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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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.
KeywordsSaccharomyces cerevisiae RAM network CBK1 ACE2 NDR kinase Polarity
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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