, Department of Molecular Biophysics and Biochemistry, 333 Cedar Street, New Haven, Connecticut 06520-8024
Received 8 November 2004/ Returned for modification 16 December 2004/ Accepted 22 February 2005
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ABSTRACT |
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cells was unphosphorylated and exhibited low protein kinase activity. Moreover, Cak1 directly phosphorylated Ctk1 in vitro. Unlike wild-type cells, cells expressing Ctk1T338A delayed growth at early stationary phase, did not show the increase in Ser2 phosphorylation that normally accompanies the transition from rapid growth to stationary phase, and had compromised transcriptional activation of two stationary-phase genes, CTT1 and SPI1. Therefore, Ctk1 phosphorylation on Thr-338 is carried out by Cak1 and is required for normal gene transcription during the transition into stationary phase. |
INTRODUCTION |
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The largest subunit of RNA pol II contains a conserved C-terminal domain (CTD) composed of tandem repeats of the consensus heptapeptide sequence YSPTSPS (7, 9). Phosphorylation of Ser2 and Ser5 within the heptapeptide functions during transcriptional initiation, elongation, and RNA processing (5, 32, 34, 40). Phosphorylations of the two serine residues have essential nonredundant functions (45). Phosphorylation of Ser5 regulates the transition from transcriptional initiation to elongation and is limited to polymerases within the promoter region. In contrast, phosphorylation of Ser2 occurs after transcriptional initiation and remains high until transcriptional termination (4, 28). Phosphorylation of Ser2 increases the affinity of the polymerase for polyadenylation factors, enabling efficient processing of the pre-mRNA 3' end (1, 38). Phosphorylated Ser5 and Ser2 also provide binding sites for the histone methyltransferases Set1 and Set2, respectively, facilitating passage of the polymerase through chromatin (25, 33, 43).
Four different cyclin-dependent kinases (CDKs), Kin28, Srb10, Bur1, and Ctk1, phosphorylate the CTD in S. cerevisiae. Kin28 phosphorylates Ser5, and this phosphorylation is required for transcription of most RNA pol II genes (6, 13, 18). Srb10 phosphorylates Ser5 and Ser2, but the effect of this phosphorylation is inhibitory (3, 17). Ctk1 is the major kinase acting on Ser2; recent findings indicate that it also phosphorylates Ser5 (20, 30, 36). Dephosphorylation of Ser5 and Ser2 is accomplished by the Ssu72 and Fcp1 phosphatases at the end of the transcription cycle (27, 29).
Phosphorylation of Ser2 by Ctk1 has been implicated in the control of transcript elongation (4, 19, 31). Recent studies revealed that it is also required for cotranscriptional recruitment of polyadenylation factors (1). In the absence of Ctk1, transcription termination and processing of RNA 3' ends are compromised. Surprisingly, CTK1 is not an essential gene but its mutation confers a conditional cold-sensitive phenotype (30). Ctk1 assembles with Ctk2 and Ctk3 into an active protein kinase complex that phosphorylates RNA pol II on Ser2 (39). Ctk2 and Ctk3 are unstable proteins degraded through the ubiquitin-proteasome pathway (15, 16). Ctk1 activity and Ser2 phosphorylation of RNA pol II increase under a variety of stress conditions (35, 36).
Like the cell cycle CDKs, most CDKs functioning during transcription undergo activating phosphorylation within their T loops. Thus, previous studies demonstrated that Cak1 phosphorylates Kin28 and Bur1 within their T loops on Thr162 and Thr240, respectively (12, 26, 44). These phosphorylations are not essential under normal conditions but provide growth advantages when CDK-cyclin interactions are weakened by additional mutations (24, 26). Substitutions within the T loops of Kin28 and Bur1 compromise CTD phosphorylation in vitro (24, 26, 44).
We noticed that Ctk1 contains a typical T loop and examined whether it is phosphorylated. We found that Ctk1 was indeed phosphorylated on a conserved Thr-338 in vivo. Mutation of Thr-338 conferred a cold-sensitive phenotype and compromised the protein kinase activity of Ctk1. Several lines of evidence indicated that this phosphorylation was carried out by Cak1. We also found that Ctk1 phosphorylation and the increased phosphorylation of RNA pol II on Ser2 are essential to activate transcription of stationary-phase genes during the diauxic shift.
