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.

FIG. 1.

Activity of Pcl5/Pho80 hybrid cyclins. (A) Sequence alignment of Pcl5 and Pho80. The initial alignment was obtained with HHpred (59) and manually curated. The position of the helices of the cyclin box (α1 to α5) and of the αNT and the start of the αCT1 helix are based on the Pho80 crystal structure (30). The position of the fusions that generated the Pcl hybrids are indicated with the number of the corresponding plasmids. Plasmid KB1301 is the exact reverse of KB1298. (B) The pcl5Δ strain KY827 carrying plasmid KB843 (GAL1-GCN4) was transformed with the indicated hybrid cyclin-expressing plasmids. Dilutions of the cell suspensions were spotted on synthetic complete drop-out plates with galactose (inducing conditions) or glucose (repressing conditions) as indicated and photographed after 3 and 2 days, respectively, of growth at 30°C. (C) The same cultures were used to measure Gcn4 stability by pulse-chase analysis, followed by immunoprecipitation (see Materials and Methods). The Gcn4 band remaining at each time point was quantitated with a phosphorimager. (D) The pcl5Δ strain KY827 carrying plasmid KB843 (GAL1-GCN4) was transformed with full-length and N-terminal truncations of Pcl5, as indicated, expressed from plasmids KB1093, KB1773, and KB1774. Dilutions of the cell suspensions were spotted and grown as for panel B. On the left, a schematic representation of the Pcl5 sequence, with the position of the predicted α-helices according to panel A. (E) The natively expressed Pho80-Pcl5-Pho80 hybrid induces Gcn4 degradation. A pcl5Δ strain expressing GCN4 from the GAL1 promoter of plasmid KB161 (37) was cotransformed with either a vector plasmid, a plasmid (KB1594) carrying the wild-type PCL5 gene, or plasmid KB1595, expressing the Pho80-Pcl5-Pho80 hybrid from the PCL5 promoter, as indicated. Gcn4 degradation was followed by pulse-chase analysis. C, no-tag control. (F) The natively expressed Pho80-Pcl5-Pho80 hybrid suppresses toxicity of Gcn4 overexpression. The same strains analyzed in panel E were grown and tested for Gcn4 overexpression toxicity as described in panel B.

FIG. 2.

Stability of Pcl5/Pho80 hybrid cyclins. The stability of the various hybrids, expressed from the inducible GAL1 promoter, was measured by pulse-chase analysis using a polyclonal anti-Pcl5 antiserum, except for Pho80, for which a hemagglutinin (HA)-tagged version was followed using the anti-HA monoclonal antibody. The structure of the hybrid cyclins is shown schematically. See Table 1 for the precise coordinates of the cyclin fragments in each hybrid.

FIG. 3.

CDS of Pcl5. (A) Full-length or N-terminal deletions of Pcl5 were fused to the C terminus of Ura3, as indicated, and expressed from the CUP1 promoter of plasmids KB1169, KB1338, KB1339, and KB1348, respectively. Degradation of the fusion proteins was measured by pulse-chase followed by immunoprecipitation, using an HA tag located at the C terminus of the Ura3 fragment. (B) The fusion proteins used in panel A were tested for their ability to complement the Ura⁻ phenotype of the host strain by monitoring growth on synthetic complete plates lacking uracil for 2 days at 30°C. (C) Pcl5 tagged at the C terminus with a Myc₃ tag was expressed from the GAL1 promoter of plasmid KB1030. The stability of the protein was monitored by pulse-chase analysis followed by immunoprecipitation with an anti-Myc antibody. C, no-tag control.

FIG. 4.

Role of residue Thr32 and of Pho85 in the degradation of the Pcl5. (A) Location of three putative CDK target sites on Pcl5, with the adjacent sequences. (B) Degradation of GAL1 promoter-overexpressed Pcl5, wild-type, triply mutated, or singly mutated at Thr32 (plasmids KB1093, KB1236, and KB1222, respectively). Stability of the protein was monitored by pulse-chase analysis, followed by immunoprecipitation with an anti-Pcl5 policlonal antibody. (C) Degradation of natively expressed Pcl5. A Myc₁₃ tag was fused to the C terminus of the chromosomal copy of PCL5 (strain KY1137). The protein was monitored by Western blotting in extracts of growing cells and in extracts treated with the translation inhibitor cycloheximide for the indicated amounts of time. (D) Stability of T32A-mutated, natively expressed Pcl5 tagged with Myc₁₃. Plasmids KB1739 (wild type) and KB1741 (T32A) were used. The membrane was exposed for a shorter time than in panel C to highlight the difference in levels between the wild-type and mutant proteins. (E) The activities of wild-type PCL5 and PCL5(T32A) were compared by testing the suppression of Gcn4 overexpression toxicity. The PCL5 wild-type and mutant genes expressed from their native promoters on plasmids KB1716 and KB1718 were transformed into strain KY827 (pcl5Δ). Gcn4 was overexpressed under the GAL1 promoter of plasmid KB843. Plates were incubated for 3 days at 30°C. (F) Degradation of Myc₁₃-tagged Pcl5 expressed from its native promoter requires Pho85 activity. A chromosomally tagged Pcl5 was visualized as in panel A, in strains KY1137 (wild type) and KY1185 (pho85Δ). (G) Degradation of overexpressed, C-terminally tagged Pcl5 requires Thr32 and co-overexpression of Pho85. Pcl5-Myc₁₃ and Pcl5_T32A-Myc₁₃ were overexpressed from the GAL1 promoter of plasmids KB1931 and KB1932, respectively, in the presence of either a vector plasmid or of plasmid KB823, which overexpresses a GST-Pho85 fusion from the GAL1 promoter. The strains harboring the indicated plasmids were induced in galactose for 3 h. The overexpressed Pcl5-Myc₁₃ protein was then monitored by Western blotting in extracts of cells treated with cycloheximide for the indicated amounts of time. The graph represents quantitation of the enhanced chemiluminescence signal of the Pcl5-Myc₁₃ bands, normalized for each extract against the actin signal.

