Nutrient sensing kinases PKA and Sch9 phosphorylate the catalytic domain of the ubiquitin-conjugating enzyme Cdc34

Abstract

Cell division is controlled in part by the timely activation of the CDK, Cdc28, through its association with G1 and G2 cyclins. Cdc28 complexes are regulated in turn by the ubiquitin conjugating enzyme Cdc34 and SCF ubiquitin ligase complexes of the ubiquitin-proteasome system (UPS) to control the initiation of DNA replication. Here we demonstrate that the nutrient sensing kinases PKA and Sch9 phosphorylate S97 of Cdc34. S97 is conserved across species and restricted to the catalytic domain of Cdc34/Ubc7-like E2s. Cdc34-S97 phosphorylation is cell cycle regulated, elevated during active cell growth and division and decreased during cell cycle arrest. Cell growth and cell division are orchestrated to maintain cell size homeostasis over a wide range of nutrient conditions. Cells monitor changes in their environment through nutrient sensing protein kinases. Thus Cdc34 phosphorylation by PKA and Sch9 provides a direct tether between G1 cell division events and cell growth.

Figure 1. Alignment of E2s: The serine/serine/insert motif is conserved in all Cdc34 family members.

(A) Partial alignment of yeast E2s and Cdc34/Ubc7 family members. Red indicates amino acid residues unique to the Cdc34 family of E2s (the regulatory triad). Asterisks represent identities to Cdc34. Dashes represent gaps. Sc sequences are from S. cerevisiae. Ube2 sequences are from humans. ASFV sequences are ASFV1 –African swine fever virus (GI:9628248) and ASFV2 –African swine fever virus (GI:450743).

Figure 2. Complementation of cdc34-2 and cdc34Δ strains by Cdc34 S97 Mutants.

A and B) YL10-1, a cdc34-2 temperature sensitive strain, bearing 2 µm plasmids encoding the indicated CDC34 mutant under control of the GAL10 promoter were spotted in tenfold serial dilutions on the indicated media and grown at the indicated temperatures for 3 days. C) YL18, a cdc34Δ strain harboring a URA3 marked plasmid encoding wild type CDC34 and a LEU2-marked plasmid encoding the indicated CDC34 allele under control of the GAL10 promoter were spotted in tenfold serial dilution on the indicated media and grown at 30°C for 3 days.

Figure 3. Cdc34 S97 is phosphorylated in vivo and its detection is prohibited by certain protein extraction conditions.

A) Soluble protein from yeast cells, expressing from the CDC34 chromosomal locus, either wild type Cdc34 (lanes 1, 3 and 5), a COOH-terminally truncated Cdc34 encoding amino acids 1–244 (lane 2) or a Cdc34^tm mutant encoding the S97D mutation (lane 4) or bacterially expressed Cdc34^6XHis (lane 6) were analyzed by western blot using the α-pS97 phospho-specific or α-Cdc34 antibodies. B) An equal amount of soluble yeast protein extract made by either the glass bead or Horvath extraction protocol (see Materials and Methods) was analyzed by western blot using the α-pS97 phospho-specific and α-Cdc34 antibodies.

Figure 4. Cdc34 S97 phosphorylation increases in the G1 phase of the cell cycle.

Strain RC150 was grown to mid-log phase and arrested with a final concentration of 0.5 µg/ml µM of α-factor. After 3 hours, >95% of the cells had arrested with a shmoo-like morphology. Cells were washed and resuspended in fresh YPD. Cells were collected at the indicated time points and total soluble protein was analyzed by western blot using the α-pS97 phospho-specific and α-Cdc34 antibody. At least 100 cells were counted per time point to determine the budding index.

Figure 5. A screen for kinases whose overexpression alters the level of Cdc34-S97 phosphorylation.

A) Total soluble protein from an array of strains each overexpressing the indicated kinase was analyzed by western blot using the α-pS97 phospho-specific and α-Cdc34 antibodies.

Figure 6. Loss of Vps34, Vps15, Sch9 and Torc1 activity reduce Cdc34 S97 phosphorylation.

Total soluble protein from strains lacking the indicated kinase or from a strain treated with 200 nM rapamycin for 1 hour was analyzed by western blot using the α-pS97 phospho-specific and α-Cdc34 antibodies.

Figure 7. The cAMP/Protein Kinase A pathway regulates Cdc34 S97 phosphorylation.

A) Total soluble protein from the strains KT945 (WT, lanes 1 and 3), RS13-58A-1 (tpk1 ^w, lane 2) and KT1126 (bcy1–14, lane 4) was analyzed by western blot using α-pS97 phospho-specific and α-Cdc34 antibodies.

Figure 8. PKA directly phosphorylates Cdc34 on S97.

A) Cdc34ΔC^6XHis and Cdc34(S97A)ΔC^6XHis were phosphorylated in vitro by addition of bovine PKA and [γ-³²P]ATP. Protein kinase A inhibitor (Sigma Aldrich, USA) was added to the indicated reactions. The reaction components were separated by SDS-PAGE and autoradiographed. To quantitate the relative amount of ³²P incorporation, Cdc34 was excised from the dried and analyzed by liquid scintillation counter. B) Cdc34ΔC^6XHis was phosphorylated in vitro by either bovine PKA, ^GSTTpk3 or ^GSTSch9. The reaction was spotted onto a PVDF membrane and probed with either α-pS97 phospho-specific or α-Cdc34 antibody.

Figure 9. The Cdc34 S73/S97/loop motif increases chronological lifespan and is required for rapamycin resistance.

A) Prototrophic strains BL2 (WT) and RRC85 (CDC34^tm) were grown in synthetic defined liquid media lacking all amino acids for the indicated number of days at 32 C and the percent viability was determined by spotting a fraction of the culture on a YPD plate and counting colonies after incubation for 3 days at 32 C. B) Strains BL2 and RRC85 were grown overnight in SD lacking all amino acids, diluted to 5×10⁷ cells/ml and spotted in ten-fold serial dilution on YPD and YPD+25 nM rapamycin plates. Plates were incubated at 32°C for 3 days and then photographed.

Figure 10. Model of Cdc34 S97 phosphorylation.