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MATERIALS AND METHODS |
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(W303a [ctk1::HIS3]), ctk2 (W303a [ctk2::URA3]), and ctk3 (W303a [ctk3::URA3]) were previously described (35). ctk1T338A and ctk1D324N were generated by QuikChange mutagenesis (Stratagene) with a YCplac22-CTK1-HA plasmid as template. The parental and mutant plasmids were transformed into ctk1 cells, and TRP+ clones corresponding to wild type and the ctk1T338A and ctk1D324N mutants were collected. To improve the resolution of Ctk1 on two-dimensional gels, a stretch of negatively charged amino acids, DEDEDEDE, was introduced at its C terminus by PCR amplification, resulting in CTK1-8DE-HA. For analysis of the cold-sensitive phenotype and of RNA pol II phosphorylation, CTK1 and ctk1T338A were used without tags. All PCR products were sequenced to verify that no additional mutations were introduced. ctk1 deletion strain 1834-2A (cdc28::HIS3 cak1::LEU2/pCDC28-169-43244B) was described previously (8). Yeast cultures were grown in YPD medium and in complete minimal medium as previously described (2). For analysis of cold-sensitive phenotypes, yeast cultures were serially diluted fivefold and spotted on YPD plates that were then incubated at 30°C and 17°C for 4 days.
Protein analyses and immunoprecipitation.
Cells from 25-ml cultures (optical density at 600 nm [OD600], 
0.6) were collected, washed with ice-cold TBS (10 mM Tris-Cl [pH 8.0], 150 mM NaCl), and suspended in 0.4 ml lysis buffer (50 mM Tris-Cl [pH 8.0], 10 mM MgCl2, 150 mM NaCl, 10 mM EDTA, 10% glycerol, 10 µg/ml each of leupeptin, chymostatin, and pepstatin [Chemicon], 1 mM dithiothreitol [DTT]). Cell lysis was achieved by shaking yeast suspensions with 0.4 g of glass beads (0.5-mm diameter; Sigma) in a bead beater (Biospec Products) for five 30-s pulses with 30 s on ice between pulses. Glass beads and cell debris were removed by centrifugation at 14,000 rpm in a Microfuge at 4°C for 10 min. The supernatant was clarified by centrifugation at 65,000 rpm in a TLA 100.2 rotor (Beckman) for 10 min at 4°C. Protein concentrations were determined using the Bradford assay (Bio-Rad) with bovine serum albumin as the standard. Yeast extracts were aliquoted, frozen in liquid nitrogen, and stored at –80°C.
For immunoprecipitation of Ctk1-HA, 1 µg of monoclonal antibody 12CA5 (Covance) was bound to 50 µl protein A-agarose (Invitrogen) at 4°C for 2 h. One hundred fifty microliters of yeast extract containing 10 mg/ml of protein was incubated with 50 µl of 12CA5 beads in 300 µl immunoprecipitation buffer (50 mM Tris-Cl [pH 8.0], 15 mM MgCl2, 10 mM EDTA, 1% NP-40, 1 mM DTT, 10 µg/ml each of leupeptin, chymostatin, and pepstatin [Chemicon], 0.5 mg/ml ovalbumin) at 4°C for 2 h. The beads were washed three times with 0.5 ml immunoprecipitation buffer for 5 min each at 4°C and stored at –20°C.
To detect Ctk1-HA by immunoblotting, 20 µg of yeast extract proteins was separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to an Immobilon-P membrane (Millipore). The membrane was incubated with rabbit anti-HA antibodies (100 ng/ml; Santa Cruz). Cak1 was detected using monoclonal anti-Cak1 antibody as described previously (21). Phosphorylated and unphosphorylated forms of RNA pol II were detected with H5 and 8WG16 antibodies (Covance) as described previously (35, 36). Proteins were visualized by chemiluminescence (SuperSignal; Pierce).
To fractionate Ctk1-containing complexes, yeast extracts were prepared from 400 ml of cells (OD600, 0.6), as described above. Yeast extracts (1 ml, 10 mg/ml protein) were fractionated on a Superdex-200 gel filtration column (Amersham Biosciences) in 25 mM HEPES (pH 7.5)-350 mM NaCl-1 mM DTT-10 µg/ml each of leupeptin, chymostatin, and pepstatin (Chemicon). A 10-µl aliquot of each 1-ml fraction was resolved by SDS-PAGE, and Ctk1-HA was detected by immunoblotting with rabbit anti-HA antibodies as described above.
