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Glucose-induced posttranslational activation of protein phosphatases PP2A and PP1 in yeast.

Castermans D, Somers I, Kriel J, Louwet W, Wera S, Versele M, Janssens V, Thevelein JM - Cell Res. (2012)

Bottom Line: Interestingly, the effect of the regulatory subunit Rts1 was consistent with its role as a subunit of both PP2A and PP1, affecting derepression and repression of SUC2, respectively.We also show that abolished phosphatase activation, except by reg1Δ, does not completely block Snf1 dephosphorylation after addition of glucose.Our results provide novel insight into the complex regulatory role of these two major protein phosphatases in glucose regulation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KULeuven, Belgium.

ABSTRACT
The protein phosphatases PP2A and PP1 are major regulators of a variety of cellular processes in yeast and other eukaryotes. Here, we reveal that both enzymes are direct targets of glucose sensing. Addition of glucose to glucose-deprived yeast cells triggered rapid posttranslational activation of both PP2A and PP1. Glucose activation of PP2A is controlled by regulatory subunits Rts1, Cdc55, Rrd1 and Rrd2. It is associated with rapid carboxymethylation of the catalytic subunits, which is necessary but not sufficient for activation. Glucose activation of PP1 was fully dependent on regulatory subunits Reg1 and Shp1. Absence of Gac1, Glc8, Reg2 or Red1 partially reduced activation while Pig1 and Pig2 inhibited activation. Full activation of PP2A and PP1 was also dependent on subunits classically considered to belong to the other phosphatase. PP2A activation was dependent on PP1 subunits Reg1 and Shp1 while PP1 activation was dependent on PP2A subunit Rts1. Rts1 interacted with both Pph21 and Glc7 under different conditions and these interactions were Reg1 dependent. Reg1-Glc7 interaction is responsible for PP1 involvement in the main glucose repression pathway and we show that deletion of Shp1 also causes strong derepression of the invertase gene SUC2. Deletion of the PP2A subunits Pph21 and Pph22, Rrd1 and Rrd2, specifically enhanced the derepression level of SUC2, indicating that PP2A counteracts SUC2 derepression. Interestingly, the effect of the regulatory subunit Rts1 was consistent with its role as a subunit of both PP2A and PP1, affecting derepression and repression of SUC2, respectively. We also show that abolished phosphatase activation, except by reg1Δ, does not completely block Snf1 dephosphorylation after addition of glucose. Finally, we show that glucose activation of the cAMP-PKA (protein kinase A) pathway is required for glucose activation of both PP2A and PP1. Our results provide novel insight into the complex regulatory role of these two major protein phosphatases in glucose regulation.

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Requirement for Reg1 and Shp1 in glucose-induced activation of PP2A. (A, B and D) At time 0, 20 mM glucose was added to glucose-deprived (glycerol-grown) cells of different deletion strains and cell extracts were used to measure specific PP2A activity. (A) WT (•), reg1Δ (○) and shp1Δ (▴). (B) WT (•), reg1Δ (○), reg1Δ + pReg1 (▴), reg1Δ + pReg1Δ8 (△) and reg1Δ + pReg1I466M F468A (▪). (C) 20 mM glucose was added to glucose-deprived (glycerol-grown) cells of BY-strains with different deletions (WT, shp1Δ and reg1Δ) that are expressing HA-tagged Pph21 or Glc7, respectively. Antibodies used for the immunopurification (IP) and immunodetection (western): HA: anti-HA; Rts1: anti-Rts1; -: no antibody. (D) WT strain (•), pig1Δ (○), pig2Δ (▴), cdc55Δ (△) and pig2Δ cdc55Δ (▪).
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fig5: Requirement for Reg1 and Shp1 in glucose-induced activation of PP2A. (A, B and D) At time 0, 20 mM glucose was added to glucose-deprived (glycerol-grown) cells of different deletion strains and cell extracts were used to measure specific PP2A activity. (A) WT (•), reg1Δ (○) and shp1Δ (▴). (B) WT (•), reg1Δ (○), reg1Δ + pReg1 (▴), reg1Δ + pReg1Δ8 (△) and reg1Δ + pReg1I466M F468A (▪). (C) 20 mM glucose was added to glucose-deprived (glycerol-grown) cells of BY-strains with different deletions (WT, shp1Δ and reg1Δ) that are expressing HA-tagged Pph21 or Glc7, respectively. Antibodies used for the immunopurification (IP) and immunodetection (western): HA: anti-HA; Rts1: anti-Rts1; -: no antibody. (D) WT strain (•), pig1Δ (○), pig2Δ (▴), cdc55Δ (△) and pig2Δ cdc55Δ (▪).

