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Essential roles of the Tap42-regulated protein phosphatase 2A (PP2A) family in wing imaginal disc development of Drosophila melanogaster.

Wang N, Leung HT, Mazalouskas MD, Watkins GR, Gomez RJ, Wadzinski BE - PLoS ONE (2012)

Bottom Line: RNAi-mediated silencing of Tap42 using the binary Gal4/UAS system and two disc drivers, pnr- and ap-Gal4, not only decreased survival rates but also hampered the development of wing discs, resulting in a remarkable thorax cleft and defective wings in adults.The Tap42(RNAi)-induced defects were the direct result of loss of regulation of Drosophila PP2A family members (MTS, PP4, and PPV), as enforced expression of wild type Tap42, but not a phosphatase binding defective Tap42 mutant, rescued fly survivorship and defects.The experimental platform described herein identifies crucial roles for Tap42•phosphatase complexes in governing imaginal disc and fly development.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.

ABSTRACT
Protein ser/thr phosphatase 2A family members (PP2A, PP4, and PP6) are implicated in the control of numerous biological processes, but our understanding of the in vivo function and regulation of these enzymes is limited. In this study, we investigated the role of Tap42, a common regulatory subunit for all three PP2A family members, in the development of Drosophila melanogaster wing imaginal discs. RNAi-mediated silencing of Tap42 using the binary Gal4/UAS system and two disc drivers, pnr- and ap-Gal4, not only decreased survival rates but also hampered the development of wing discs, resulting in a remarkable thorax cleft and defective wings in adults. Silencing of Tap42 also altered multiple signaling pathways (HH, JNK and DPP) and triggered apoptosis in wing imaginal discs. The Tap42(RNAi)-induced defects were the direct result of loss of regulation of Drosophila PP2A family members (MTS, PP4, and PPV), as enforced expression of wild type Tap42, but not a phosphatase binding defective Tap42 mutant, rescued fly survivorship and defects. The experimental platform described herein identifies crucial roles for Tap42•phosphatase complexes in governing imaginal disc and fly development.

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Tap42 interacts with all three PP2A members and is required for normal wing disc development.Panel A: FLAG immunoprecipitations (FLAG IPs) were performed from extracts of Drosophila S2 cells expressing HA3-Mts, HA3-PP4, or HA3-PPV alone or together with wildtype (FLAG3-Tap42WT) or mutant Tap42 (FLAG3-Tap42ED). The FLAG immune complexes and corresponding cell extracts (lysates) were analyzed by Western blotting using the indicated epitope tag antibodies. Panel B: Adult flies expressing Tap42RNAi in the ap domain displayed a marked thorax cleft (red arrow, B1) and shriveled wings (B5). Expression of Tap42WT in this background completely rescued both thorax (B2) and wing defects (B6). However, introduction of the Tap42ED mutant in this background failed to rescue the defects and the flies lacked the scutum (B3) and formed blistered wings (B7). Expression of Tap42ED alone resulted in a mild defect around the scutum (B4) and the formation of a forked wing vein (B8). Genotypes: (B1 & B5) ap-Gal4/UAS-Tap42RNAi; +/+. (B2 & B6) ap-Gal4/UAS-Tap42RNAi; +/UAS-Tap42WT. (B3 & B7) ap-Gal4/UAS-Tap42RNAi; +/UAS-Tap42ED. (A4 & B4) ap-Gal4/+; +/UAS-Tap42ED.
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pone-0038569-g006: Tap42 interacts with all three PP2A members and is required for normal wing disc development.Panel A: FLAG immunoprecipitations (FLAG IPs) were performed from extracts of Drosophila S2 cells expressing HA3-Mts, HA3-PP4, or HA3-PPV alone or together with wildtype (FLAG3-Tap42WT) or mutant Tap42 (FLAG3-Tap42ED). The FLAG immune complexes and corresponding cell extracts (lysates) were analyzed by Western blotting using the indicated epitope tag antibodies. Panel B: Adult flies expressing Tap42RNAi in the ap domain displayed a marked thorax cleft (red arrow, B1) and shriveled wings (B5). Expression of Tap42WT in this background completely rescued both thorax (B2) and wing defects (B6). However, introduction of the Tap42ED mutant in this background failed to rescue the defects and the flies lacked the scutum (B3) and formed blistered wings (B7). Expression of Tap42ED alone resulted in a mild defect around the scutum (B4) and the formation of a forked wing vein (B8). Genotypes: (B1 & B5) ap-Gal4/UAS-Tap42RNAi; +/+. (B2 & B6) ap-Gal4/UAS-Tap42RNAi; +/UAS-Tap42WT. (B3 & B7) ap-Gal4/UAS-Tap42RNAi; +/UAS-Tap42ED. (A4 & B4) ap-Gal4/+; +/UAS-Tap42ED.

