Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 May;25(10):3906-13.
doi: 10.1128/MCB.25.10.3906-3913.2005.

Phosphorylation by Cak1 regulates the C-terminal domain kinase Ctk1 in Saccharomyces cerevisiae

Denis Ostapenko  1 , Mark J Solomon
Affiliations

Phosphorylation by Cak1 regulates the C-terminal domain kinase Ctk1 in Saccharomyces cerevisiae

Denis Ostapenko et al. Mol Cell Biol. 2005 May.

Abstract

Ctk1 is a Saccharomyces cerevisiae cyclin-dependent protein kinase (CDK) that assembles with Ctk2 and Ctk3 to form an active protein kinase complex, CTDK-I. CTDK-I phosphorylates Ser2 within the RNA polymerase II C-terminal domain, an activity that is required for efficient transcriptional elongation and 3' RNA processing. Ctk1 contains a conserved T loop, which undergoes activating phosphorylation in other CDKs. We show that Ctk1 is phosphorylated on Thr-338 within the T loop. Mutation of this residue abolished Ctk1 kinase activity in vitro and resulted in a cold-sensitive phenotype. As with other yeast CDKs undergoing T-loop phosphorylation, Ctk1 phosphorylation on Thr-338 was dependent on the Cak1 protein kinase. Ctk1 isolated from cak1Delta cells was unphosphorylated and exhibited low protein kinase activity. Moreover, Cak1 directly phosphorylated Ctk1 in vitro. Unlike wild-type cells, cells expressing Ctk1(T338A) 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.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Ctk1 is phosphorylated on Thr-338. (A) Protein sequence alignment of T-loop regions in four S. cerevisiae CDKs. Grey shading denotes conserved residues. Numbers indicate positions of the T loops within the individual CDKs. The alignment was done using the Clustal algorithm. The site of activating phosphorylation is indicated at the top. (B) Serial dilutions of wild-type (WT) and ctk1T338A and ctk1Δ mutant cultures were spotted onto YPD plates. The plates were incubated at 30°C and at 17°C and photographed after 4 days. (C) Two-dimensional gel analysis of Ctk1. Yeast proteins were separated by nonequilibrium pH gel electrophoresis according to their charges and molecular weights. Ctk1-8DE-HA contained eight negatively charged amino acids to compensate for the large positive charge of the protein. Ctk1-8DE-HA was detected by immunoblotting with rabbit anti-HA antibodies. In wild-type yeast extracts, Ctk1-8DE-HA migrated as a doublet (spots 1 and 2, top). Yeast extracts were treated with λ phosphatase (PPase) in the absence (second panel) or presence (third panel) of phosphatase inhibitors (Inh). Ctk1T338A-8DE-HA is shown in the lower panel. A yeast extract containing Ctk1-HA with no added acidic residues was included in the loading mixtures to provide a reference (Ref) mark to monitor the positions of the Ctk1-8DE-HA spots. Ctk1-HA is more positively charged and smaller than Ctk1-8DE-HA. Ctk1-HA was not treated with phosphatase, and its position remained constant in the various experiments.
FIG. 2.
FIG. 2.
Thr-338 phosphorylation is required for Ctk1 activity. (A) Ctk1-HA was immunoprecipitated from yeast strains expressing vector (−HA), wild-type (WT) Ctk1, Ctk1T338A, and Ctk1D324A (lanes 1 to 4). The expression levels and efficiencies of immunoprecipitation were verified by immunoblotting with rabbit anti-HA antibodies (top). Immunoprecipitated Ctk1 complexes were assayed for their CTD kinase activity using a CTD4 peptide as the substrate in the presence of [γ-32P]ATP (bottom). The reaction products were resolved by SDS-PAGE and visualized by autoradiography. (B) Yeast strains corresponding to CTK1, ctk1T338A (lanes 1 and 2) and the wild type and ctk1Δ, ctk2Δ, and ctk3Δ mutants (lanes 3 to 6) were examined for their levels of RNA pol II phosphorylation. Proteins were resolved by SDS-PAGE and immunoblotted for the presence of phosphorylated serine 2 (Ser2-P) using H5 antibodies; unphosphorylated RNA pol II was detected using 8WG16 antibodies (bottom). (C) Yeast extracts from CTK1, ctk1T338A, and ctk3Δ strains were fractionated on a Sephadex-200 gel filtration column and resolved by SDS-PAGE. The distribution of Ctk1-HA among the fractions was determined by immunoblotting with rabbit anti-HA antibodies. The positions of molecular weight markers are indicated at the top.