Two-dimensional gel electrophoresis. Ctk1-8DE-HA was resolved by nonequilibrium pH gel electrophoresis. One hundred microliters of yeast extract containing 1 mg of protein was treated with 2 µl DNase and 2 µl RNase (10 U/µl each; Roche) in 80 mM ß-glycerophosphate-60 mM NaCl-10 mM MgSO4-10 mM EDTA-10% NP-40-10 µg/ml each of leupeptin, chymostatin, and pepstatin (Chemicon) for 30 min on ice. The proteins were precipitated with 9 volumes of cold acetone for 1 h at –20°C, centrifuged at 7,000 rpm for 10 min in a Microfuge at 4°C, and air dried. The protein pellets were dissolved in 200 µl 9.5 M urea-2% Triton X-100-1% ampholyte 3/10-1% ampholyte 7/9 (Bio-Rad)-100 mM DTT. Ten-microliter samples were loaded onto 8-mm tube gels (Bio-Rad) containing 9.8 M urea, 2% Triton, 4% acrylamide-bisacrylamide (19:1), 1% ampholyte 3/10, and 1% ampholyte 7/9 (Bio-Rad). Proteins were focused for 1,900 V-h with 10 mM phosphoric acid and 20 mM NaOH in the upper and lower buffer chambers, respectively. Following separation in the first dimension, gels were equilibrated in 62 mM Tris-Cl (pH 8.0)-2% SDS-10% glycerol, and proteins were separated by SDS-PAGE. Ctk1-8DE-HA was detected by immunoblotting using rabbit anti-HA antibodies as described above.
Protein kinase assays. (i) CTD kinase assay.
Immunoprecipitated Ctk1-HA complexes (8 µl; see above) were incubated with 4 µg CTD peptide (YSPTSPS)4 in the presence of 5 µCi [
-32P]ATP and 10 µM ATP in 20 µl KB buffer (50 mM Tris-Cl [pH 8.0], 15 mM MgCl2, 150 mM NaCl, 0.1% Tween, 10 mM DTT, 0.1 mg/ml ovalbumin, 10 µg/ml each of leupeptin, chymostatin, and pepstatin [Chemicon]) for 60 min at 23°C. The reaction products were separated by SDS-PAGE and analyzed by PhosphorImaging (Molecular Dynamics) and by autoradiography.
(ii) Phosphorylation of Ctk1.
Ctk1-HA was expressed in the Coupled Transcription/Translation System (Promega) according to the manufacturer's protocol. Synthesized Ctk1-HA was immunoprecipitated (see above). GST-Cak1 was purified from insect cells as described previously (42). Ctk1-HA beads (8 µl) were incubated with 0.4 µg recombinant GST-Cak1 in the presence of 5 µCi [-32P]ATP and 10 µM ATP in 20 µl KB buffer for 90 min at 23°C. The reaction products were separated by SDS-PAGE and analyzed by PhosphorImaging (Molecular Dynamics) and autoradiography.
RNA isolation and analysis.
Total RNA was isolated from 100-ml cultures (OD600, 0.6) by an acid lysis protocol. Briefly, cells were pelleted, resuspended in 1.5 ml lysis buffer (10 mM Tris-Cl [pH 7.5], 10 mM EDTA, 0.5% SDS), and incubated with 1.5 ml acidic phenol, pH 4.5 (Gibco BRL) for 1 h at 65°C with brief vortexing every 10 min. Aqueous phases were extracted with phenol-chloroform-isoamyl alcohol (25:24:1) and chloroform-isoamyl alcohol (24:1). The RNA was ethanol precipitated for 3 h at –20°C, centrifuged at 10,000 rpm for 15 min at 4°C, washed with 70% ethanol, and resuspended in 300 µl diethyl pyrocarbonate-treated H2O. RNA concentrations were determined spectrophotometrically. For hybridization analysis, 20 µg of RNA was electrophoresed in 1% formaldehyde-agarose gels and transferred to GeneScreen membranes (NEN Life Science Products). DNA probes were labeled with [
-32P]dCTP using a random priming kit (Roche). Hybridizations were carried out for 10 h at 42°C according to the manufacturer's protocol. Signals were detected by PhosphorImaging (Molecular Dynamics) and by autoradiography.