Mentions: Our analysis of ap-Gal4>UAS-Tap42RNAi; mtsXE2258 flies implicates a crucial role for Tap42 and Mts in normal fly development; however, Drosophila PP4 and PP6 (PPV) may also be involved in this process as the mammalian homolog of Tap42, α4, interacts with all three PP2A family members [4], [5]. To test if Tap42 interacts with Mts, PP4, and PPV, we performed FLAG immunoprecipitations from lysates of Drosophila S2 cells expressing the HA3-tagged phosphatase alone or together with FLAG3-Tap42WT. Western analysis of the immune complexes revealed that Tap42 interacts with all three Drosophila phosphatase catalytic subunits (Fig. 6-A). Since prior studies have identified a double point mutant of α4 that lacks the PP2Ac binding determinants [10], [42], we mutated the corresponding residues in Tap42, R152E152 and K155D155, and monitored the ability of this mutant (Tap42ED) to interact with Mts, PP4, and PPV. In contrast to wild type Tap42 (Tap42WT), Tap42ED failed to interact with the Drosophila phosphatases (Fig. 6-A).


Essential roles of the Tap42-regulated protein phosphatase 2A (PP2A) family in wing imaginal disc development of Drosophila melanogaster.

Wang N, Leung HT, Mazalouskas MD, Watkins GR, Gomez RJ, Wadzinski BE - PLoS ONE (2012)

