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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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Tob and Orb2 are phosphorylated, and phosphorylation regulates Tob-Orb2 association.(A) Orb2 is phosphorylated in the adult fly head. Blotting of Orb2 immunoprecipitate with phospho-tag™ detects phosphorylated Orb2B. Treatment with calf-intestinal phosphatase (PPase) that removes phosphate groups from proteins shows specificity of phospho-tag™. (B) Both Orb2A and Orb2B are phosphorylated at a low level in S2cells. Orb2 immunoprecipitate from the S2 cell is probed with phospho-tag™. (C) Tob is phosphorylated in the adult fly brain. (Left panel) Approximately 2 mg of total head extracts were immunoprecipitated with preimmune (pre) or immune Tob serum. The phospho-tag™ detects a band at the position of Tob only in the immune but not in preimmune lane. Treatment with λ-phosphatase (PPase) reduces phospho- tag™ signal. (Right panel, top) Total adult head extracts were treated with calf-intestinal phosphatase (PPase). Change in phosphorylation status of Tob was assessed as a change in mobility by Western blot analysis. (Right panel, bottom) Exogenously expressed Flag-tagged Tob protein is also phosphorylated in the adult brain. (D) The addition of the phosphatase inhibitor, calyculin (CY), dissociates the Orb2-Tob complex. S2 cells were transfected with Orb2A with and without Tob and treated with 10 µm CY for 1 h prior to Tob immunoprecipitation. CY treatment almost completely abolished Tob association with Orb2A oligomers and reduced association with the monomers. (E) Unphosphorylated Tob has a greater affinity for Orb2A. S2 cell lysates were first treated with the indicated units of phosphatase (uPPase), and subsequently the Orb2A-Tob complex was immunoprecipitated. More Orb2A was found to be associated with Tob following phosphatase treatment. Western blots of the lysates show the level of Orb2A and Tob (input). The 4%–12% gradient gels were used in these experiments. (F) Hyperphosphorylated Tob does not associate with Orb2. Untagged Orb2 and FLAG-tagged Tob complex was immunopurified using anti-Orb2 antibodies and probed with phosphor-tag™ (top panel). Phosphorylated proteins correspond to the size of Orb2 (bottom panel) but not Tob (middle panel). The hyperphosphorylated Tob proteins are visible in the total extract (input), however they are absent in the Orb2-Tob complex. The 8% gel in Tris-Glycine buffer was used in this experiment.
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pbio-1001786-g004: Tob and Orb2 are phosphorylated, and phosphorylation regulates Tob-Orb2 association.(A) Orb2 is phosphorylated in the adult fly head. Blotting of Orb2 immunoprecipitate with phospho-tag™ detects phosphorylated Orb2B. Treatment with calf-intestinal phosphatase (PPase) that removes phosphate groups from proteins shows specificity of phospho-tag™. (B) Both Orb2A and Orb2B are phosphorylated at a low level in S2cells. Orb2 immunoprecipitate from the S2 cell is probed with phospho-tag™. (C) Tob is phosphorylated in the adult fly brain. (Left panel) Approximately 2 mg of total head extracts were immunoprecipitated with preimmune (pre) or immune Tob serum. The phospho-tag™ detects a band at the position of Tob only in the immune but not in preimmune lane. Treatment with λ-phosphatase (PPase) reduces phospho- tag™ signal. (Right panel, top) Total adult head extracts were treated with calf-intestinal phosphatase (PPase). Change in phosphorylation status of Tob was assessed as a change in mobility by Western blot analysis. (Right panel, bottom) Exogenously expressed Flag-tagged Tob protein is also phosphorylated in the adult brain. (D) The addition of the phosphatase inhibitor, calyculin (CY), dissociates the Orb2-Tob complex. S2 cells were transfected with Orb2A with and without Tob and treated with 10 µm CY for 1 h prior to Tob immunoprecipitation. CY treatment almost completely abolished Tob association with Orb2A oligomers and reduced association with the monomers. (E) Unphosphorylated Tob has a greater affinity for Orb2A. S2 cell lysates were first treated with the indicated units of phosphatase (uPPase), and subsequently the Orb2A-Tob complex was immunoprecipitated. More Orb2A was found to be associated with Tob following phosphatase treatment. Western blots of the lysates show the level of Orb2A and Tob (input). The 4%–12% gradient gels were used in these experiments. (F) Hyperphosphorylated Tob does not associate with Orb2. Untagged Orb2 and FLAG-tagged Tob complex was immunopurified using anti-Orb2 antibodies and probed with phosphor-tag™ (top panel). Phosphorylated proteins correspond to the size of Orb2 (bottom panel) but not Tob (middle panel). The hyperphosphorylated Tob proteins are visible in the total extract (input), however they are absent in the Orb2-Tob complex. The 8% gel in Tris-Glycine buffer was used in this experiment.