Mentions: For PP2A, we found that deletion of REG1 or SHP1, up to now considered to encode PP1 subunits, resulted in abolished or partially reduced activation, respectively (Figure 5A). Hence, Reg1 and Shp1 seem to play a role in the activation of this phosphatase as well. Since GLC7 is an essential gene, we were unable to investigate the effect of reg1Δ or shp1Δ on PP2A activation in the absence of PP1. However, to check whether the effect of Reg1 on PP2A activation is an indirect effect of the absence of PP1 activation, we used specific mutant alleles of REG1. The Reg1Δ8 and Reg1I466M F468A mutant proteins, which are unable to interact with the PP1 catalytic subunit Glc7 65, caused either abolished or reduced activation of PP2A (Figure 5B). This suggests that the loss of PP2A activation due to malfunctioning of Reg1 is at least in part dependent on Reg1 interaction with PP1. The two Reg1 mutant proteins also showed the expected loss of PP1 activation upon addition of glucose (Supplementary information, Figure S8A, expression of these constructs was confirmed in Supplementary information, Figure S8B). The Reg1I466M F468A mutant protein still showed nearly half of the WT PP2A activation (Figure 5B) in spite of more strongly reduced PP1 activation (Supplementary information, Figure S8A), supporting that the effect on PP2A does not completely act through PP1. We were unable to demonstrate any direct Reg1-Pph21 interaction (data not shown), suggesting that the interaction may be weak or indirect. Interestingly, strains lacking Reg1 showed significantly reduced interaction of Rts1 with both Glc7 and Pph21 (Figure 5C), which supports the importance of Reg1 for PP2A activation and suggests that it may involve the Rts1 subunit. Strains lacking Shp1, on the other hand, were not affected in these interactions (Figure 5C).


Glucose-induced posttranslational activation of protein phosphatases PP2A and PP1 in yeast.

Castermans D, Somers I, Kriel J, Louwet W, Wera S, Versele M, Janssens V, Thevelein JM - Cell Res. (2012)

Requirement for Reg1 and Shp1 in glucose-induced activation of PP2A. (A, B and D) At time 0, 20 mM glucose was added to glucose-deprived (glycerol-grown) cells of different deletion strains and cell extracts were used to measure specific PP2A activity. (A) WT (•), reg1Δ (○) and shp1Δ (▴). (B) WT (•), reg1Δ (○), reg1Δ + pReg1 (▴), reg1Δ + pReg1Δ8 (△) and reg1Δ + pReg1I466M F468A (▪). (C) 20 mM glucose was added to glucose-deprived (glycerol-grown) cells of BY-strains with different deletions (WT, shp1Δ and reg1Δ) that are expressing HA-tagged Pph21 or Glc7, respectively. Antibodies used for the immunopurification (IP) and immunodetection (western): HA: anti-HA; Rts1: anti-Rts1; -: no antibody. (D) WT strain (•), pig1Δ (○), pig2Δ (▴), cdc55Δ (△) and pig2Δ cdc55Δ (▪).
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Related In: Results  -  Collection

