Modulation of the Enzymatic Properties of Protein Phosphatase 2A Catalytic Subunit by the Recombinant 65-kDa Regulatory Subunit PR65alpha

Existing evidence implicates regulatory roles for protein phosphatase 2A (PP2A) in a variety of cellular functions, including cytoskeletal remodeling, hormone secretion, and apoptosis. We report here activation of PP2A in normal rat islets and insulin-secreting INS-1 832/13 cells under the duress of hyperglycemic (HG) conditions. Small interfering RNA-mediated knockdown of the catalytic subunit of PP2A (PP2Ac) markedly attenuated glucose-induced activation of PP2A. HG, but not nonmetabolizable 3-O-methyl glucose or mannitol (osmotic control), significantly stimulated the methylation of PP2Ac at its C-terminal Leu-309, suggesting a novel role for this posttranslational modification in glucose-induced activation of PP2A. Moreover, knockdown of the cytosolic leucine carboxymethyl transferase 1 (LCMT1), which carboxymethylates PP2Ac, significantly attenuated PP2A activation under HG conditions. In addition, HG conditions, but not 3-O-methyl glucose or mannitol, markedly increased the expression of LCMT1. Furthermore, HG conditions significantly increased the expression of B55α, a regulatory subunit of PP2A, which has been implicated in islet dysfunction under conditions of oxidative stress and diabetes. Thapsigargin, a known inducer of endoplasmic reticulum stress, failed to exert any discernible effects on the carboxymethylation of PP2Ac, expression of LCMT1 and B55α, or PP2A activity, suggesting no clear role for endoplasmic reticulum stress in HG-induced activation of PP2A. Based on these findings, we conclude that exposure of the islet β-cell to HG leads to accelerated PP2A signaling pathway, leading to loss in glucose-induced insulin secretion.

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Molecular cloning and developmental expression of the catalytic and 65-kDa regulatory subunits of protein phosphatase 2A in Drosophila.

Molecular Biology of the Cell ◽

10.1091/mbc.3.3.287 ◽

1992 ◽

Vol 3 (3) ◽

pp. 287-298 ◽

Cited By ~ 35

Author(s):

R E Mayer-Jaekel ◽

S Baumgartner ◽

G Bilbe ◽

H Ohkura ◽

D M Glover ◽

...

Keyword(s):

Protein Phosphatase ◽

Developmental Stages ◽

Protein Phosphatase 2A ◽

Catalytic Subunit ◽

Developmental Expression ◽

Regulatory Subunit ◽

Predicted Amino Acid Sequence ◽

Cdna Clones ◽

Transcript Levels ◽

Phosphatase 2A

cDNA clones encoding the catalytic subunit and the 65-kDa regulatory subunit of protein phosphatase 2A (PR65) from Drosophila melanogaster have been isolated by homology screening with the corresponding human cDNAs. The Drosophila clones were used to analyze the spatial and temporal expression of the transcripts encoding these two proteins. The Drosophila PR65 cDNA clones contained an open reading frame of 1773 nucleotides encoding a protein of 65.5 kDa. The predicted amino acid sequence showed 75 and 71% identity to the human PR65 alpha and beta isoforms, respectively. As previously reported for the mammalian PR65 isoforms, Drosophila PR65 is composed of 15 imperfect repeating units of approximately 39 amino acids. The residues contributing to this repeat structure show also the highest sequence conservation between species, indicating a functional importance for these repeats. The gene encoding Drosophila PR65 was located at 29B1,2 on the second chromosome. A major transcript of 2.8 kilobase (kb) encoding the PR65 subunit and two transcripts of 1.6 and 2.5 kb encoding the catalytic subunit could be detected throughout Drosophila development. All of these mRNAs were most abundant during early embryogenesis and were expressed at lower levels in larvae and adult flies. In situ hybridization of different developmental stages showed a colocalization of the PR65 and catalytic subunit transcripts. The mRNA expression is high in the nurse cells and oocytes, consistent with a high equally distributed expression in early embryos. In later embryonal development, the expression remains high in the nervous system and the gonads but the overall transcript levels decrease. In third instar larvae, high levels of mRNA could be observed in brain, imaginal discs, and in salivary glands. These results indicate that protein phosphatase 2A transcript levels change during development in a tissue and in a time-specific manner.

