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Mutations in the C-terminus of the conserved NDR kinase, Cbk1p of Saccharomyces cerevisiae, make the protein independent of upstream activators

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Abstract

In Saccharomyces cerevisiae, the RAM network is involved in cell separation after cytokinesis, cell integrity and cell polarity. The key function of this network is the regulation of the activity of the protein kinase Cbk1p, which is a member of the conserved NDR kinase family. Cbk1p function is controlled by its sub-cellular localization and at least two phosphorylation events: an auto phosphorylation in the kinase domain (S570) and the phosphorylation of a C-terminal hydrophobic motif by an upstream kinase (T743). After a UV mutagenesis, we have isolated 115 independent extragenic suppressors of four ∆ram mutations: ∆tao3, ∆hym1, ∆kic1 and ∆sog2. Over 50% of the suppressors affect a single residue in Cbk1p (S745F), which is close to the phosphorylation site in the hydrophobic motif. Our results show that the CBK1-S745F allele leads to a constitutively active form of Cbk1p that is independent of the upstream RAM network. We hypothesize that the mutant Cbk1-S745Fp mimics the effect of the phosphorylation of T743.

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Acknowledgments

We would like to thank Madame A-M. Bécam for invaluable technical assistance, Dr G. Dujardin critical reading of the manuscript and many helpful discussions and Dr B. Séraphin for the SSD1 plasmid. This work was financed by the CNRS, a “Subvention Fixe” from the ARC and an ACI-BCMS grant from the French ministry of Research. M. B. would like to thank the Institut de Chimie des Substances Naturelles du CNRS, Gif-sur-Yvette, for financial support.

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Author notes

  1. Aleksandra Nowacka

    Present address: Institut de Génétique et Microbiologie UMR8621, Université de Paris-Sud 11, 91405, Orsay, France

Authors and Affiliations

  1. Centre de Génétique Moléculaire du CNRS, FRE3144, FRC3115, Ave de la Terrasse, 91198, Gif-sur-Yvette, France

    Cristina Panozzo, Myriam Bourens, Aleksandra Nowacka & Christopher James Herbert

  2. Université de Paris-Sud 11, 91405, Orsay, France

    Cristina Panozzo, Myriam Bourens, Aleksandra Nowacka & Christopher James Herbert

  3. Université Pierre et Marie Curie-Paris 6, 75005, Paris, France

    Myriam Bourens, Aleksandra Nowacka & Christopher James Herbert

Authors
  1. Cristina Panozzo
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  2. Myriam Bourens
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  3. Aleksandra Nowacka
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  4. Christopher James Herbert
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Corresponding author

Correspondence to Christopher James Herbert.

Additional information

Communicated by M. Collart.

Electronic supplementary material

Below is the link to the electronic supplementary material.

438_2009_501_MOESM1_ESM.doc

Supplementary Table 1 S. cerevisiae strains used in this study. All the strains used in this study are derived from the homozygous diploid W303 (MAT a/MAT alpha leu2 3,112 trp1 1 can1 100 ura3 1 ade2 1 his3 11,15 ssd1 d). Only the markers that are different from the parent strain are listed. (DOC 56 kb)

Supplementary Table 2 Oligonucleotides used in the study. (DOC 26 kb)

438_2009_501_MOESM3_ESM.doc

Supplementary Table 3 List of the mutations isolated in this study. The different suppressor mutations obtained are shown with the corresponding nucleotide changes and the parent strains from which the suppressors were isolated. (DOC 50 kb)

438_2009_501_MOESM4_ESM.ppt

Supplementary Figure 1 Distribution of aggregate size in wild type and mutant strains. Cells were grown to early logarithmic phase and observed by DIC microscopy; the numbers of aggregates of different sizes were counted. Over 100 aggregates were counted in each experiment and the results show the average and standard deviation for three independent experiments. (PPT 98 kb)

438_2009_501_MOESM5_ESM.ppt

Supplementary Figure 2 Distribution of aggregate size in wild type and CBK1 mutant strains. Cells were grown to early logarithmic phase and observed by DIC microscopy; the numbers of aggregates of different sizes were counted. Over 100 aggregates were counted in each experiment and the results show the average and standard deviation for three independent experiments. (PPT 89 kb)

438_2009_501_MOESM6_ESM.ppt

Supplementary Figure 3 Localization of Mob2 GFP and Ace2 GFP in the CBK1 S745Y mutant. Overnight cultures of haploid cells were diluted to OD600 0.2, grown for 5h at 28°C and observed by DIC and fluorescence microscopy. The cells of the CBK1 S745Y are hyperpolarized compared to wild type and show the presence of large vacuoles. The localization of the Mob2 GFP signal in CBK1 S745Y is essentially the same as in the wild type, although the signal is a little weaker. However, the daughter cell specific localization of Ace2 GFP is lost and Ace2 GFP accumulates in both the mother and daughter nuclei. (PPT 1230 kb)

438_2009_501_MOESM7_ESM.ppt

Supplementary Figure 4 Phenotype of CBK1 T743E and CBK1 S745F in a wild type SSD1 background. To ensure that the truncated form of Ssd1p present in our strains did not interfere with the experiment the gene was completely deleted at the chromosomal locus. The resulting strains were then transformed by either the vector control or a centromeric plasmid carrying the wild type SSD1 gene. The axial ratios were determined for >200 cells and the standard deviation was calculated from three independent experiments. All cultures were plated and 75 92% of the cells contained the respective plasmid. The results show that the presence of a wild type SSD1 gene does not modify the polarity or aggregation phenotype of the CBK1 T743E and CBK1 S745F alleles. (PPT 244 kb)

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Panozzo, C., Bourens, M., Nowacka, A. et al. Mutations in the C-terminus of the conserved NDR kinase, Cbk1p of Saccharomyces cerevisiae, make the protein independent of upstream activators. Mol Genet Genomics 283, 111–122 (2010). https://doi.org/10.1007/s00438-009-0501-3

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