Abstract #21

Phosphoregulation of Cbk1 integrates RAM network control of gene expression and morphogenesis. Jaclyn Jansen, Margaret Barry, Eric Weiss. Dept BMBCB, Northwestern Univ, Evanston, IL.
   The budding yeast RAM network is a conserved but poorly understood signaling pathway required for maintenance of polarized growth. It also controls Ace2, an asymmetrically segregated transcription factor that drives daughter cell specific gene expression after mitotic exit. The network’s role in cell morphology control is independent of its transcriptional function. It thus far comprises six proteins, including the Ndr/warts family kinase Cbk1. We found that two conserved phosphorylation sites in Cbk1 are important in RAM network function. One is an autophosphorylation site; while critical for the kinase’s in vitro activity, its mutation gives a partial loss of function phenotype. The other site is in a C-terminal hydrophobic motif and is phosphorylated by another kinase. In contrast, it is absolutely required for in vivo function but is not required for in vitro activity. Cbk1 that cannot be phosphorylated at the C-terminal site is heavily modified at other sites in vivo, suggesting that the modification promotes a change in the extent of these modifications. Using phosphospecific antibodies, we determined that the autophosphorylation site is constitutively modified throughout the cell cycle, while the C-terminal site is modified during bud emergence and during the M/G1 transition. Phosphorylation of the C-terminal site requires all of the other RAM network components. Surprisingly, this modification is also greatly reduced in cells lacking Ace2. This is unexpected because Cbk1’s polarized growth function is independent of Ace2, but absolutely dependent on C-terminal site modification. Thus, a feedback loop may exist that increases or protects the M/G1 phosphorylation of Cbk1’s C-terminal regulatory site upon Ace2-Cbk1 association. The kinase’s role in polarized growth, in contrast, may require only a small amount of C-terminally modified protein.

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