Mentions: Because Tob is constitutively present in the adult fly brain, we wondered how Tob-mediated oligomerization of Orb2 could be temporally regulated by neuronal activity. Phosphorylation is known to regulate the activity of both Btg/Tob [30]–[32] as well as the CPEB family members [33]–[35]. Consistent with these observations, protein phosphatase 1 (PP1-87B) and protein phosphatase 2A (PP2A) regulatory subunit twins were found in the Orb2 protein complex (Table S1), suggesting that Orb2 may also be regulated via phosphorylation and/or that Orb2 recruits these phosphatases to regulate phosphorylation of other proteins (such as Tob) in the complex. Blotting of Orb2 immunoprecipitates from the adult brain with phospho-tag™ [36], a biotin-tagged dinuclear metal complex that selectively binds to phospho-proteins, detected a small amount of phosphorylated monomeric Orb2B protein (Figure 4A). Similar to the fly brain, when expressed ectopically in S2 cells, both Orb2A and Orb2B are phosphorylated, albeit at very low levels (Figure 4B), suggesting Orb2 proteins are transiently phosphorylated in a regulated manner or kept primarily in an unphosphorylated state by the phosphatase. We observed that Tob is also phosphorylated in the adult fly brain (Figure 4C). To avoid a secondary consequence of prolonged inhibition or activation of phosphatases or kinases in the nervous system, we took advantage of the phosphorylation of Orb2 and Tob in S2 cells to determine the acute role of phosphorylation.


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)

Tob and Orb2 are phosphorylated, and phosphorylation regulates Tob-Orb2 association.(A) Orb2 is phosphorylated in the adult fly head. Blotting of Orb2 immunoprecipitate with phospho-tag™ detects phosphorylated Orb2B. Treatment with calf-intestinal phosphatase (PPase) that removes phosphate groups from proteins shows specificity of phospho-tag™. (B) Both Orb2A and Orb2B are phosphorylated at a low level in S2cells. Orb2 immunoprecipitate from the S2 cell is probed with phospho-tag™. (C) Tob is phosphorylated in the adult fly brain. (Left panel) Approximately 2 mg of total head extracts were immunoprecipitated with preimmune (pre) or immune Tob serum. The phospho-tag™ detects a band at the position of Tob only in the immune but not in preimmune lane. Treatment with λ-phosphatase (PPase) reduces phospho- tag™ signal. (Right panel, top) Total adult head extracts were treated with calf-intestinal phosphatase (PPase). Change in phosphorylation status of Tob was assessed as a change in mobility by Western blot analysis. (Right panel, bottom) Exogenously expressed Flag-tagged Tob protein is also phosphorylated in the adult brain. (D) The addition of the phosphatase inhibitor, calyculin (CY), dissociates the Orb2-Tob complex. S2 cells were transfected with Orb2A with and without Tob and treated with 10 µm CY for 1 h prior to Tob immunoprecipitation. CY treatment almost completely abolished Tob association with Orb2A oligomers and reduced association with the monomers. (E) Unphosphorylated Tob has a greater affinity for Orb2A. S2 cell lysates were first treated with the indicated units of phosphatase (uPPase), and subsequently the Orb2A-Tob complex was immunoprecipitated. More Orb2A was found to be associated with Tob following phosphatase treatment. Western blots of the lysates show the level of Orb2A and Tob (input). The 4%–12% gradient gels were used in these experiments. (F) Hyperphosphorylated Tob does not associate with Orb2. Untagged Orb2 and FLAG-tagged Tob complex was immunopurified using anti-Orb2 antibodies and probed with phosphor-tag™ (top panel). Phosphorylated proteins correspond to the size of Orb2 (bottom panel) but not Tob (middle panel). The hyperphosphorylated Tob proteins are visible in the total extract (input), however they are absent in the Orb2-Tob complex. The 8% gel in Tris-Glycine buffer was used in this experiment.
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Related In: Results  -  Collection