FIG. 5.

Pcl5 is phosphorylated on Thr32 by Pho85. (A) The upper panel shows in vitro phosphorylation of GST-Pcl5, either wild-type or mutant T32A, with GST-Pho85. The lower panel shows the same Pho85-Pcl5 recombinant complexes were mixed with myelin basic protein (MBP). See Materials and Methods for the reaction conditions. (B) Kinetics of the same type of Pcl5 phosphorylation reaction shown in panel A. For each time point, 20 ng (each) of GST-Pho85 and GST-Pcl5 were preincubated for 5 min at 30°C and then diluted into increasing volumes of kinase buffer containing 1 μM cold ATP and 1 μCi of [γ-³²P]ATP/10 μl. Aliquots were obtained at the indicated times and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the GST-Pcl5 label was quantitated with a phosphorimager.

FIG. 6.

A catalytically inactive Pcl5 mutant is significantly stabilized. (A) In vitro phosphorylation of GST-Gcn4 with Pho85 and either wild-type Pcl5 or Pcl5 carrying the T32A mutation or inactivating mutation L158P. See Materials and Methods for reaction conditions. (B) Degradation of Myc₁₃-tagged Pcl5 expressed from its native promoter requires cyclin activity. Plasmids carrying the wild-type, 13-Myc-tagged Pcl5 (KB1739) or the inactive Pcl5 L158P (KB1740) were transformed into strain W303, together with either a plasmid expressing wild-type Pcl5 under the strong constitutive ADH1 promoter (KB1310) or a vector plasmid. Cells were grown at 30°C and treated as for Fig. 5A. Blots were reacted with both anti-Myc antibody (9E10) and anti-actin antibody. C, no-tag control.

FIG. 7.

Degradation of Pcl5 depends on the SCF ubiquitin ligase. The stability of Myc₁₃-tagged Pcl5 expressed from its native promoter (plasmid KB1739) was tested in the SCF mutant cdc53-1 and in the E2 mutant cdc34-2 (A) and in the F-box protein mutant grr1Δ (B). Cells were grown at 30°C and treated as in Fig. 4C. A second, longer exposure of the anti-Myc reaction is shown in panel A (middle panel) to better visualize Pcl5-Myc₁₃ in the wild-type cells. The same blots were reacted with an antiactin antibody to ascertain equal loading (bottom panels).

FIG. 8.

Effect of Pcl5 mutation T32A on regulation of Gcn4 degradation and amino acid analog resistance. (A) pcl5Δ cells (KY827) were transformed with a plasmid overexpressing Myc-tagged Gcn4 (KB1281) and with either a vector plasmid, a plasmid carrying wild-type PCL5 (KB1716), or a plasmid carrying PCL5(T32A) (KB1718). The cells were subjected to amino acid starvation for 45 min as previously described (37). Gcn4 decay rate was measured by pulse-chase analysis followed by immunoprecipitation. (B) pcl5Δ cells (KY827) transformed with either a vector plasmid, a plasmid carrying wild-type PCL5 (KB1716), or a plasmid carrying PCL5(T32A) (KB1718) were grown in synthetic complete medium lacking histidine, with the amino acid analog 3AT. The left panel shows the cell density after overnight growth in liquid medium with increasing 3AT concentrations (mean ± the standard deviation of quadruplicate cultures). The right panel shows growth after 3 days on a 30 mM 3AT plate versus 2 days on a YPD plate.

FIG. 9.

Predicted Pcl5-Pho85 structure. The model includes the Pho85 structure (2pmi [30]) and Pcl5 (residues 26 to 189) modeled after Pho80 (see Materials and Methods for the modeling protocol). Pho85 is colored in yellow, and the ATP analogue ATP-γ-S is depicted as blue spheres. The Pcl5 cyclin box domain (residues 75 to 189) is colored dark green, the N-terminal domain including helix αNT (residues 41 to 74) is colored light green, and residues 26 to 40, predicted to be disordered, are colored red. Thr32 is shown as red spheres. The image was produced by using the Chimera package (University of California at San Francisco).