Microarray hybridization and analysis. For microarray hybridization, mRNA was isolated using an Oligotex column (QIAGEN). cDNA probes were synthesized using Superscript II reverse transcriptase (Life Technologies) in the presence of Cy3- and Cy5-conjugated dCTP (Amersham). Cy3- and Cy5-labeled probes corresponding to wild-type and mutant samples were combined and hybridized with custom-made yeast whole-genome microarrays (Yale Microarray Facility). The microarrays were read on a laser scanner GenePix4000A (Axon Instruments Inc.). The fold change for every gene was calculated as an average of pixel-by-pixel ratios using the GenePix Pro software (Axon Instruments Inc.).
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RESULTS |
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strain, which was inviable (Fig. 1B). We also noticed that ctk1T338A cells had a specific growth delay during the diauxic shift, when yeast cells prepare to enter stationary phase (see below). Taken together, the phenotypes of ctk1T338A cells suggested that Thr-338 was essential for Ctk1 activity under suboptimal conditions.
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cells and analyzed on high-resolution gels by immunoblotting. Unfortunately, Ctk1-HA migrated through one-dimensional gels as a single band, which was consistent with previous reports. We then attempted to resolve Ctk1 on two-dimensional gels, separating proteins according to their charge in the first dimension and by their molecular weight in the second dimension. We found in preliminary experiments that the extremely basic isoelectric point of Ctk1-HA (pI = 9.4) significantly reduced resolution in the first dimension. To compensate for this effect, we appended eight negatively charged amino acids to the C terminus of Ctk1. The resulting protein, Ctk1-8DE-HA, was judged functional based on its ability to complement the cold-sensitive phenotype of ctk1 cells. Ctk1-8DE-HA could be resolved into two distinct spots, suggestive of posttranslational modification (Fig. 1C, top). A reference protein (Ctk1-HA without the introduced acidic residues) was added from a parallel extract to allow comparison of Ctk1-8DE-HA mobilities on different gels. Yeast extract containing Ctk1-8DE-HA was then treated with 
phosphatase in the absence or presence of phosphatase inhibitors. The acidic spot disappeared after phosphatase treatment, indicating that it corresponded to phosphorylated Ctk1-8DE-HA (Fig. 1C, spot 1, middle panel). The reference protein was mock treated, so that its mobility was unaltered. Conversely, Ctk1T338-8DE-HA migrated as a single spot with the same mobility as phosphatase-treated wild-type Ctk1 (Fig. 1C, spot 2, lower panel and second panel). More of the Ctk1-8DE-HA was present in the acidic spot after phosphatase inhibitor treatment (Fig. 1C, third panel) than in the untreated sample (Fig. 1C, first panel), suggesting that the inhibitors also prevented dephosphorylation by endogenous phosphatases (see also Fig. 3A). We concluded that spots 1 and 2 correspond to phosphorylated and unphosphorylated forms of Ctk1-8DE-HA, respectively. Taken together, these experiments indicated that Ctk1 is phosphorylated in vivo at a unique site corresponding to Thr-338. A recent proteomic study also identified Thr-338 as a site of Ctk1 phosphorylation (14).
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cells. The three Ctk1 proteins were expressed at comparable levels (Fig. 2A, top; data not shown).
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cells to grow at low temperatures.
Because phosphorylation of the T loop increases cyclin binding to some CDKs, we wondered whether Ctk2 and Ctk3 could associate stably with unphosphorylated Ctk1. To address this question, we fractionated yeast extracts from wild-type and ctk1T338A mutant cells on gel filtration columns and compared the sizes of Ctk1-containing complexes. We found that Ctk1 eluted as a complex of 160 kDa (Fig. 2C) that probably contained Ctk2 and Ctk3. Ctk1T338A showed the same elution pattern. In contrast, Ctk1 isolated from ctk3 cells appeared in fractions of lower molecular weight, indicating that Ctk3 was a component of the 160-kDa complex and that binding of Ctk3 was not affected by the absence of T-loop phosphorylation in cells expressing Ctk1T338A. Therefore, it is unlikely that loss of Ctk1T338A activity in vitro resulted from dissociation of its cyclin subunits. It is worth noting that the level of Ctk1 in ctk2 and ctk3 mutant cells was significantly lower than that in wild-type cells, suggesting that binding to Ctk2 and Ctk3 stabilized Ctk1 (Fig. 2C and data not shown).
Cak1 phosphorylates Ctk1 within the T loop.
