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Nutrient control of yeast PKA activity involves opposing effects on phosphorylation of the Bcy1 regulatory subunit.

Budhwar R, Lu A, Hirsch JP - Mol. Biol. Cell (2010)

Bottom Line: The BCY1(S145A) mutation eliminates the effect of gpb1Δ gpb2Δ on Bcy1 stability but maintains their effect on phosphorylation and signaling, indicating that modulation of PKA activity by Gpb1 and Gpb2 is not solely due to increased levels of Bcy1.When PKA is inhibited, gpb1Δ gpb2Δ mutations have no effect on Bcy1 phosphorylation.Stimulation of Bcy1 phosphorylation by Gpb1 and Gpb2 produces a form of Bcy1 that is more stable and is a more effective PKA inhibitor.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY 10029, USA.

ABSTRACT
GPB1 and GPB2 encode kelch repeat-containing proteins that regulate protein kinase A (PKA) in yeast by a cAMP-independent process. Here we show that Gpb1 and Gpb2 stimulate phosphorylation of PKA regulatory subunit Bcy1 in low glucose concentrations, thereby promoting the inhibitory function of Bcy1 when nutrients are scarce and PKA activity is expected to be low. Gpb1 and Gpb2 stimulate Bcy1 phosphorylation at an unknown site, and this modification stabilizes Bcy1 that has been phosphorylated by PKA catalytic subunits at serine-145. The BCY1(S145A) mutation eliminates the effect of gpb1Δ gpb2Δ on Bcy1 stability but maintains their effect on phosphorylation and signaling, indicating that modulation of PKA activity by Gpb1 and Gpb2 is not solely due to increased levels of Bcy1. Inhibition of PKA catalytic subunits that are ATP analog-sensitive causes increased Bcy1 phosphorylation at the unknown site in high glucose. When PKA is inhibited, gpb1Δ gpb2Δ mutations have no effect on Bcy1 phosphorylation. Therefore, Gpb1 and Gpb2 oppose PKA activity by blocking the ability of PKA to inhibit Bcy1 phosphorylation at a site other than serine-145. Stimulation of Bcy1 phosphorylation by Gpb1 and Gpb2 produces a form of Bcy1 that is more stable and is a more effective PKA inhibitor.

