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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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Model for control of PKA activity by Gpb1 and Gpb2. The PKA regulatory subunit Bcy1 is phosphorylated on an unknown site by a kinase that is inhibited by PKA catalytic activity. This phosphorylation stabilizes Bcy1 that has been phosphorylated at serine-145 by PKA catalytic subunits and causes Bcy1 to be a more efficient inhibitor of Tpk1, Tpk2, and Tpk3. In low glucose, Gpb1 and Gpb2 block the inhibitory effect of PKA on the kinase that phosphorylates Bcy1. Under these conditions, more Bcy1 is bound to PKA catalytic subunits and the signal output is low. In the absence of Gpb1 and Gpb2, PKA inhibits the kinase that phosphorylates Bcy1, and Bcy1 that is phosphorylated at serine-145 becomes unstable. Under these conditions, less Bcy1 is bound to PKA catalytic subunits and the signal output is high.
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Figure 7: Model for control of PKA activity by Gpb1 and Gpb2. The PKA regulatory subunit Bcy1 is phosphorylated on an unknown site by a kinase that is inhibited by PKA catalytic activity. This phosphorylation stabilizes Bcy1 that has been phosphorylated at serine-145 by PKA catalytic subunits and causes Bcy1 to be a more efficient inhibitor of Tpk1, Tpk2, and Tpk3. In low glucose, Gpb1 and Gpb2 block the inhibitory effect of PKA on the kinase that phosphorylates Bcy1. Under these conditions, more Bcy1 is bound to PKA catalytic subunits and the signal output is low. In the absence of Gpb1 and Gpb2, PKA inhibits the kinase that phosphorylates Bcy1, and Bcy1 that is phosphorylated at serine-145 becomes unstable. Under these conditions, less Bcy1 is bound to PKA catalytic subunits and the signal output is high.

Mentions: The model shown in Figure 7incorporates the following findings. In gpb1Δ gpb2Δ mutants, Bcy1 abundance is low because its rate of degradation is increased (Figure 3). When the abundance of Bcy1 is low, the amount of free, active PKA catalytic subunits is expected to be high. The effect of gpb1Δ gpb2Δ mutations on Bcy1 stability requires either serine or a negatively charged amino acid at position 145 of Bcy1. Serine-145 is the site at which Bcy1 is phosphorylated by Tpk proteins (Kuret et al., 1988; Werner-Washburne et al., 1991). Therefore, these observations suggest that phosphorylation of Bcy1 by Tpk proteins targets it for degradation. Phosphorylated Bcy1 could be recognized by F-box subunits of the SCF E3 ubiquitin ligase, which target proteins that contain a phosphorylated motif (Vodermaier, 2004).


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)

Model for control of PKA activity by Gpb1 and Gpb2. The PKA regulatory subunit Bcy1 is phosphorylated on an unknown site by a kinase that is inhibited by PKA catalytic activity. This phosphorylation stabilizes Bcy1 that has been phosphorylated at serine-145 by PKA catalytic subunits and causes Bcy1 to be a more efficient inhibitor of Tpk1, Tpk2, and Tpk3. In low glucose, Gpb1 and Gpb2 block the inhibitory effect of PKA on the kinase that phosphorylates Bcy1. Under these conditions, more Bcy1 is bound to PKA catalytic subunits and the signal output is low. In the absence of Gpb1 and Gpb2, PKA inhibits the kinase that phosphorylates Bcy1, and Bcy1 that is phosphorylated at serine-145 becomes unstable. Under these conditions, less Bcy1 is bound to PKA catalytic subunits and the signal output is high.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 7: Model for control of PKA activity by Gpb1 and Gpb2. The PKA regulatory subunit Bcy1 is phosphorylated on an unknown site by a kinase that is inhibited by PKA catalytic activity. This phosphorylation stabilizes Bcy1 that has been phosphorylated at serine-145 by PKA catalytic subunits and causes Bcy1 to be a more efficient inhibitor of Tpk1, Tpk2, and Tpk3. In low glucose, Gpb1 and Gpb2 block the inhibitory effect of PKA on the kinase that phosphorylates Bcy1. Under these conditions, more Bcy1 is bound to PKA catalytic subunits and the signal output is low. In the absence of Gpb1 and Gpb2, PKA inhibits the kinase that phosphorylates Bcy1, and Bcy1 that is phosphorylated at serine-145 becomes unstable. Under these conditions, less Bcy1 is bound to PKA catalytic subunits and the signal output is high.
Mentions: The model shown in Figure 7incorporates the following findings. In gpb1Δ gpb2Δ mutants, Bcy1 abundance is low because its rate of degradation is increased (Figure 3). When the abundance of Bcy1 is low, the amount of free, active PKA catalytic subunits is expected to be high. The effect of gpb1Δ gpb2Δ mutations on Bcy1 stability requires either serine or a negatively charged amino acid at position 145 of Bcy1. Serine-145 is the site at which Bcy1 is phosphorylated by Tpk proteins (Kuret et al., 1988; Werner-Washburne et al., 1991). Therefore, these observations suggest that phosphorylation of Bcy1 by Tpk proteins targets it for degradation. Phosphorylated Bcy1 could be recognized by F-box subunits of the SCF E3 ubiquitin ligase, which target proteins that contain a phosphorylated motif (Vodermaier, 2004).

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