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Contribution of Orb2A stability in regulated amyloid-like oligomerization of Drosophila Orb2.

White-Grindley E, Li L, Mohammad Khan R, Ren F, Saraf A, Florens L, Si K - PLoS Biol. (2014)

Bottom Line: Mutation of LimK abolishes activity-dependent Orb2 oligomerization in the adult brain.Moreover, Tob-Orb2 association is modulated by neuronal activity and Tob activity in the mushroom body is required for stable memory formation.These observations suggest that the interplay between PP2A and Tob-LimK activity may dynamically regulate Orb2 amyloid-like oligomer formation and the stabilization of memories.

View Article: PubMed Central - PubMed

Affiliation: Stowers Institute for Medical Research, Kansas City, Missouri, United States of America.

ABSTRACT
How learned experiences persist as memory for a long time is an important question. In Drosophila the persistence of memory is dependent upon amyloid-like oligomers of the Orb2 protein. However, it is not clear how the conversion of Orb2 to the amyloid-like oligomeric state is regulated. The Orb2 has two protein isoforms, and the rare Orb2A isoform is critical for oligomerization of the ubiquitous Orb2B isoform. Here, we report the discovery of a protein network comprised of protein phosphatase 2A (PP2A), Transducer of Erb-B2 (Tob), and Lim Kinase (LimK) that controls the abundance of Orb2A. PP2A maintains Orb2A in an unphosphorylated and unstable state, whereas Tob-LimK phosphorylates and stabilizes Orb2A. Mutation of LimK abolishes activity-dependent Orb2 oligomerization in the adult brain. Moreover, Tob-Orb2 association is modulated by neuronal activity and Tob activity in the mushroom body is required for stable memory formation. These observations suggest that the interplay between PP2A and Tob-LimK activity may dynamically regulate Orb2 amyloid-like oligomer formation and the stabilization of memories.

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PP2A regulates Tob-Orb2 phosphorylation and stability.(A–C) Tob is destabilized upon PP2A inhibition when in complex with Orb2. (A) The phosphatase inhibitor calyculinA (CY) does not significantly affect the half-life of Tob alone. (B and C) In the presence of CY, the increase in Tob stability normally observed in the presence of Orb2A (B) or Orb2B (C) was significantly reduced. (D) In S2 cells Tob is phosphorylated when coexpressed with Orb2A or Orb2B. Changes in Tob phosphorylation was confirmed by λ-phosphatase (PPase) treatment. (E) PP2A inhibitor CY, okadaic acid, but not PP1 inhibitor tautomycin increases Orb2A and Orb2B phosphorylation, as evident in shift electrophoretic mobility. (F) The Orb2 proteins are phosphorylated in multiple sites. (G) PP2A inhibitor CY increases the half-life of Orb2A and Orb2B. (H) Overexpression of PP2A catalytic subunit Mts destabilizes Orb2A and Orb2B. The RNA binding protein Hrp36 serves as loading control. Statistical significance was measured with two-tailed t test (*) p≤0.05, (***) p≤0.001. n, the number of independent experiments for each experimental group. The shift in molecular weight associated with phosphorylation was assayed in 8% SDS-PAGE. The half-life determination experiments were assayed in 4%–12% SDS-PAGE. Also see Figure S5.
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pbio-1001786-g005: PP2A regulates Tob-Orb2 phosphorylation and stability.(A–C) Tob is destabilized upon PP2A inhibition when in complex with Orb2. (A) The phosphatase inhibitor calyculinA (CY) does not significantly affect the half-life of Tob alone. (B and C) In the presence of CY, the increase in Tob stability normally observed in the presence of Orb2A (B) or Orb2B (C) was significantly reduced. (D) In S2 cells Tob is phosphorylated when coexpressed with Orb2A or Orb2B. Changes in Tob phosphorylation was confirmed by λ-phosphatase (PPase) treatment. (E) PP2A inhibitor CY, okadaic acid, but not PP1 inhibitor tautomycin increases Orb2A and Orb2B phosphorylation, as evident in shift electrophoretic mobility. (F) The Orb2 proteins are phosphorylated in multiple sites. (G) PP2A inhibitor CY increases the half-life of Orb2A and Orb2B. (H) Overexpression of PP2A catalytic subunit Mts destabilizes Orb2A and Orb2B. The RNA binding protein Hrp36 serves as loading control. Statistical significance was measured with two-tailed t test (*) p≤0.05, (***) p≤0.001. n, the number of independent experiments for each experimental group. The shift in molecular weight associated with phosphorylation was assayed in 8% SDS-PAGE. The half-life determination experiments were assayed in 4%–12% SDS-PAGE. Also see Figure S5.

