Limits...
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.

Show MeSH

Related in: MedlinePlus

Glucose activation of PP2A and PP1 protein phosphatase activity. At time 0 a given concentration of the indicated sugar was added to glucose-deprived (glycerol-grown) cells of BY-strains and cell extracts were used to measure protein phosphatase activity in the presence of the appropriate inhibitors: 0.2 μM inhibitor-2 to measure specific PP2A activity and 20 nM okadaic acid to measure specific PP1 activity. (A) Total phosphatase activity after addition of different glucose concentrations: 1 mM (•), 5 mM (○), 20 mM (▴), 60 mM (△) and 100 mM (▪). (B) Total phosphatase activity after addition of 20 mM of the indicated sugar: glucose (•), sucrose (○), fructose (▴), galactose (△) and sorbitol (▪). (C) Total (•) or specific PP2A (○) and PP1 (▴) phosphatase activity after addition of 20 mM glucose. Control: presence of both PP2A and PP1 inhibitor (△). (D) PP2A activity after addition of 20 mM glucose to WT strain (•), pph21Δ (○), pph22Δ (▴) and pph21Δ pph22Δ (△). (E) 20 mM glucose was added and activity was measured with HA-Pph21 immunoprecipitated from cell extracts: pph21Δ pph22Δ + pHA-Pph21 in the absence of inhibitors (•), in the presence of inhibitor-2 (○), in the presence of okadaic acid (▴) and pph21Δ pph22Δ + pEMPTY in the absence of inhibitors (△). (F) 20 mM glucose was added and activity was measured with HA-Glc7 immunoprecipitated from cell extracts: glc7Δ + pHA-Glc7 in the absence of inhibitors (•), in the presence of inhibitor-2 (○), in the presence of okadaic acid (▴) and WT + pEMPTY in the absence of inhibitors (△).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3367521&req=5

fig1: Glucose activation of PP2A and PP1 protein phosphatase activity. At time 0 a given concentration of the indicated sugar was added to glucose-deprived (glycerol-grown) cells of BY-strains and cell extracts were used to measure protein phosphatase activity in the presence of the appropriate inhibitors: 0.2 μM inhibitor-2 to measure specific PP2A activity and 20 nM okadaic acid to measure specific PP1 activity. (A) Total phosphatase activity after addition of different glucose concentrations: 1 mM (•), 5 mM (○), 20 mM (▴), 60 mM (△) and 100 mM (▪). (B) Total phosphatase activity after addition of 20 mM of the indicated sugar: glucose (•), sucrose (○), fructose (▴), galactose (△) and sorbitol (▪). (C) Total (•) or specific PP2A (○) and PP1 (▴) phosphatase activity after addition of 20 mM glucose. Control: presence of both PP2A and PP1 inhibitor (△). (D) PP2A activity after addition of 20 mM glucose to WT strain (•), pph21Δ (○), pph22Δ (▴) and pph21Δ pph22Δ (△). (E) 20 mM glucose was added and activity was measured with HA-Pph21 immunoprecipitated from cell extracts: pph21Δ pph22Δ + pHA-Pph21 in the absence of inhibitors (•), in the presence of inhibitor-2 (○), in the presence of okadaic acid (▴) and pph21Δ pph22Δ + pEMPTY in the absence of inhibitors (△). (F) 20 mM glucose was added and activity was measured with HA-Glc7 immunoprecipitated from cell extracts: glc7Δ + pHA-Glc7 in the absence of inhibitors (•), in the presence of inhibitor-2 (○), in the presence of okadaic acid (▴) and WT + pEMPTY in the absence of inhibitors (△).

Mentions: Addition of glucose to glucose-deprived (glucose-derepressed) yeast cells triggered rapid activation of protein phosphatase activity (within 1 min), as measured by the dephosphorylation of 32P-phosphorylated mammalian glycogen phosphorylase a (Figure 1A). Protein phosphatase activity was measured in cell extracts, protein concentration was determined for normalization and the measured relative phosphatase activity is shown as 'nmol phosphate released per min and per mg protein'. In spite of the variability in the basal level, the rapid increase in activity after addition of glucose could always be clearly recognized. For more details and comments concerning the reproducibility, please see Supplementary information, Data S1.


