A cyclin-dependent kinase that promotes cytokinesis through modulating phosphorylation of the carboxy terminal domain of the RNA Pol II Rpb1p sub-unit

Abstract

In Schizosaccharomyces pombe, the nuclear-localized kinase, Lsk1p, promotes cytokinesis by positively regulating the Septation Initiation Network (SIN). Although a member of the cyclin-dependent kinase (CDK) family, neither a cyclin partner nor a physiological target has been identified. In this report we identify a cyclin, Lsc1p, that physically interacts and co-localizes with Lsk1p. Furthermore, lsk1Delta, lsc1Delta, as well as kinase-dead lsk1-K306R mutants, display highly similar cytokinesis defects. Lsk1p is related to CDKs that phosphorylate the carboxy-terminal domain (CTD) of the largest sub-unit of RNA polymerase II (Rpb1p). Interestingly, we find that Lsk1p and Lsc1p are required for phosphorylation of Ser-2 residues found in the heptad repeats of the CTD. To determine if Rpb1p could be a physiological target, we replaced the native rpb1 gene with a synthetic gene encoding a Rpb1p protein in which Ser-2 was substituted with the non-phosphorylatable amino-acid alanine in all heptads. Cells carrying this allele were similar to lsk1Delta mutants: They were viable, displayed genetic interactions with the SIN, and were unable to complete cytokinesis upon perturbation of the cell division machinery. We conclude that Ser-2 phosphorylation of the CTD heptads plays a novel physiological role in the regulation of cytokinesis.

Figure 1. Strains bearing the lsc1Δ or lsk1-K306R mutations display highly similar cytokinesis defects as compared to lsk1Δ mutants.

(A) A phylogeny analyzing the relationship between S. pombe cyclins (black type) and the cyclin partners of Lsk1p relatives in budding yeast, mouse, human, and Drosophila (blue type). The most likely cyclin partner of Lsk1p based on this analysis, SPCC530.13, is shown in red. The phylogeny was created by MEGA version 3.1 software using the neighbor-joining algorithm. Sp, Schizosacharomyces pombe; Dm, Drosophila melanogaster; Hs Homo sapiens; Mm Mus musculus; Sc Saccharomyces cerevisiae. (B) ClustalW alignment comparing S. pombe SPCC530.13, S. cerevisiae Ctk2p, and human cyclin T. (C) Ten-fold serial dilutions of logarithmically growing cultures were plated onto YES plates containing 0.5 µM LatA or DMSO (solvent control) and incubated at 32°C for 3 days. (D) Cells of the indicated genotype were grown to mid-log phase at 32°C and then treated with 0.3 µM LatA for 5 hours before being fixed and stained with DAPI (nuclei) and aniline blue (cell wall/septa). Bar, 10 µM. (E) Cells of the indicated genotype were freshly streaked to YES plates and incubated for 24 hours at 36°C. Bar, 50 µM.

Figure 2. Lsk1p and Lsc1p co-localize to the nucleus and physically interact in a manner dependent on Lsk1p kinase activity.

(A) Cells expressing Lsk1-CFP or Lsc1-GFP under the control of the thiamine repressible nmt1 promoter were grown for 12 hours in minimal media in the absence of thiamine, and then imaged in the CFP and YFP channels, respectively. Bar, 3 µM. (B) Wild-type, Lsk1-HA, Lsc1-myc, and Lsk1-HA Lsc1-myc cells were grown to mid-log phase and lysed under native conditions. A portion of the lysates were subjected to anti-HA and anti-myc immunoprecipitations. Both total lysates and immunoprecipitates were resolved by SDS-PAGE and immunoblotted with antibodies specific for the myc epitope. (C) Cells of the indicated genotype, carrying integrated GFP tagged versions of Lsk1p or Lsc1p under the control of their native promoter, were grown to mid-log phase in YES media, fixed, and then stained with DAPI (nuclei) and antibodies specific for microtubules and GFP. Bar, 3 µM. (D) Lsk1-HA, Lsk1-K306R-HA, Lsk1-HA Lsc1-myc, and Lsk1-K306R-HA Lsc1-myc cells were grown to mid-log phase in YES and lysed under native conditions. A portion of the lysates were subjected to anti-HA and anti-myc immunoprecipitations. Both total lysates and immunoprecipitates were resolved by SDS-PAGE and immunoblotted with antibodies specific for the myc epitope.

Figure 3. Lsk1p and Lsc1p are required for Ser-2 phosphorylation of the heptad repeats found in the carboxy-terminal domain of the Rpb1p sub-unit of RNA pol II.

Cells were grown to mid-log phase in YES, and then treated either with 0.3 µM LatA or DMSO (solvent control) for 2 hours. Total lysates were resolved by SDS-PAGE and immunoblotted with antibodies specific for the unphosphorylated form of the CTD (8WG16), the Ser-5 phosphorylated form of the CTD (H14), the Ser-2 phosphorylated form of the CTD (H5), as well as antibodies specific for the Arp3 protein (loading control). Arrowheads indicate bands of increased mobility derived from rpb1-12xCTD and rpb1-12xS2ACTD mutants, while arrows indicate Rpb1p protein bands derived from the wild-type rpb1 locus.

Figure 4. Over-expression of the CTD phosphatase, Fcp1p, causes defects in cytokinesis.

(A) Cells of the indicated genotype were grown to mid-log phase in minimal media containing thiamine and then re-cultured in the presence or absence of thiamine for a further 18 hours. Cells were then treated with 0.3 µM LatA or DMSO (solvent control) for 5 hours and subsequently fixed and stained with DAPI (nuclei) and aniline blue (cell wall/septa). Bar, 10 µM. (B) Cells of the indicated genotype were grown to mid-log phase in minimal media containing thiamine and then re-cultured in the presence or absence of thiamine for a further 18 hours. Cells were then treated with 0.3 µM LatA or DMSO (solvent control) for a further 2 hours. Total lysates were resolved by SDS-PAGE and immunoblotted with antibodies specific for the unphosphorylated form of the CTD (8WG16), the Ser-5 phosphorylated form of the CTD (H14), the Ser-2 phosphorylated form of the CTD (H5), as well as antibodies specific for the Arp3 protein (loading control).

Figure 5. Schematic describing the creation of rpb1-12xCTD and rpb1-12xS2ACTD strains.

(See Materials and Methods for details.)

Figure 6. Mutation of Ser-2 to alanine in the heptad repeats of the carboxy-terminal domain of Rpb1p results in cytokinesis defects.

(A) Ten-fold serial dilutions of logarithmically growing cultures were plated onto YES plates containing 0.5 µM LatA or DMSO (solvent control) and incubated at 32°C for 3 days. (B) Cells of the indicated genotype were grown to mid-log phase at 32°C and then treated with 0.3 µM LatA for 5 hours before being fixed and stained with DAPI (nuclei) and aniline blue (cell wall/septa). Bar, 10 µM. (C) Cells of the indicated genotype were freshly streaked to YES plates and incubated for 24 hours at 34°C. Bar, 50 µM. (D) Cells of the indicated genotype were grown to mid-log phase at 24°C and then shifted to 34°C for 5 hours before being fixed and stained with DAPI (nuclei) and aniline blue (cell wall/septa). Bar, 10 µM.