FIG. 3.
FIG. 3.
Cak1p phosphorylates Ctk1 on Thr-338. (A) Ctk1-8DE-HA was expressed in wild-type (WT) and cak1Δ cells and resolved on two-dimensional gels as described in the legend to Fig. 1C. Spots 1 and 2 correspond to phosphorylated and unphosphorylated forms of Ctk1-8DE-HA, respectively. Ctk1-HA was included as a reference (Ref) protein. (B) Yeast extracts were prepared from wild-type, cak1Δ, and cak1Δ/CAK1 cells also containing an empty plasmid (−HA, lane 1) or a plasmid expressing Ctk1-HA (lanes 2 to 4). The presence of Cak1p was determined by immunoblotting with an anti-Cak1p monoclonal antibody (middle). Ctk1-HA-containing complexes were immunoprecipitated. The level of precipitated Ctk1-HA was determined by immunoblotting using rabbit anti-HA antibodies (top). The immunoprecipitated Ctk1 was also used to phosphorylate CTD4 in the presence of [γ-32P]ATP (bottom). (C) Coupled transcription/translation reactions were programmed with an empty vector (−), ctk1T338A-HA, and CTK1-HA. The expressed proteins were immunoprecipitated with 12CA5 antibodies and incubated with [γ-32P]ATP in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of recombinant GST-Cak1p (bottom). The abundance of synthesized Ctk1-HA was verified by immunoblotting with rabbit anti-HA antibodies (top). Note that we used GST-Cak1p in this experiment, which has a molecular mass of 67 kDa and runs close to Ctk1-HA (63 kDa).
FIG. 4.
FIG. 4.
Thr-338 phosphorylation is important for entry into stationary phase. (A) Growth rates of CTK1 and ctk1T338A cells. Yeast cultures were grown in YPD medium at 30°C, and cell density was measured during the transition from exponential to early stationary phase. The average of three independent experiments is presented. Similar results were obtained by direct counting of yeast cells. The time course started when cells reached an OD600 of 0.1. (B) Yeast extracts were prepared from CTK1 and ctk1T338A cells at different times during late exponential growth and early stationary phase. The indicated times are the same as in panel A. The proteins were resolved by SDS-PAGE and immunoblotted for the presence of Ser2-phosphorylated RNA pol II (Ser2-P) using H5 antibodies and for unphosphorylated RNA pol II (CTD) using 8WG16 antibodies. (C) Δcak1 cells were grown into early stationary phase and analyzed for the presence of RNA pol II isoforms as for panel B. WT, wild type.
FIG. 5.
FIG. 5.
Phosphorylation of Ctk1 is required for normal transcription of diauxic-shift genes. Total RNA was isolated from CTK1 (lanes 1 to 7) and ctk1T338A cells (lanes 8 to 14) during late exponential phase and early stationary phase (same time points as in Fig. 4A and B). Equal amounts of RNA were separated on a formaldehyde-agarose gel and probed for the presence of specific transcripts by Northern blot hybridization. TPK1, CTT1, and SPI1 transcripts were selected for analysis due to their differential expression in CTK1 and ctk1T338A cells in preliminary microarray hybridization experiments. ADH1 is a housekeeping gene and was used as a loading control. Its level is known to decline during stationary-phase growth. WT, wild type.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ahn, S. H., M. Kim, and S. Buratowski. 2004. Phosphorylation of serine 2 within the RNA polymerase II C-terminal domain couples transcription and 3′ end processing. Mol. Cell 13:67-76. - PubMed
    1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1995. Current protocols in molecular biology. John Wiley & Sons, Inc., New York, N.Y.
    1. Borggrefe, T., R. Davis, H. Erdjument-Bromage, P. Tempst, and R. D. Kornberg. 2002. A complex of the srb8, -9, -10, and -11 transcriptional regulatory proteins from yeast. J. Biol. Chem. 277:44202-44207. - PubMed
    1. Cho, E. J., M. S. Kobor, M. Kim, J. Greenblatt, and S. Buratowski. 2001. Opposing effects of Ctk1 kinase and Fcp1 phosphatase at Ser 2 of the RNA polymerase II C-terminal domain. Genes Dev. 15:3319-3329. - PMC - PubMed
    1. Cho, E. J., T. Takagi, C. R. Moore, and S. Buratowski. 1997. mRNA capping enzyme is recruited to the transcription complex by phosphorylation of the RNA polymerase II carboxy-terminal domain. Genes Dev. 11:3319-3326. - PMC - PubMed

Publication types

MeSH terms