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The BCY1S145A mutation eliminates the effect of gpb1Δ gpb2Δ mutations on Bcy1 stability. (A) Cell lysates were prepared from the following strains: strain HS287-2C (bcy1Δ) carrying plasmid 313pBHB1-416 (HA-BCY1, WT), strain HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid 313pBHB1-416, strain HS287-2C carrying plasmid pBHBSA-313.4 (HA-BCY1, SA), strain HS293-10D carrying plasmid pBHBSA-313.4, and wild-type strain SKY762 carrying vector pRS313. Lysates were analyzed by SDS-PAGE and immunoblotting with anti-HA and anti-PGK antibodies. Protein levels were quantified by infrared imaging, and numbers under the lanes are the relative amount of HA-Bcy1 normalized to PGK. (B) Cell lysates from the following strains were analyzed as described in A: strain HS287-2C (bcy1Δ) carrying plasmid 313pBHB1-416 (HA-BCY1, WT), strain HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid 313pBHB1-416, strain HS287-2C carrying plasmid pBCS145E-313.3 (HA-BCY1, SE), strain HS293-10D carrying plasmid pBCS145E-313.3, strain HS287-2C carrying plasmid pBCS145D-313.4 (HA-BCY1, SD), and strain HS293-10D carrying plasmid pBCS145D-313.4. (C) Strains HS287-2C (bcy1Δ) and HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid 313pBHB1-416 were treated with cycloheximide (100 μg/ml) at time 0, and lysates were made from samples removed at the indicated times. Lysates were analyzed by SDS-PAGE and immunoblotting with anti-HA and anti-PGK antibodies. (D) Strains HS287-2C (bcy1Δ) and HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid pBHBSA-313.4 were treated as described in C. (E) Strains HS287-2C (bcy1Δ) and HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid pBCS145E-313.3 were treated as described in C. (F) Protein levels from the blots shown in C–E were quantified by infrared imaging, and the relative amount of HA-Bcy1 normalized to PGK was plotted as the percent remaining over time, using the following symbols: ▵, 313pBHB1-416 in HS287-2C; ▴, 313pBHB1-416 in HS293-10D; □, pBHBSA-313.4 in HS287-2C; ■, pBHBSA-313.4 in HS293-10D; ○, pBCS145E-313.3 in HS287-2C; •, pBCS145E-313.3 in HS293-10D.
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Figure 3: The BCY1S145A mutation eliminates the effect of gpb1Δ gpb2Δ mutations on Bcy1 stability. (A) Cell lysates were prepared from the following strains: strain HS287-2C (bcy1Δ) carrying plasmid 313pBHB1-416 (HA-BCY1, WT), strain HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid 313pBHB1-416, strain HS287-2C carrying plasmid pBHBSA-313.4 (HA-BCY1, SA), strain HS293-10D carrying plasmid pBHBSA-313.4, and wild-type strain SKY762 carrying vector pRS313. Lysates were analyzed by SDS-PAGE and immunoblotting with anti-HA and anti-PGK antibodies. Protein levels were quantified by infrared imaging, and numbers under the lanes are the relative amount of HA-Bcy1 normalized to PGK. (B) Cell lysates from the following strains were analyzed as described in A: strain HS287-2C (bcy1Δ) carrying plasmid 313pBHB1-416 (HA-BCY1, WT), strain HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid 313pBHB1-416, strain HS287-2C carrying plasmid pBCS145E-313.3 (HA-BCY1, SE), strain HS293-10D carrying plasmid pBCS145E-313.3, strain HS287-2C carrying plasmid pBCS145D-313.4 (HA-BCY1, SD), and strain HS293-10D carrying plasmid pBCS145D-313.4. (C) Strains HS287-2C (bcy1Δ) and HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid 313pBHB1-416 were treated with cycloheximide (100 μg/ml) at time 0, and lysates were made from samples removed at the indicated times. Lysates were analyzed by SDS-PAGE and immunoblotting with anti-HA and anti-PGK antibodies. (D) Strains HS287-2C (bcy1Δ) and HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid pBHBSA-313.4 were treated as described in C. (E) Strains HS287-2C (bcy1Δ) and HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid pBCS145E-313.3 were treated as described in C. (F) Protein levels from the blots shown in C–E were quantified by infrared imaging, and the relative amount of HA-Bcy1 normalized to PGK was plotted as the percent remaining over time, using the following symbols: ▵, 313pBHB1-416 in HS287-2C; ▴, 313pBHB1-416 in HS293-10D; □, pBHBSA-313.4 in HS287-2C; ■, pBHBSA-313.4 in HS293-10D; ○, pBCS145E-313.3 in HS287-2C; •, pBCS145E-313.3 in HS293-10D.

Mentions: In wild-type cells grown in a high concentration of glucose, Bcy1 is present at an approximately three- to fourfold higher level in GPB1 GPB2 cells compared with gpb1Δ gpb2Δ cells (Figures 3A, lanes 1 and 2). However, the Bcy1S145A variant is present at the same level in both GPB1 GPB2 and gpb1Δ gpb2Δ cells (Figure 3A, lanes 3 and 4). Thus, changing the PKA phosphorylation site in Bcy1 to a site that cannot be phosphorylated eliminates the effect of the gpb1Δ gpb2Δ mutations on Bcy1 abundance. The phenotype of cells containing the Bcy1S145A variant is slow growth (Werner-Washburne et al., 1991), which probably results from the failure of PKA to become fully activated due to excess levels of the altered regulatory subunit.


