Abstract #19

Multiple homeostatic mechanisms regulate the activity of the Pho85 cyclin Pcl5. Tsvia Gildor, Sharon Aviram, Revital Shemer, Daniel Kornitzer. Dept Molec Microbiol, Fac Med, Technion-IIT, Haifa, Israel.
   The cyclin-dependent kinase (CDK) Pho85 in conjunction with a specific cyclin, Pcl5, phosphorylates the yeast transcription factor Gcn4, leading to its degradation. We find that PCL5 is tightly regulated at multiple levels. First, PCL5 is itself a transcription target of Gcn4, resulting in a negative feedback loop between Gcn4 and its cyclin. PCL5 is also regulated at the level of translation due to an extended 5’ UTR containing two uORFs, which repress PCL5 translation some 10-fold. At the protein level, Pcl5 is very short-lived. In order to identify degradation signals, hybrid cyclins were constructed between Pcl5 and the stable Pho85 cyclin, Pho80. These hybrids reveal three functional domains of Pcl5: the cyclin box, carrying the substrate specificity determinant, and two distinct degradation signals. A C-terminal, Pho85-independent degradation signal is responsible for turnover of free Pcl5, whereas an N-terminal degradation signal that depends on phosphorylation of a specific residue by Pho85, is responsible for turnover of CDK-bound cyclin. Finally, in vitro kinase assays of Pho85/Pcl5 activity revealed a novel mechanism of cyclin/CDK regulation, namely, modulation of specific substrate recognition. Autophosphorylation of Pcl5 by Pho85 leads to a gradual loss of specificity of the Pho85/Pcl5 complex for its specific substrate Gcn4, but not to loss of CDK activity against non-specific substrates. This observation suggests that the phosphorylated cyclin remains bound to the CDK, but has phosphorylation interferes with specific substrate recognition. The last two mechanisms ensure that activation of Pho85 by Pcl5 is inherently self-limiting: first, following autophosphorylation, catalytic activity towards the specific substrate is disabled, and second, autophosphorylation induces degradation of the cyclin, thereby ensuring efficient turnover of inactive Pho85 complexes. These homeostatic mechanisms, that ensure exquisite regulation of Pcl5/Pho85 activity, appear largely conserved among Pcl5 orthologs and, at least to some extent, also among other Pho85 cyclins.

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