A Functional Genomics Analysis of the B56 Isoforms of Drosophila Protein Phosphatase 2A

Liu, Wei; Silverstein, Adam M.; Shu, Hongjun; Martinez, Bobbie; Mumby, Marc C.

doi:10.1074/mcp.m600272-mcp200

Cited by 13 publications

(22 citation statements)

References 61 publications

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“…Complimentary DNA clones corresponding to Drosophila PP2A subunits and PP4 have been described [31,32]. A plasmid containing the full-length sequence of Drosophila PP6 (SD01279) was obtained from the Drosophila Genomics Resource Center.…”

Section: Production Of Double-stranded Rnamentioning

confidence: 99%

“…A full-length Drosophila Tap42 (dTap42/CG31852) cDNA was generated by PCR from a 4-8 hour Drosophila embryo cDNA library (a gift of Dr. Gregory Tall) using the following primers; 5'-ATGGCTGAGGGTAATACCGCTGG-3' (sense strand) and 5'-GACGGCAACCGTCATAACCGTAGTTAA-3' (anti-sense strand). Double-stranded RNA was produced from PCR fragments generated from these templates as described [31,32]. The nucleotide sequences of primers used for individual PCR reactions are listed in Table 1.…”

Section: Production Of Double-stranded Rnamentioning

confidence: 99%

“…Samples were heated for 3 minutes at 95°C, vortexed for 30 seconds to shear genomic DNA, centrifuged for 10 minutes at 14,000 × g, and stored at −20°C. In some experiments, the dsRNA treatments and cell incubations were conducted in the presence of the caspase inhibitor Z-VAD-fmk (30 µM) to inhibit apoptosis as described [32].…”

Section: Cell Treatment and Lysismentioning

confidence: 99%

“…Total RNA was isolated using the Tripure isolation reagent (Roche) followed by RT-PCR with subunit-specific primers using the Superscript One-Step RT-PCR kit (Invitrogen) as described [38]. The integrity of the RNA used was verified in PCR reactions using primers for the stubarista gene (Table 1) whose mRNA levels are not affected by knockdown of PP2A subunits [32]. Knockdown of Drosophila Tap42 was confirmed using quantitative real-time PCR.…”

Section: Rt-pcr and Real Time Quantitative Pcrmentioning

confidence: 99%

“…The mammalian PP4 catalytic subunit also interacts with scaffold and regulatory subunits [27,28], while PP6 regulatory proteins have been identified in yeast [29]. Due to decreased complexity relative to mammalian cells, Drosophila has been a valuable system to study functions of individual catalytic and regulatory subunits within the PP2A subfamily [30][31][32]. The Drosophila genome contains homologs of the PP2A catalytic, scaffold, and regulatory subunits [30,31], as well as homologs of PP4 [33], PP6 [34], and Tap42 [18].…”

Section: Introductionmentioning

confidence: 99%

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Functional analysis of the PP2A subfamily of protein phosphatases in regulating Drosophila S6 kinase

Bielinski¹,

Mumby²

2007

Experimental Cell Research

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Phosphorylation and activation of ribosomal S6 protein kinase is an important link in the regulation of cell size by the target of rapamycin (TOR) protein kinase. A combination of selective inhibition and RNA interference were used to test the roles of members of the PP2A subfamily of protein phosphatases in dephosphorylation of Drosophila S6 kinase (dS6K). Treatment of Drosophila Schneider 2 cells with calyculin A, a selective inhibitor of PP2A-like phosphatases, resulted in a 7-fold increase in the basal level of dS6K phosphorylation at the TOR phosphorylation site (Thr398) and blocked dephosphorylation following inactivation of TOR by amino acid starvation or rapamycin treatment. Knockdown of the PP2A catalytic subunit increased basal dS6K phosphorylation and inhibited dephosphorylation induced by amino acid withdrawal. In contrast, depletion of the catalytic subunits of the other two members of the subfamily did not enhance dS6K phosphorylation. Knockdown of PP4 caused a 20% decrease in dS6K phosphorylation and knockdown of PP6 had no effect. Knockdown of the Drosophila B56-2 subunit resulted in enhanced dephosphorylation of dS6K following removal of amino acids. In contrast, knockdown of the homologs of the other PP2A regulatory subunits had no effects. Knockdown of the Drosophila homolog of the PP2A/PP4/PP6 interaction protein α4/Tap42 did not affect S6K phosphorylation, but did induce apoptosis. These results indicate that PP2A, but not other members of this subfamily, is likely to be a major S6K phosphatase in intact cells and is consistent with an important role for this phosphatase in the TOR pathway.

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Section: Production Of Double-stranded Rnamentioning

confidence: 99%

Section: Production Of Double-stranded Rnamentioning

confidence: 99%

Section: Cell Treatment and Lysismentioning

confidence: 99%

Section: Rt-pcr and Real Time Quantitative Pcrmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Functional analysis of the PP2A subfamily of protein phosphatases in regulating Drosophila S6 kinase

Bielinski¹,

Mumby²

2007

Experimental Cell Research

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AccERK2, A MAP KINASE GENE FROM Apis cerana cerana, PLAYS ROLES IN STRESS RESPONSES, DEVELOPMENTAL PROCESSES, AND THE NERVOUS SYSTEM

Zhang

Kang

et al. 2012

Arch Insect Biochem Physiol

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Extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinase (MAPK), plays roles in a variety of cellular responses. However, limited information is available on the relationship between ERKs and environmental stresses. In this report, an ERK gene, AccERK2, was cloned and characterized from Apis cerana cerana. Polypeptide sequence alignment revealed that the single-copied AccERK2 shares high identity with other known ERKs and contains the typical conserved Thr-Glu-Tyr (TEY) motif in its activation loop. Genomic sequence analysis revealed that the seven exons of AccERK2 are interrupted by six introns, and the seventh intron is located in the 3' untranslated region. Semi-quantitative reverse transcription (RT-PCR) indicated that AccERK2 was expressed at higher levels in the larval and pupal stages than in the adult stage. AccERK2 was also most highly expressed in the brain. The expression of AccERK2 was induced by abiotic stresses, including heat, ion irradiation, oxidative stress, and heavy metal ions. Based on these results, it appears that AccERK2 in A. cerana cerana participates in developmental processes, the nervous system, and responses to environmental stressors.

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Proteomics and phosphoproteomics for the mapping of cellular signalling networks

et al. 2008

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Proteomics is transitioning from inventory mapping to the mapping of functional cellular contexts. This has been enabled by progress in technologies as well as conceptual strategies. Here, we review recent advances in this area with focus on cellular signalling pathways. We discuss genetics-based methods such as yeast two hybrid methods as well as biochemistry-based methods such as two-dimensional gel electrophoresis, quantitative proteomics, interaction proteomics, and phosphoproteomics. A central tenet is that by its ability to capture dynamic changes in protein expression, localisation and modification modern proteomics has become a powerful tool to map signal transduction pathways and deliver the functional information that will promote insights in cell biology and systems biology.

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A Functional Genomics Analysis of the B56 Isoforms of Drosophila Protein Phosphatase 2A

Cited by 13 publications

References 61 publications

Functional analysis of the PP2A subfamily of protein phosphatases in regulating Drosophila S6 kinase

Functional analysis of the PP2A subfamily of protein phosphatases in regulating Drosophila S6 kinase

AccERK2, A MAP KINASE GENE FROM Apis cerana cerana, PLAYS ROLES IN STRESS RESPONSES, DEVELOPMENTAL PROCESSES, AND THE NERVOUS SYSTEM

Proteomics and phosphoproteomics for the mapping of cellular signalling networks

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