Nutrient control of yeast PKA activity involves opposing effects on phosphorylation of the Bcy1 regulatory subunit.

Budhwar R, Lu A, Hirsch JP - Mol. Biol. Cell (2010)

The BCY1S145A mutation eliminates the effect of gpb1Δ gpb2Δ mutations on Bcy1 stability. (A) Cell lysates were prepared from the following strains: strain HS287-2C (bcy1Δ) carrying plasmid 313pBHB1-416 (HA-BCY1, WT), strain HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid 313pBHB1-416, strain HS287-2C carrying plasmid pBHBSA-313.4 (HA-BCY1, SA), strain HS293-10D carrying plasmid pBHBSA-313.4, and wild-type strain SKY762 carrying vector pRS313. Lysates were analyzed by SDS-PAGE and immunoblotting with anti-HA and anti-PGK antibodies. Protein levels were quantified by infrared imaging, and numbers under the lanes are the relative amount of HA-Bcy1 normalized to PGK. (B) Cell lysates from the following strains were analyzed as described in A: strain HS287-2C (bcy1Δ) carrying plasmid 313pBHB1-416 (HA-BCY1, WT), strain HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid 313pBHB1-416, strain HS287-2C carrying plasmid pBCS145E-313.3 (HA-BCY1, SE), strain HS293-10D carrying plasmid pBCS145E-313.3, strain HS287-2C carrying plasmid pBCS145D-313.4 (HA-BCY1, SD), and strain HS293-10D carrying plasmid pBCS145D-313.4. (C) Strains HS287-2C (bcy1Δ) and HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid 313pBHB1-416 were treated with cycloheximide (100 μg/ml) at time 0, and lysates were made from samples removed at the indicated times. Lysates were analyzed by SDS-PAGE and immunoblotting with anti-HA and anti-PGK antibodies. (D) Strains HS287-2C (bcy1Δ) and HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid pBHBSA-313.4 were treated as described in C. (E) Strains HS287-2C (bcy1Δ) and HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid pBCS145E-313.3 were treated as described in C. (F) Protein levels from the blots shown in C–E were quantified by infrared imaging, and the relative amount of HA-Bcy1 normalized to PGK was plotted as the percent remaining over time, using the following symbols: ▵, 313pBHB1-416 in HS287-2C; ▴, 313pBHB1-416 in HS293-10D; □, pBHBSA-313.4 in HS287-2C; ■, pBHBSA-313.4 in HS293-10D; ○, pBCS145E-313.3 in HS287-2C; •, pBCS145E-313.3 in HS293-10D.
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Figure 3: The BCY1S145A mutation eliminates the effect of gpb1Δ gpb2Δ mutations on Bcy1 stability. (A) Cell lysates were prepared from the following strains: strain HS287-2C (bcy1Δ) carrying plasmid 313pBHB1-416 (HA-BCY1, WT), strain HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid 313pBHB1-416, strain HS287-2C carrying plasmid pBHBSA-313.4 (HA-BCY1, SA), strain HS293-10D carrying plasmid pBHBSA-313.4, and wild-type strain SKY762 carrying vector pRS313. Lysates were analyzed by SDS-PAGE and immunoblotting with anti-HA and anti-PGK antibodies. Protein levels were quantified by infrared imaging, and numbers under the lanes are the relative amount of HA-Bcy1 normalized to PGK. (B) Cell lysates from the following strains were analyzed as described in A: strain HS287-2C (bcy1Δ) carrying plasmid 313pBHB1-416 (HA-BCY1, WT), strain HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid 313pBHB1-416, strain HS287-2C carrying plasmid pBCS145E-313.3 (HA-BCY1, SE), strain HS293-10D carrying plasmid pBCS145E-313.3, strain HS287-2C carrying plasmid pBCS145D-313.4 (HA-BCY1, SD), and strain HS293-10D carrying plasmid pBCS145D-313.4. (C) Strains HS287-2C (bcy1Δ) and HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid 313pBHB1-416 were treated with cycloheximide (100 μg/ml) at time 0, and lysates were made from samples removed at the indicated times. Lysates were analyzed by SDS-PAGE and immunoblotting with anti-HA and anti-PGK antibodies. (D) Strains HS287-2C (bcy1Δ) and HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid pBHBSA-313.4 were treated as described in C. (E) Strains HS287-2C (bcy1Δ) and HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid pBCS145E-313.3 were treated as described in C. (F) Protein levels from the blots shown in C–E were quantified by infrared imaging, and the relative amount of HA-Bcy1 normalized to PGK was plotted as the percent remaining over time, using the following symbols: ▵, 313pBHB1-416 in HS287-2C; ▴, 313pBHB1-416 in HS293-10D; □, pBHBSA-313.4 in HS287-2C; ■, pBHBSA-313.4 in HS293-10D; ○, pBCS145E-313.3 in HS287-2C; •, pBCS145E-313.3 in HS293-10D.
Mentions: In wild-type cells grown in a high concentration of glucose, Bcy1 is present at an approximately three- to fourfold higher level in GPB1 GPB2 cells compared with gpb1Δ gpb2Δ cells (Figures 3A, lanes 1 and 2). However, the Bcy1S145A variant is present at the same level in both GPB1 GPB2 and gpb1Δ gpb2Δ cells (Figure 3A, lanes 3 and 4). Thus, changing the PKA phosphorylation site in Bcy1 to a site that cannot be phosphorylated eliminates the effect of the gpb1Δ gpb2Δ mutations on Bcy1 abundance. The phenotype of cells containing the Bcy1S145A variant is slow growth (Werner-Washburne et al., 1991), which probably results from the failure of PKA to become fully activated due to excess levels of the altered regulatory subunit.