The primary candidate for Ctk1 phosphorylation was Cak1, as it phosphorylates other yeast CDKs within their T loops. Therefore, we first examined whether Ctk1 phosphorylation is Cak1 dependent. Although CAK1 is normally an essential gene, it can be rendered nonessential in the presence of a form of Cdc28 (Cdc28-169-43244B) containing a number of mutations, including a T169E mutation of its site of activating phosphorylation, making its activation by cyclins independent of Cak1 (8). In this experiment, Ctk1-8DE-HA was expressed in wild-type and cak1 mutant cells and analyzed by two-dimensional gel electrophoresis. As demonstrated earlier, Ctk1-8DE-HA isolated from wild-type cells migrated as a doublet (Fig. 1C and 3A, spots 1 and 2). In contrast, Ctk1-8DE-HA expressed in cak1 cells migrated as a single spot corresponding to the unphosphorylated form of Ctk1-8DE-HA (Fig. 3A, bottom).
Using a similar approach, we then assessed whether Cak1 is required for Ctk1 activity. To this end, Ctk1 was immunoprecipitated from cak1 cells and assayed for its CTD kinase activity in vitro. We also immunoblotted cell extracts to determine their levels of Cak1 and Ctk1. As expected, Cak1 was absent from cak1 cells, whereas expression of Ctk1-HA was comparable in CAK1 and cak1 cells (Fig. 3B, lanes 2 and 3). Deletion of CAK1 eliminated detectable Ctk1 activity (Fig. 3B, bottom, compare lanes 2 and 3), and reintroduction of CAK1 restored Ctk1 activity (Fig. 3B, lane 4). Similar results were obtained using a conditional cak1-23 mutant at the nonpermissive temperature (data not shown). We conclude that Ctk1 activity depends strictly on the integrity of its T loop and the presence of Cak1.
Because Cak1 also phosphorylates Kin28 and Bur1, two CDKs that interact with RNA pol II transcription complexes and function in close proximity to Ctk1, it was formally possible that the Cak1-dependent phosphorylation of Ctk1 was mediated by Kin28 or Bur1. To rule out these possibilities, we asked whether Ctk1 activity requires the integrity of the Kin28 and Bur1 pathways. Ctk1 activity was examined after isolation from conditional kin28-ts and bur1-1 mutant strains, each of which lacks the corresponding protein kinase activity. Ctk1 retained its full activity in both strains (data not shown), suggesting that neither of these kinases is responsible for Ctk1 phosphorylation and that Cak1 phosphorylates Ctk1 directly.
We tested whether Cak1 could phosphorylate Ctk1 in vitro. In spite of numerous attempts, we were unable to detect Cak1-mediated phosphorylation or activation of Ctk1 isolated from cak1 cells (data not shown). Because Cak1 preferentially phosphorylates CDKs unbound to cyclin subunits (37), we suspected that Ctk1 isolated from yeast cells might be inaccessible for Cak1-mediated phosphorylation. To test whether a Ctk1 monomer might be a better substrate for Cak1, we expressed Ctk1-HA by translation in a reticulocyte lysate, immunoprecipitated it, and incubated it with recombinant Cak1 in the presence of radioactive ATP (Fig. 3C). In addition to Cak1 autophosphorylation (Fig. 3C, lane 2, bottom), we detected phosphorylation of wild-type Ctk1 (Fig. 3C, lane 6) but not of Ctk1T338A (Fig. 3C, lane 4). Thus, Cak1 can phosphorylate Ctk1 directly but its accessibility to the T loop appears to be restricted in the presence of Ctk2 and Ctk3.
Phosphorylation of Ctk1 promotes the transition into stationary phase.
Phosphorylation of Ser2 of RNA pol II by Ctk1 increases during the diauxic shift, when nutrients become limiting and cells prepare to enter stationary phase (36). We wondered whether the role of T-loop phosphorylation might become more prominent under these conditions. To examine this possibility, we compared the growth of wild-type and ctk1T338A cells. The two strains grew at nearly identical rates during exponential phase, but their growth started to diverge as nutrients became limited (Fig. 4A). The ctk1T338A cells delayed growth and reached stationary phase later than wild-type cells. This small but reproducible difference in growth rates suggested that the activity of Ctk1T338A was limiting for cell growth during this period. We then examined the level of RNA pol II phosphorylation during the diauxic shift. In agreement with previous reports, we found that phosphorylation of Ser2 in wild-type cells increased and remained elevated during the transition from rapid growth to stationary phase (Fig. 4B, lanes 1 to 4). This effect was specific for Ser2 phosphorylation, as the amount of unphosphorylated RNA pol II changed little. The level of Ser2 phosphorylation and the total amount of RNA pol II declined at later times (data not shown). In contrast, ctk1T338A cells showed little change in Ser2 phosphorylation during the diauxic shift (Fig. 4B, lanes 5 to 8). The difference between wild-type and ctk1T338A cells was most apparent at the beginning of this interval (Fig. 4B, compare lanes 2 and 6), which just preceded the growth delay of ctk1T338A cells (Fig. 4A). cak1 cells also showed no change in RNA pol II phosphorylation at the diauxic shift (Fig. 4C). Thus, Cak1-mediated phosphorylation of Ctk1 on Thr-338 is essential for the elevated Ser2 phosphorylation and rapid cell growth during the transition into stationary phase.