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The Role of Protein Phosphatase 2A Catalytic Subunit Cα in Embryogenesis: Evidence from Sequence Analysis and Localization Studies

Biological Chemistry ◽

10.1515/bc.1999.139 ◽

1999 ◽

Vol 380 (9) ◽

pp. 1117-1120 ◽

Cited By ~ 12

Author(s):

Jürgen Götz ◽

Wilfried Kues

Keyword(s):

Protein Phosphatase ◽

Genomic Organization ◽

Protein Phosphatase 2A ◽

Catalytic Subunit ◽

Regulatory Subunit ◽

Catalytic Subunits ◽

Regulatory Subunits ◽

Phosphatase 2A ◽

The Brain

AbstractProtein phosphatase 2A (PP2A) constitutes one of the major families of protein serine/threonine phosphatases found in all eukaryotic cells. PP2A holoenzymes are composed of a catalytic subunit complexed with a structural regulatory subunit of 65 kDa. These core subunits associate with regulatory subunits of various sizes to form different heterotrimers which have been purified and evaluated with regard to substrate specificity. In fully differentiated tissues PP2A expression levels are highest in the brain, however, relatively little is known about expression in the developing embryo.In order to determine the composition of PP2A catalytic subunits in the mouse, cDNAs were cloned and the genomic organization of PP2A Cα was determined.By a gene targeting approach in the mouse, we have previously shown that the absence of the major catalytic subunit of PP2A, Cα, resulted in embryonic lethality around embryonic day E6.5. No mesoderm was formed which implied that PP2A plays a crucial role in gastrulation.Here, we extended our studies and analyzed wildtype embryos for Cα expression at subsequent stages of development. After gastrulation is completed, we find high expression of Cα restricted to the neural folds, which suggests that PP2A plays an additional pivotal role in neurulation.

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Developmental Expression of Protein Phosphatase 2A in the Kidney

Journal of the American Society of Nephrology ◽

10.1681/asn.v1081737 ◽

1999 ◽

Vol 10 (8) ◽

pp. 1737-1745

Author(s):

ALLEN D. EVERETT ◽

CHUN XUE ◽

TAMARA STOOPS

Keyword(s):

Signal Transduction ◽

Protein Phosphatase ◽

Kidney Development ◽

Developmental Regulation ◽

Protein Phosphatase 2A ◽

Catalytic Subunit ◽

Developmental Expression ◽

Regulatory Subunit ◽

Northern Analysis ◽

Phosphatase 2A

Abstract. Although a number of growth and transcription factors are known to regulate renal growth and development, the signal transduction molecules necessary to mediate these developmental signals are relatively unknown. Therefore, the activity and mRNA and protein expression of the signal transduction molecule protein phosphatase 2A (PP2A) were examined during rat kidney development. Northern analysis of total kidney RNA or Western analysis of kidney protein homogenates from embryonic day 15 to 90-d-old adults demonstrated developmental regulation of the catalytic, major 55-kD B regulatory subunit and A structural subunit with the highest levels of expression in late embryonic and newborn kidneys. Similarly, okadaic acid-inhibitable phosphatase enzyme activity was highest in the embryonic and newborn kidney. To map cell-specific expression of PP2A in the developing kidney, in situ hybridization with a catalytic subunit digoxigenin-labeled cRNA was performed on embryonic day 20 and newborn kidneys. PP2A was found predominately in the nephrogenic cortex and particularly in the developing glomeruli and nonbrush border tubules in the embryonic day 20 and newborn kidneys. Similarly, immunocytochemistry with a specific PP2A catalytic subunit polyclonal anti-peptide antibody demonstrated catalytic subunit protein particularly concentrated in the podocytes of glomeruli in the newborn kidney. In the adult kidney, PP2A protein was no longer detectable except in the nuclei of distal tubular cells. Therefore, the developmental regulation of PP2A activity and protein during kidney development and its mapping to the nephrogenic cortex, developing glomeruli, and tubules suggests a role for PP2A in the regulation of nephron growth and differentiation.

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A Newborn with Severe Ventriculomegaly: Expanding the PPP2R1A Gene Mutation Phenotype

Journal of Pediatric Genetics ◽

10.1055/s-0039-1692414 ◽

2019 ◽

Vol 08 (04) ◽

pp. 240-243

Author(s):

Alexandra Wallace ◽

Paul Caruso ◽

Amel Karaa

Keyword(s):

Corpus Callosum ◽

Gene Mutation ◽

Protein Phosphatase ◽

Protein Phosphatase 2A ◽

Catalytic Subunit ◽

Regulatory Subunit ◽

Pontocerebellar Hypoplasia ◽

Genetic Condition ◽

Diverse Range ◽

Phosphatase 2A

AbstractProtein phosphatase 2A (PP2A) is a heterotrimeric protein serine/threonine phosphatase that regulates a diverse range of cellular activities. The PPP2R1A gene on chromosome 19 (19q13.41) encodes the α isoform of the scaffolding subunit of the PP2A holoenzyme, which functions to link the catalytic subunit to the regulatory subunit. Here we present a case of a newborn boy with a novel PPP2R1A gene mutation (c.548G>A; p.Arg183Gln) with severe lateral and third ventriculomegaly, hypoplastic corpus callosum, and pontocerebellar hypoplasia. To our knowledge, this is the sixth case reported in the literature, thus expanding the phenotype of this rare genetic condition.

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