Mentions: Because the Tob-Orb2 association alters the half-life of both proteins and phosphorylation affects their association, we examined the effect of phosphatase inhibition on the half-life of both proteins. When Tob was expressed by itself there was modest change in stability in the presence of CY (Table S2) compared to the untreated samples (Figure 5A). Interestingly, the increase in Tob stability that occurred when co-expressed with either Orb2A (Figure 5B) or Orb2B (Figure 5C) was ∼50% reduced when the phosphatases were inhibited (Table S2). The destabilization of Tob was observed only in the presence of the PP2A/PP1 inhibitor CY or okadaic acid (1 nM) but not the PP1 selective inhibitor tautomycin (10 nM) (Figure S5A) [37],[38]. Moreover, the extent of Tob phosphorylation appears to be specifically linked to Orb2 complex formation (Figure 5D). The Orb2 proteins, but not the other homologue of CPEB in Drosophila, Orb1, enhance phosphorylation of Tob, although Tob interacts with both Orb2 and Orb1 (Figure S5B and C). These results suggest un- or hypophosphorylated Tob binds Orb2. Association of Tob with Orb2 and PP2A inactivation leads to additional phosphorylation of Tob-Orb2, which results in dissociation and eventual destabilization of Tob.


Contribution of Orb2A stability in regulated amyloid-like oligomerization of Drosophila Orb2.

White-Grindley E, Li L, Mohammad Khan R, Ren F, Saraf A, Florens L, Si K - PLoS Biol. (2014)

PP2A regulates Tob-Orb2 phosphorylation and stability.(A–C) Tob is destabilized upon PP2A inhibition when in complex with Orb2. (A) The phosphatase inhibitor calyculinA (CY) does not significantly affect the half-life of Tob alone. (B and C) In the presence of CY, the increase in Tob stability normally observed in the presence of Orb2A (B) or Orb2B (C) was significantly reduced. (D) In S2 cells Tob is phosphorylated when coexpressed with Orb2A or Orb2B. Changes in Tob phosphorylation was confirmed by λ-phosphatase (PPase) treatment. (E) PP2A inhibitor CY, okadaic acid, but not PP1 inhibitor tautomycin increases Orb2A and Orb2B phosphorylation, as evident in shift electrophoretic mobility. (F) The Orb2 proteins are phosphorylated in multiple sites. (G) PP2A inhibitor CY increases the half-life of Orb2A and Orb2B. (H) Overexpression of PP2A catalytic subunit Mts destabilizes Orb2A and Orb2B. The RNA binding protein Hrp36 serves as loading control. Statistical significance was measured with two-tailed t test (*) p≤0.05, (***) p≤0.001. n, the number of independent experiments for each experimental group. The shift in molecular weight associated with phosphorylation was assayed in 8% SDS-PAGE. The half-life determination experiments were assayed in 4%–12% SDS-PAGE. Also see Figure S5.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3921104&req=5