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)

Glucose activation of PP2A and PP1 protein phosphatase activity. At time 0 a given concentration of the indicated sugar was added to glucose-deprived (glycerol-grown) cells of BY-strains and cell extracts were used to measure protein phosphatase activity in the presence of the appropriate inhibitors: 0.2 μM inhibitor-2 to measure specific PP2A activity and 20 nM okadaic acid to measure specific PP1 activity. (A) Total phosphatase activity after addition of different glucose concentrations: 1 mM (•), 5 mM (○), 20 mM (▴), 60 mM (△) and 100 mM (▪). (B) Total phosphatase activity after addition of 20 mM of the indicated sugar: glucose (•), sucrose (○), fructose (▴), galactose (△) and sorbitol (▪). (C) Total (•) or specific PP2A (○) and PP1 (▴) phosphatase activity after addition of 20 mM glucose. Control: presence of both PP2A and PP1 inhibitor (△). (D) PP2A activity after addition of 20 mM glucose to WT strain (•), pph21Δ (○), pph22Δ (▴) and pph21Δ pph22Δ (△). (E) 20 mM glucose was added and activity was measured with HA-Pph21 immunoprecipitated from cell extracts: pph21Δ pph22Δ + pHA-Pph21 in the absence of inhibitors (•), in the presence of inhibitor-2 (○), in the presence of okadaic acid (▴) and pph21Δ pph22Δ + pEMPTY in the absence of inhibitors (△). (F) 20 mM glucose was added and activity was measured with HA-Glc7 immunoprecipitated from cell extracts: glc7Δ + pHA-Glc7 in the absence of inhibitors (•), in the presence of inhibitor-2 (○), in the presence of okadaic acid (▴) and WT + pEMPTY in the absence of inhibitors (△).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367521&req=5

fig1: Glucose activation of PP2A and PP1 protein phosphatase activity. At time 0 a given concentration of the indicated sugar was added to glucose-deprived (glycerol-grown) cells of BY-strains and cell extracts were used to measure protein phosphatase activity in the presence of the appropriate inhibitors: 0.2 μM inhibitor-2 to measure specific PP2A activity and 20 nM okadaic acid to measure specific PP1 activity. (A) Total phosphatase activity after addition of different glucose concentrations: 1 mM (•), 5 mM (○), 20 mM (▴), 60 mM (△) and 100 mM (▪). (B) Total phosphatase activity after addition of 20 mM of the indicated sugar: glucose (•), sucrose (○), fructose (▴), galactose (△) and sorbitol (▪). (C) Total (•) or specific PP2A (○) and PP1 (▴) phosphatase activity after addition of 20 mM glucose. Control: presence of both PP2A and PP1 inhibitor (△). (D) PP2A activity after addition of 20 mM glucose to WT strain (•), pph21Δ (○), pph22Δ (▴) and pph21Δ pph22Δ (△). (E) 20 mM glucose was added and activity was measured with HA-Pph21 immunoprecipitated from cell extracts: pph21Δ pph22Δ + pHA-Pph21 in the absence of inhibitors (•), in the presence of inhibitor-2 (○), in the presence of okadaic acid (▴) and pph21Δ pph22Δ + pEMPTY in the absence of inhibitors (△). (F) 20 mM glucose was added and activity was measured with HA-Glc7 immunoprecipitated from cell extracts: glc7Δ + pHA-Glc7 in the absence of inhibitors (•), in the presence of inhibitor-2 (○), in the presence of okadaic acid (▴) and WT + pEMPTY in the absence of inhibitors (△).
Mentions: Addition of glucose to glucose-deprived (glucose-derepressed) yeast cells triggered rapid activation of protein phosphatase activity (within 1 min), as measured by the dephosphorylation of 32P-phosphorylated mammalian glycogen phosphorylase a (Figure 1A). Protein phosphatase activity was measured in cell extracts, protein concentration was determined for normalization and the measured relative phosphatase activity is shown as 'nmol phosphate released per min and per mg protein'. In spite of the variability in the basal level, the rapid increase in activity after addition of glucose could always be clearly recognized. For more details and comments concerning the reproducibility, please see Supplementary information, Data S1.

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