| The Ndr/Lats kinase Cbk1 is an essential component of the RAM network, a conserved signaling pathway that integrates cell fate determination and morphogenesis in budding yeast. As the downstream-most component of the network, Cbk1 controls aspects of cell polarity, cell cycle-regulated transcription, and cell integrity. Loss of Cbk1 function is lethal in the presence of SSD1, a gene that is polymorphic in common lab strain backgrounds. Therefore, I sought to identify both upstream regulatory inputs and downstream targets of this kinase to better understand how Cbk1 functions in differentiation and morphogenesis.;I have shown that phosphoregulation of Cbk1 at conserved sites is critical for RAM network function. Intramolecular autophosphorylation within the activation loop is critical for kinase activity but is only partially required for cell separation and polarized growth. In contrast, phosphorylation of a C-terminal hydrophobic motif is required for Cbk1 function in vivo, but not for its kinase activity, suggesting a novel mechanism for control of this family of kinases. Phosphorylation of the C-terminal site is regulated over the cell cycle and requires all upstream RAM network components. In addition, phosphorylation at this site requires the Cbk1 target, Ace2, so that the downstream target reinforces the activation of its upstream regulator, suggesting a positive feedback loop.;I have also sought to identify downstream targets of Cbk1. I have demonstrated that Ssd1, a protein of unknown function, is a novel target of Cbk1. Mutation of the eight putative Cbk1 phosphorylation sites found in Ssd1 abolishes phosphorylation. Overexpression of this mutant Ssd1 protein is lethal, suggesting that Cbk1 antagonizes an unknown cell-lethal function of Ssd1. I find that Ssd1 binds to a specific subset of RNAs in vivo and represses the translation of these messages. In addition, Ssd1 acts as a potent stimulator global mRNA decay. Cbk1 inhibits both these functions. Many Ssd1 targets are daughter specific genes, whose expression are controlled in part by the transcription factor Ace2. Thus, Cbk1 functions at multiple points to precisely control gene expression in the daughter cell, acting as a novel mechanism for the integration of cell fate determination and morphogenesis. |