pbio-1001786-g005: PP2A regulates Tob-Orb2 phosphorylation and stability.(A–C) Tob is destabilized upon PP2A inhibition when in complex with Orb2. (A) The phosphatase inhibitor calyculinA (CY) does not significantly affect the half-life of Tob alone. (B and C) In the presence of CY, the increase in Tob stability normally observed in the presence of Orb2A (B) or Orb2B (C) was significantly reduced. (D) In S2 cells Tob is phosphorylated when coexpressed with Orb2A or Orb2B. Changes in Tob phosphorylation was confirmed by λ-phosphatase (PPase) treatment. (E) PP2A inhibitor CY, okadaic acid, but not PP1 inhibitor tautomycin increases Orb2A and Orb2B phosphorylation, as evident in shift electrophoretic mobility. (F) The Orb2 proteins are phosphorylated in multiple sites. (G) PP2A inhibitor CY increases the half-life of Orb2A and Orb2B. (H) Overexpression of PP2A catalytic subunit Mts destabilizes Orb2A and Orb2B. The RNA binding protein Hrp36 serves as loading control. Statistical significance was measured with two-tailed t test (*) p≤0.05, (***) p≤0.001. n, the number of independent experiments for each experimental group. The shift in molecular weight associated with phosphorylation was assayed in 8% SDS-PAGE. The half-life determination experiments were assayed in 4%–12% SDS-PAGE. Also see Figure S5.
Mentions: Because the Tob-Orb2 association alters the half-life of both proteins and phosphorylation affects their association, we examined the effect of phosphatase inhibition on the half-life of both proteins. When Tob was expressed by itself there was modest change in stability in the presence of CY (Table S2) compared to the untreated samples (Figure 5A). Interestingly, the increase in Tob stability that occurred when co-expressed with either Orb2A (Figure 5B) or Orb2B (Figure 5C) was ∼50% reduced when the phosphatases were inhibited (Table S2). The destabilization of Tob was observed only in the presence of the PP2A/PP1 inhibitor CY or okadaic acid (1 nM) but not the PP1 selective inhibitor tautomycin (10 nM) (Figure S5A) [37],[38]. Moreover, the extent of Tob phosphorylation appears to be specifically linked to Orb2 complex formation (Figure 5D). The Orb2 proteins, but not the other homologue of CPEB in Drosophila, Orb1, enhance phosphorylation of Tob, although Tob interacts with both Orb2 and Orb1 (Figure S5B and C). These results suggest un- or hypophosphorylated Tob binds Orb2. Association of Tob with Orb2 and PP2A inactivation leads to additional phosphorylation of Tob-Orb2, which results in dissociation and eventual destabilization of Tob.

Bottom Line: Mutation of LimK abolishes activity-dependent Orb2 oligomerization in the adult brain.Moreover, Tob-Orb2 association is modulated by neuronal activity and Tob activity in the mushroom body is required for stable memory formation.These observations suggest that the interplay between PP2A and Tob-LimK activity may dynamically regulate Orb2 amyloid-like oligomer formation and the stabilization of memories.

View Article: PubMed Central - PubMed

Affiliation: Stowers Institute for Medical Research, Kansas City, Missouri, United States of America.

ABSTRACT
How learned experiences persist as memory for a long time is an important question. In Drosophila the persistence of memory is dependent upon amyloid-like oligomers of the Orb2 protein. However, it is not clear how the conversion of Orb2 to the amyloid-like oligomeric state is regulated. The Orb2 has two protein isoforms, and the rare Orb2A isoform is critical for oligomerization of the ubiquitous Orb2B isoform. Here, we report the discovery of a protein network comprised of protein phosphatase 2A (PP2A), Transducer of Erb-B2 (Tob), and Lim Kinase (LimK) that controls the abundance of Orb2A. PP2A maintains Orb2A in an unphosphorylated and unstable state, whereas Tob-LimK phosphorylates and stabilizes Orb2A. Mutation of LimK abolishes activity-dependent Orb2 oligomerization in the adult brain. Moreover, Tob-Orb2 association is modulated by neuronal activity and Tob activity in the mushroom body is required for stable memory formation. These observations suggest that the interplay between PP2A and Tob-LimK activity may dynamically regulate Orb2 amyloid-like oligomer formation and the stabilization of memories.

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