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DISCUSSION |
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Is Thr-338 phosphorylation essential for Ctk1 activity? We demonstrated that mutation of Thr-338 compromised Ctk1 activity in vitro, which is similar to the effect of the equivalent mutations in Kin28 and Bur1, two other CDKs regulating CTD phosphorylation (26, 44). Surprisingly, the lack of T-loop phosphorylation in Ctk1 or Kin28 had little effect on cell growth. However, T-loop phosphorylation became essential when additional mutations were introduced into Kin28 or its cyclin subunit, indicating that activating phosphorylation was required for stability of the kinase complex (24, 26). In contrast, examination of Ctk1T338A-containing complexes by gel filtration chromatography indicated that mutation did not affect binding of Ctk2 and Ctk3. Ctk1 function was more sensitive to mutation of Thr-338 in vitro than in vivo. It is possible that the increased requirement for Ctk1 phosphorylation in vitro is due to substrate differences, as the CTD peptide used in vitro contained fewer repeats than the natural RNA pol II CTD and may not adopt its normal conformation. Alternatively, unstressed cells may require very little Ctk1 activity to phosphorylate the CTD to sufficient levels for normal growth. Regarding the need for Thr-338 phosphorylation for activity in vitro, Hautbergue and Goguel reported the reconstitution of a functional Ctk1-Ctk2-Ctk3 complex from recombinant proteins, suggesting that T-loop phosphorylation might not be required for activity (15). However, a comparison of their results and ours is difficult without knowing the specific activities of the respective complexes. It seems likely that a high concentration of unphosphorylated Ctk1 could display detectable activity in vitro but that its kinase activity could still be increased significantly upon T-loop phosphorylation.
Several lines of evidence indicate that Cak1 is responsible for Ctk1 phosphorylation. First, Ctk1 isolated from cak1 cells is unphosphorylated. Second, like Ctk1T338A from wild-type cells, Ctk1 isolated from cak1 cells was catalytically inactive in vitro. Third, Cak1 directly phosphorylated monomeric Ctk1 on Thr-338. Because Cak1 has a strong preference for cyclin-free CDKs, and because gel filtration experiments did not reveal the presence of any Ctk1 monomer, we suspect that our inability to phosphorylate Ctk1 isolated from yeast cells was due to its incorporation into Ctk1-Ctk2-Ctk3 complexes.
Ctk1 joins a growing list of Cak1 substrates. Phosphorylation by Cak1 is clearly essential for Cdc28 activity and cell cycle progression (11, 22). However, phosphorylation by Cak1 has more modest effects on the functioning of three other CDKs, Kin28, Bur1, and Ctk1. An intriguing possibility is that activating phosphorylation of these kinases becomes important under specific growth conditions. For example, Yao and Prelich demonstrated that cak1 cells are sensitive to caffeine, sharing this phenotype with a conditional bur1 mutant (44). Presumably, Cak1-mediated phosphorylation of Bur1 is important for the transcriptional activation of genes required for cell growth in the presence of caffeine. We found that ctk1T338A cells delayed growth during the diauxic shift. At a molecular level, ctk1T338A was unable to stimulate RNA pol II phosphorylation on Ser2 and displayed aberrant activation of diauxic-shift genes. Thus, a lack of activating phosphorylation resulted in diverse conditional phenotypes. Cak1-mediated phosphorylation of Bur1, Ctk1, and Kin28 may have been preserved to support transcriptional adaptation to variable growth conditions.
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ACKNOWLEDGMENTS |
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The microarray facility was supported by grant CA77808 from the National Institutes of Health to Michael Snyder. This work was supported by grant GM47830 (to M.J.S.) from the National Institutes of Health.
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FOOTNOTES |
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