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.Cyclin-dependent kinases (CDKs) are central cellular regulators that are involved in cell proliferation (46), as well as other processes, e.g., regulation of transcription via the phosphorylation of RNA polymerase II (7). CDKs are activated by binding of an ancillary subunit, the cyclin (32, 44). The cyclins also participate in targeting the kinase to specific substrates (28, 55, 65; reviewed in reference 43). Cyclins were so named because of their cyclic appearance and disappearance from the cells, in correlation with cell cycle progression (16). Their rapid and coordinated disappearance depends on the ubiquitin system, with the anaphase-promoting complex/cyclosome ubiquitin ligase being responsible for targeting mitotic cyclins, and SCF-type ubiquitin ligases often being involved in the degradation of G1 cyclins (68). However, not all cyclins are directly involved in cell cycle regulation, and not all cyclins are unstable. Common to all cyclins is that they possess a region of moderate sequence conservation, the cyclin box. The cyclin box was shown to assume a typical five-helix structure (8, 32), with helix 5 contacting the CDK near its catalytic site and helix 1 facing away from the CDK (32).The yeast CDK Pho85, a regulator of cell morphogenesis and of several metabolic pathways, is structurally and functionally related to the mammalian kinase CDK5 (29, 48). Of 10 Pho85 cyclin (Pcl) paralogs identified in yeast (40), many were associated with the phosphorylation of specific target proteins

Regulation of Amino Acid, Nucleotide, and Phosphate Metabolism in Saccharomyces cerevisiae

“…Some of these responses impinge directly on Gcn2 (Cherkasova et al 2010;Zaborske et al 2009Zaborske et al , 2010 and some function independently, apparently in parallel. Notably, Gcn4 stability is increased under amino acid starvation (Kornitzer et al 1994;Shemer et al 2002;Bomeke et al 2006;Aviram et al 2008;Streckfuss-Bomeke et al 2009). …”

The Ubiquitin Ligase Hul5 Promotes Proteasomal Processivity

“…Pcl5 is a yeast cyclin-like protein that is rapidly degraded via two distinct degradation signals, an N-terminal phosphorylation-dependent signal and a C-terminal signal that requires a free carboxy-terminal end (1). Fusion of Pcl5 C-terminally to the normally stable enzyme Ura3 causes destabilization of the fusion construct (1).…”

“…Fusion of Pcl5 C-terminally to the normally stable enzyme Ura3 causes destabilization of the fusion construct (1). We used the instability of Ura3-Pcl5 to screen for yeast mutants that stabilize Pcl5.…”

Degradation of Saccharomyces cerevisiae Transcription Factor Gcn4 Requires a C-Terminal Nuclear Localization Signal in the Cyclin Pcl5

“…This reduced activity may be due to dissociation of Pcl5 from Pho85 under amino acid starvation conditions (3) combined with autophosphorylation-induced instability of Pcl5 (2,52).…”

“…Here, we analyzed Pcl5-green fluorescent protein (GFP) hybrids to test whether nuclear localization represents a distinct domain of this cyclin and compared them to the localization of Pcl5-Pho80-GFP chimera (2).…”