Tap42 interacts with all three PP2A members and is required for normal wing disc development.Panel A: FLAG immunoprecipitations (FLAG IPs) were performed from extracts of Drosophila S2 cells expressing HA3-Mts, HA3-PP4, or HA3-PPV alone or together with wildtype (FLAG3-Tap42WT) or mutant Tap42 (FLAG3-Tap42ED). The FLAG immune complexes and corresponding cell extracts (lysates) were analyzed by Western blotting using the indicated epitope tag antibodies. Panel B: Adult flies expressing Tap42RNAi in the ap domain displayed a marked thorax cleft (red arrow, B1) and shriveled wings (B5). Expression of Tap42WT in this background completely rescued both thorax (B2) and wing defects (B6). However, introduction of the Tap42ED mutant in this background failed to rescue the defects and the flies lacked the scutum (B3) and formed blistered wings (B7). Expression of Tap42ED alone resulted in a mild defect around the scutum (B4) and the formation of a forked wing vein (B8). Genotypes: (B1 & B5) ap-Gal4/UAS-Tap42RNAi; +/+. (B2 & B6) ap-Gal4/UAS-Tap42RNAi; +/UAS-Tap42WT. (B3 & B7) ap-Gal4/UAS-Tap42RNAi; +/UAS-Tap42ED. (A4 & B4) ap-Gal4/+; +/UAS-Tap42ED.
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pone-0038569-g006: Tap42 interacts with all three PP2A members and is required for normal wing disc development.Panel A: FLAG immunoprecipitations (FLAG IPs) were performed from extracts of Drosophila S2 cells expressing HA3-Mts, HA3-PP4, or HA3-PPV alone or together with wildtype (FLAG3-Tap42WT) or mutant Tap42 (FLAG3-Tap42ED). The FLAG immune complexes and corresponding cell extracts (lysates) were analyzed by Western blotting using the indicated epitope tag antibodies. Panel B: Adult flies expressing Tap42RNAi in the ap domain displayed a marked thorax cleft (red arrow, B1) and shriveled wings (B5). Expression of Tap42WT in this background completely rescued both thorax (B2) and wing defects (B6). However, introduction of the Tap42ED mutant in this background failed to rescue the defects and the flies lacked the scutum (B3) and formed blistered wings (B7). Expression of Tap42ED alone resulted in a mild defect around the scutum (B4) and the formation of a forked wing vein (B8). Genotypes: (B1 & B5) ap-Gal4/UAS-Tap42RNAi; +/+. (B2 & B6) ap-Gal4/UAS-Tap42RNAi; +/UAS-Tap42WT. (B3 & B7) ap-Gal4/UAS-Tap42RNAi; +/UAS-Tap42ED. (A4 & B4) ap-Gal4/+; +/UAS-Tap42ED.
Mentions: Our analysis of ap-Gal4>UAS-Tap42RNAi; mtsXE2258 flies implicates a crucial role for Tap42 and Mts in normal fly development; however, Drosophila PP4 and PP6 (PPV) may also be involved in this process as the mammalian homolog of Tap42, α4, interacts with all three PP2A family members [4], [5]. To test if Tap42 interacts with Mts, PP4, and PPV, we performed FLAG immunoprecipitations from lysates of Drosophila S2 cells expressing the HA3-tagged phosphatase alone or together with FLAG3-Tap42WT. Western analysis of the immune complexes revealed that Tap42 interacts with all three Drosophila phosphatase catalytic subunits (Fig. 6-A). Since prior studies have identified a double point mutant of α4 that lacks the PP2Ac binding determinants [10], [42], we mutated the corresponding residues in Tap42, R152E152 and K155D155, and monitored the ability of this mutant (Tap42ED) to interact with Mts, PP4, and PPV. In contrast to wild type Tap42 (Tap42WT), Tap42ED failed to interact with the Drosophila phosphatases (Fig. 6-A).

Bottom Line: RNAi-mediated silencing of Tap42 using the binary Gal4/UAS system and two disc drivers, pnr- and ap-Gal4, not only decreased survival rates but also hampered the development of wing discs, resulting in a remarkable thorax cleft and defective wings in adults.The Tap42(RNAi)-induced defects were the direct result of loss of regulation of Drosophila PP2A family members (MTS, PP4, and PPV), as enforced expression of wild type Tap42, but not a phosphatase binding defective Tap42 mutant, rescued fly survivorship and defects.The experimental platform described herein identifies crucial roles for Tap42•phosphatase complexes in governing imaginal disc and fly development.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.

ABSTRACT
Protein ser/thr phosphatase 2A family members (PP2A, PP4, and PP6) are implicated in the control of numerous biological processes, but our understanding of the in vivo function and regulation of these enzymes is limited. In this study, we investigated the role of Tap42, a common regulatory subunit for all three PP2A family members, in the development of Drosophila melanogaster wing imaginal discs. RNAi-mediated silencing of Tap42 using the binary Gal4/UAS system and two disc drivers, pnr- and ap-Gal4, not only decreased survival rates but also hampered the development of wing discs, resulting in a remarkable thorax cleft and defective wings in adults. Silencing of Tap42 also altered multiple signaling pathways (HH, JNK and DPP) and triggered apoptosis in wing imaginal discs. The Tap42(RNAi)-induced defects were the direct result of loss of regulation of Drosophila PP2A family members (MTS, PP4, and PPV), as enforced expression of wild type Tap42, but not a phosphatase binding defective Tap42 mutant, rescued fly survivorship and defects. The experimental platform described herein identifies crucial roles for Tap42•phosphatase complexes in governing imaginal disc and fly development.

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