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

pbio-1001786-g004: Tob and Orb2 are phosphorylated, and phosphorylation regulates Tob-Orb2 association.(A) Orb2 is phosphorylated in the adult fly head. Blotting of Orb2 immunoprecipitate with phospho-tag™ detects phosphorylated Orb2B. Treatment with calf-intestinal phosphatase (PPase) that removes phosphate groups from proteins shows specificity of phospho-tag™. (B) Both Orb2A and Orb2B are phosphorylated at a low level in S2cells. Orb2 immunoprecipitate from the S2 cell is probed with phospho-tag™. (C) Tob is phosphorylated in the adult fly brain. (Left panel) Approximately 2 mg of total head extracts were immunoprecipitated with preimmune (pre) or immune Tob serum. The phospho-tag™ detects a band at the position of Tob only in the immune but not in preimmune lane. Treatment with λ-phosphatase (PPase) reduces phospho- tag™ signal. (Right panel, top) Total adult head extracts were treated with calf-intestinal phosphatase (PPase). Change in phosphorylation status of Tob was assessed as a change in mobility by Western blot analysis. (Right panel, bottom) Exogenously expressed Flag-tagged Tob protein is also phosphorylated in the adult brain. (D) The addition of the phosphatase inhibitor, calyculin (CY), dissociates the Orb2-Tob complex. S2 cells were transfected with Orb2A with and without Tob and treated with 10 µm CY for 1 h prior to Tob immunoprecipitation. CY treatment almost completely abolished Tob association with Orb2A oligomers and reduced association with the monomers. (E) Unphosphorylated Tob has a greater affinity for Orb2A. S2 cell lysates were first treated with the indicated units of phosphatase (uPPase), and subsequently the Orb2A-Tob complex was immunoprecipitated. More Orb2A was found to be associated with Tob following phosphatase treatment. Western blots of the lysates show the level of Orb2A and Tob (input). The 4%–12% gradient gels were used in these experiments. (F) Hyperphosphorylated Tob does not associate with Orb2. Untagged Orb2 and FLAG-tagged Tob complex was immunopurified using anti-Orb2 antibodies and probed with phosphor-tag™ (top panel). Phosphorylated proteins correspond to the size of Orb2 (bottom panel) but not Tob (middle panel). The hyperphosphorylated Tob proteins are visible in the total extract (input), however they are absent in the Orb2-Tob complex. The 8% gel in Tris-Glycine buffer was used in this experiment.
Mentions: Because Tob is constitutively present in the adult fly brain, we wondered how Tob-mediated oligomerization of Orb2 could be temporally regulated by neuronal activity. Phosphorylation is known to regulate the activity of both Btg/Tob [30]–[32] as well as the CPEB family members [33]–[35]. Consistent with these observations, protein phosphatase 1 (PP1-87B) and protein phosphatase 2A (PP2A) regulatory subunit twins were found in the Orb2 protein complex (Table S1), suggesting that Orb2 may also be regulated via phosphorylation and/or that Orb2 recruits these phosphatases to regulate phosphorylation of other proteins (such as Tob) in the complex. Blotting of Orb2 immunoprecipitates from the adult brain with phospho-tag™ [36], a biotin-tagged dinuclear metal complex that selectively binds to phospho-proteins, detected a small amount of phosphorylated monomeric Orb2B protein (Figure 4A). Similar to the fly brain, when expressed ectopically in S2 cells, both Orb2A and Orb2B are phosphorylated, albeit at very low levels (Figure 4B), suggesting Orb2 proteins are transiently phosphorylated in a regulated manner or kept primarily in an unphosphorylated state by the phosphatase. We observed that Tob is also phosphorylated in the adult fly brain (Figure 4C). To avoid a secondary consequence of prolonged inhibition or activation of phosphatases or kinases in the nervous system, we took advantage of the phosphorylation of Orb2 and Tob in S2 cells to determine the acute role of phosphorylation.

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.

Show MeSH
Related in: MedlinePlus