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Show All Figures
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fig5: Requirement for Reg1 and Shp1 in glucose-induced activation of PP2A. (A, B and D) At time 0, 20 mM glucose was added to glucose-deprived (glycerol-grown) cells of different deletion strains and cell extracts were used to measure specific PP2A activity. (A) WT (•), reg1Δ (○) and shp1Δ (▴). (B) WT (•), reg1Δ (○), reg1Δ + pReg1 (▴), reg1Δ + pReg1Δ8 (△) and reg1Δ + pReg1I466M F468A (▪). (C) 20 mM glucose was added to glucose-deprived (glycerol-grown) cells of BY-strains with different deletions (WT, shp1Δ and reg1Δ) that are expressing HA-tagged Pph21 or Glc7, respectively. Antibodies used for the immunopurification (IP) and immunodetection (western): HA: anti-HA; Rts1: anti-Rts1; -: no antibody. (D) WT strain (•), pig1Δ (○), pig2Δ (▴), cdc55Δ (△) and pig2Δ cdc55Δ (▪).
Mentions: For PP2A, we found that deletion of REG1 or SHP1, up to now considered to encode PP1 subunits, resulted in abolished or partially reduced activation, respectively (Figure 5A). Hence, Reg1 and Shp1 seem to play a role in the activation of this phosphatase as well. Since GLC7 is an essential gene, we were unable to investigate the effect of reg1Δ or shp1Δ on PP2A activation in the absence of PP1. However, to check whether the effect of Reg1 on PP2A activation is an indirect effect of the absence of PP1 activation, we used specific mutant alleles of REG1. The Reg1Δ8 and Reg1I466M F468A mutant proteins, which are unable to interact with the PP1 catalytic subunit Glc7 65, caused either abolished or reduced activation of PP2A (Figure 5B). This suggests that the loss of PP2A activation due to malfunctioning of Reg1 is at least in part dependent on Reg1 interaction with PP1. The two Reg1 mutant proteins also showed the expected loss of PP1 activation upon addition of glucose (Supplementary information, Figure S8A, expression of these constructs was confirmed in Supplementary information, Figure S8B). The Reg1I466M F468A mutant protein still showed nearly half of the WT PP2A activation (Figure 5B) in spite of more strongly reduced PP1 activation (Supplementary information, Figure S8A), supporting that the effect on PP2A does not completely act through PP1. We were unable to demonstrate any direct Reg1-Pph21 interaction (data not shown), suggesting that the interaction may be weak or indirect. Interestingly, strains lacking Reg1 showed significantly reduced interaction of Rts1 with both Glc7 and Pph21 (Figure 5C), which supports the importance of Reg1 for PP2A activation and suggests that it may involve the Rts1 subunit. Strains lacking Shp1, on the other hand, were not affected in these interactions (Figure 5C).

Bottom Line: Interestingly, the effect of the regulatory subunit Rts1 was consistent with its role as a subunit of both PP2A and PP1, affecting derepression and repression of SUC2, respectively.We also show that abolished phosphatase activation, except by reg1Δ, does not completely block Snf1 dephosphorylation after addition of glucose.Our results provide novel insight into the complex regulatory role of these two major protein phosphatases in glucose regulation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KULeuven, Belgium.

ABSTRACT
The protein phosphatases PP2A and PP1 are major regulators of a variety of cellular processes in yeast and other eukaryotes. Here, we reveal that both enzymes are direct targets of glucose sensing. Addition of glucose to glucose-deprived yeast cells triggered rapid posttranslational activation of both PP2A and PP1. Glucose activation of PP2A is controlled by regulatory subunits Rts1, Cdc55, Rrd1 and Rrd2. It is associated with rapid carboxymethylation of the catalytic subunits, which is necessary but not sufficient for activation. Glucose activation of PP1 was fully dependent on regulatory subunits Reg1 and Shp1. Absence of Gac1, Glc8, Reg2 or Red1 partially reduced activation while Pig1 and Pig2 inhibited activation. Full activation of PP2A and PP1 was also dependent on subunits classically considered to belong to the other phosphatase. PP2A activation was dependent on PP1 subunits Reg1 and Shp1 while PP1 activation was dependent on PP2A subunit Rts1. Rts1 interacted with both Pph21 and Glc7 under different conditions and these interactions were Reg1 dependent. Reg1-Glc7 interaction is responsible for PP1 involvement in the main glucose repression pathway and we show that deletion of Shp1 also causes strong derepression of the invertase gene SUC2. Deletion of the PP2A subunits Pph21 and Pph22, Rrd1 and Rrd2, specifically enhanced the derepression level of SUC2, indicating that PP2A counteracts SUC2 derepression. Interestingly, the effect of the regulatory subunit Rts1 was consistent with its role as a subunit of both PP2A and PP1, affecting derepression and repression of SUC2, respectively. We also show that abolished phosphatase activation, except by reg1Δ, does not completely block Snf1 dephosphorylation after addition of glucose. Finally, we show that glucose activation of the cAMP-PKA (protein kinase A) pathway is required for glucose activation of both PP2A and PP1. Our results provide novel insight into the complex regulatory role of these two major protein phosphatases in glucose regulation.

Show MeSH
Related in: MedlinePlus