Bottom Line: The BCY1(S145A) mutation eliminates the effect of gpb1Δ gpb2Δ on Bcy1 stability but maintains their effect on phosphorylation and signaling, indicating that modulation of PKA activity by Gpb1 and Gpb2 is not solely due to increased levels of Bcy1.When PKA is inhibited, gpb1Δ gpb2Δ mutations have no effect on Bcy1 phosphorylation.Stimulation of Bcy1 phosphorylation by Gpb1 and Gpb2 produces a form of Bcy1 that is more stable and is a more effective PKA inhibitor.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY 10029, USA.

ABSTRACT
GPB1 and GPB2 encode kelch repeat-containing proteins that regulate protein kinase A (PKA) in yeast by a cAMP-independent process. Here we show that Gpb1 and Gpb2 stimulate phosphorylation of PKA regulatory subunit Bcy1 in low glucose concentrations, thereby promoting the inhibitory function of Bcy1 when nutrients are scarce and PKA activity is expected to be low. Gpb1 and Gpb2 stimulate Bcy1 phosphorylation at an unknown site, and this modification stabilizes Bcy1 that has been phosphorylated by PKA catalytic subunits at serine-145. The BCY1(S145A) mutation eliminates the effect of gpb1Δ gpb2Δ on Bcy1 stability but maintains their effect on phosphorylation and signaling, indicating that modulation of PKA activity by Gpb1 and Gpb2 is not solely due to increased levels of Bcy1. Inhibition of PKA catalytic subunits that are ATP analog-sensitive causes increased Bcy1 phosphorylation at the unknown site in high glucose. When PKA is inhibited, gpb1Δ gpb2Δ mutations have no effect on Bcy1 phosphorylation. Therefore, Gpb1 and Gpb2 oppose PKA activity by blocking the ability of PKA to inhibit Bcy1 phosphorylation at a site other than serine-145. Stimulation of Bcy1 phosphorylation by Gpb1 and Gpb2 produces a form of Bcy1 that is more stable and is a more effective PKA inhibitor.

Show MeSH
Related in: MedlinePlus