Autophosphorylation-Induced Degradation of the Pho85 Cyclin Pcl5 Is Essential for Response to Amino Acid Limitation

Abstract

Pho85 cyclins (Pcls), activators of the yeast cyclin-dependent kinase (CDK) Pho85, belong together with the p35 activator of mammalian CDK5 to a distinct structural cyclin class. Different Pcls target Pho85 to distinct substrates. Pcl5 targets Pho85 specifically to Gcn4, a yeast transcription factor involved in the response to amino acid starvation, eventually causing the degradation of Gcn4. Pcl5 is itself highly unstable, an instability that was postulated to be important for regulation of Gcn4 degradation. We used hybrids between different Pcls to circumscribe the substrate recognition function to the core cyclin box domain of Pcl5. Furthermore, the cyclin hybrids revealed that Pcl5 degradation is uniquely dependent on two distinct degradation signals: one N-terminal and one C-terminal to the cyclin box domain. Whereas the C-terminal degradation signal is independent of Pho85, the N-terminal degradation signal requires phosphorylation of a specific threonine residue by the Pho85 molecule bound to the cyclin. This latter mode of degradation depends on the SCF ubiquitin ligase. Degradation of Pcl5 after self-catalyzed phosphorylation ensures that activity of the Pho85/Pcl5 complex is self-limiting in vivo. We demonstrate the importance of this mechanism for the regulation of Gcn4 degradation and for cell growth under conditions of amino acid starvation.

ACKNOWLEDGMENTS

We thank Brenda Andrews, Erin O'Shea, Mike Tyers, Masafumi Nishizawa, and Dick Kulka for plasmids and strains; Yael Mandel-Gutfreund for advice with protein structure prediction; and Sara Selig for critical reading of the manuscript.

This research project was supported by grants from the Israel Science Foundation and the Niedersachsen-Israel Volkswagen Foundation. The Chimera package from the Computer Graphics Laboratory, University of California, San Francisco, is supported by NIH P41 RR-01081.