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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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Deletion of GPB1 and GPB2 affects phosphorylation and abundance of Bcy1. (A) Cell lysates were prepared from strains HS287-2C (bcy1Δ) and HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid 313pBHB1-416 that were grown in 2% glucose to log phase and then switched to the indicated concentrations of glucose for 3 h. Lysates were analyzed by SDS-PAGE and immunoblotting with anti-HA and anti-PGK antibodies. (B) Cell lysates were prepared from wild-type strain SKY762 carrying vector YCplac33 and from the following strains carrying plasmid 313pBHB1-416: wild-type strain SKY762, strain HS182-3B.k2T (gpb1Δ gpb2Δ), strain HS275-9B (gpa2Δ ras2Δ), and strain HS282-23C (gpb1Δ gpb2Δ gpa2Δ ras2Δ). Cells were grown in 2.0% glucose to log phase, and lysates were analyzed by SDS-PAGE and immunoblotting with anti-HA and anti-PGK antibodies.
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Figure 2: Deletion of GPB1 and GPB2 affects phosphorylation and abundance of Bcy1. (A) Cell lysates were prepared from strains HS287-2C (bcy1Δ) and HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid 313pBHB1-416 that were grown in 2% glucose to log phase and then switched to the indicated concentrations of glucose for 3 h. Lysates were analyzed by SDS-PAGE and immunoblotting with anti-HA and anti-PGK antibodies. (B) Cell lysates were prepared from wild-type strain SKY762 carrying vector YCplac33 and from the following strains carrying plasmid 313pBHB1-416: wild-type strain SKY762, strain HS182-3B.k2T (gpb1Δ gpb2Δ), strain HS275-9B (gpa2Δ ras2Δ), and strain HS282-23C (gpb1Δ gpb2Δ gpa2Δ ras2Δ). Cells were grown in 2.0% glucose to log phase, and lysates were analyzed by SDS-PAGE and immunoblotting with anti-HA and anti-PGK antibodies.

Mentions: To determine whether Gpb1 and Gpb2 are involved in the response to low glucose, wild-type and gpb1Δ gpb2Δ cells grown to log phase in 2% glucose were transferred to medium containing lower concentrations of glucose, and protein levels of HA-Bcy1 were measured by immunoblot. For the wild-type strain, transferring cells to 0.2, 0.05, or 0.005% glucose caused a substantial increase in Bcy1 levels (Figure 2A, lanes 1–4). The degree of Bcy1 phosphorylation also increased in response to lower concentrations of glucose. For the gpb1Δ gpb2Δ strain, transferring cells to low glucose concentrations resulted in Bcy1 levels that were more than 10-fold lower than the levels present in wild-type cells grown at the same glucose concentrations (Figure 2A, lanes 5–8). Moreover, the degree of Bcy1 phosphorylation was not substantially changed in gpb1Δ gpb2Δ cells in response to lower concentrations of glucose. These results strongly suggest that Gpb1 and Gpb2 play a role in reducing PKA activity in response to low levels of extracellular glucose.


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)

Deletion of GPB1 and GPB2 affects phosphorylation and abundance of Bcy1. (A) Cell lysates were prepared from strains HS287-2C (bcy1Δ) and HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid 313pBHB1-416 that were grown in 2% glucose to log phase and then switched to the indicated concentrations of glucose for 3 h. Lysates were analyzed by SDS-PAGE and immunoblotting with anti-HA and anti-PGK antibodies. (B) Cell lysates were prepared from wild-type strain SKY762 carrying vector YCplac33 and from the following strains carrying plasmid 313pBHB1-416: wild-type strain SKY762, strain HS182-3B.k2T (gpb1Δ gpb2Δ), strain HS275-9B (gpa2Δ ras2Δ), and strain HS282-23C (gpb1Δ gpb2Δ gpa2Δ ras2Δ). Cells were grown in 2.0% glucose to log phase, and lysates were analyzed by SDS-PAGE and immunoblotting with anti-HA and anti-PGK antibodies.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2965690&req=5

Figure 2: Deletion of GPB1 and GPB2 affects phosphorylation and abundance of Bcy1. (A) Cell lysates were prepared from strains HS287-2C (bcy1Δ) and HS293-10D (gpb1Δ gpb2Δ bcy1Δ) carrying plasmid 313pBHB1-416 that were grown in 2% glucose to log phase and then switched to the indicated concentrations of glucose for 3 h. Lysates were analyzed by SDS-PAGE and immunoblotting with anti-HA and anti-PGK antibodies. (B) Cell lysates were prepared from wild-type strain SKY762 carrying vector YCplac33 and from the following strains carrying plasmid 313pBHB1-416: wild-type strain SKY762, strain HS182-3B.k2T (gpb1Δ gpb2Δ), strain HS275-9B (gpa2Δ ras2Δ), and strain HS282-23C (gpb1Δ gpb2Δ gpa2Δ ras2Δ). Cells were grown in 2.0% glucose to log phase, and lysates were analyzed by SDS-PAGE and immunoblotting with anti-HA and anti-PGK antibodies.
Mentions: To determine whether Gpb1 and Gpb2 are involved in the response to low glucose, wild-type and gpb1Δ gpb2Δ cells grown to log phase in 2% glucose were transferred to medium containing lower concentrations of glucose, and protein levels of HA-Bcy1 were measured by immunoblot. For the wild-type strain, transferring cells to 0.2, 0.05, or 0.005% glucose caused a substantial increase in Bcy1 levels (Figure 2A, lanes 1–4). The degree of Bcy1 phosphorylation also increased in response to lower concentrations of glucose. For the gpb1Δ gpb2Δ strain, transferring cells to low glucose concentrations resulted in Bcy1 levels that were more than 10-fold lower than the levels present in wild-type cells grown at the same glucose concentrations (Figure 2A, lanes 5–8). Moreover, the degree of Bcy1 phosphorylation was not substantially changed in gpb1Δ gpb2Δ cells in response to lower concentrations of glucose. These results strongly suggest that Gpb1 and Gpb2 play a role in reducing PKA activity in response to low levels of extracellular glucose.

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