Pkc1p regulates CLB2 cluster gene expression. (A) The association of transcription factors with differential mRNA expression levels in wild-type (WT) and pkc1^ts cells. Gene expression changes in WT and pkc1^ts cells treated with hydroxyurea (HU) and released for 60 min were compared by microarray analysis, and changes in gene expression levels associated with transcription factor binding activities to produce a transcription factor activation strength signal (TFAS) were analyzed. (B and C) Real-time RT-PCR analysis of the indicated genes in DZ3 cells in the presence or absence of the PKC inhibitor (PKCi) cercosporamide following release from HU block at the indicated times (B) or W303-1a cells expressing either WT or a nuclear export mutant form of Pkc1p following release from α-factor block at the indicated times (C). Data are normalized to 18S rRNA levels. Cell cycle profiles were obtained by fluorescence-activated cell sorter (FACS) analysis at equivalent time points and are shown beneath panel B. (D) Real-time RT-PCR analysis of CLB2 and HTB2 expression in wild-type or Δbck1 BY4741 cells grown in sorbitol for the indicated times following release from α-factor block.

Pkc1p is dynamically recruited to CLB2 cluster gene promoters. (A) GFP fluorescence was used to detect GFP-tagged Pkc1p (middle) and DAPI staining to detect DNA (right). Nuclear Pkc1p is indicated by the arrows. A differential inference contrast (DIC) image of the cells is shown (left). (B and C) ChIP analysis of Pkc1p binding to the indicated gene promoters using either anti-HA antibody to detect HA epitope-tagged Pkc1p in DZ2 cells and analyzed by semiquantitative PCR (B) or anti-Myc antibody in cells expressing Myc epitope-tagged Pkc1p from its natural locus (KD592) measured by real-time PCR (C). Binding was analyzed following release from α-factor block in G₁ phase at the indicated times. The control (con) is ChIP with either anti-HA antibody from logarithmically growing WT (W303-1a) cells (B) or with nonspecific IgG from cells expressing Myc epitope-tagged Pkc1p (KD592) (C). Data in panel C are shown relative to the control background binding to the CLB2 promoter (con) (taken as 1). Cell cycle profiles were obtained by FACS analysis at equivalent time points and are shown beneath each figure.

Pkc1p phosphorylates Ndd1p. In vitro kinase assays using HA epitope-tagged Pkc1p immunoprecipitated from DZ2 cells and the following substrates: GST-Fkh2p(458-862) and GST-Ndd1p(1-554) (A), GST-Ndd1p(1-554) (B), the indicated truncation series of GST-Ndd1p constructs (C), or the indicated wild-type and mutant forms of GST-Ndd1p(1-554) (D). Mock HA-immunoprecipitated material from W303-1a cells was used as a control (A; lanes 1 and 2). (B) Increasing concentrations of HA-Pkc1p (relative molar amounts, 1, 2, and 4) were added in the presence and absence of GF109203X. The top panels in each case are phosphorimages, and the bottom panels are Coomassie-stained gels of the input GST fusion proteins. The data from four experiments were quantified and are shown graphically below panel D. The schematics in C and D show the structures of the truncation mutant versions of Ndd1p and the location and local sequence contexts of the Pkc1p target sites. (E) Product ion spectra for the triply charged precursor ion, [544.23]³⁺. The spectra include full annotation of product ions detected, which include both b- and y-ions on either side of the site of modification, demonstrating that the phosphate is located on Ser527.

Ndd1p(S520A/S527A) causes aberrant expression of CLB2 cluster genes. (A) ChIP analysis of Ndd1p binding to the indicated gene promoters using anti-HA antibody to detect HA epitope-tagged Ndd1p in AP179 cells expressing either wild-type (WT) Ndd1p or Ndd1p(S520A/527A). Cells were analyzed following release from the HU block at the indicated times. The dotted line represents a control ChIP with anti-HA antibody from logarithmically growing WT W303-1a cells. Data are shown relative to this control level (taken as 1). (B and C) Western blot (WB) analysis of Ndd1p expression from DZ3 cells expressing wild-type (WT) Ndd1p or DZ4 cells expressing Ndd1p(S520A/S527A) at the indicated times following release from the HU block. Tubulin levels are shown as a control. In panel C, cells also ectopically expressed WT Pkc1p (top) or a nuclear export mutant form of Pkc1p (Pkc1-nuc) (bottom). (D) Real-time RT-PCR analysis of the expression of the indicated genes in AP179 cells expressing either wild-type (WT) Ndd1p or Ndd1p(S520A/S527A) at the indicated times following release from the HU block. Data are normalized to 18S rRNA levels and are shown relative to the levels of expression in the strain expressing Ndd1p(WT) in HU-treated cells at time zero (taken as 1). (E) Real-time RT-PCR analysis of the indicated genes in DZ4 cells [expressing Ndd1p(S520A/S527A)] in the presence or absence of the PKC inhibitor (PKCi) cercosporamide following release from the HU block at the indicated times.

Mutation of the Pkc1p-dependent phosphorylation sites of Ndd1p causes cell cycle defects and different stress-sensitive phenotypes. (A) Tenfold serial dilutions of DZ3 cells expressing WT Ndd1p or DZ4 cells expressing Ndd1p(S520A/S527A) were plated onto SD medium in the presence or absence of 1 M sorbitol. Where indicated, HU was added to the medium or cells were grown at 37°C. (B) DNA content analysis of AP179 cells expressing either WT Ndd1p or Ndd1p(S520A/S527A) at the indicated times after release from an HU block. (C) Nuclear spindles were stained with antitubulin antibody (red), and nuclei were detected with DAPI (blue) in AP179 cells expressing either WT Ndd1p or Ndd1p(S520A/S527A) maintained in HU for 3 h. The percentage of cells containing elongated spindles is shown on the right. (D) Model depicting the kinases acting on the Mcm1p-Fkh2p-Ndd1p complex during the cell cycle. Pkc1p phosphorylates Ndd1p to inhibit activation during S and early G₂ phases. Subsequent phosphorylation by cyclin-Cdk and Cdc5p kinases results in activation of the complex and hence CLB2 cluster gene transcription during late G₂ phase.