Deregulation of translational control of the 65-kDa regulatory subunit (PR65 alpha) of protein phosphatase 2A leads to multinucleated cells.

Efficient translation of the mRNA encoding the 65-kDa regulatory subunit (PR65 alpha) of protein phosphatase 2A (PP2A) is prevented by an out of frame upstream AUG and a stable stem-loop structure (delta G = -55.9 kcal/mol) in the 5'-untranslated region (5'-UTR). Deletion of the 5'-UTR allows efficient translation of the PR65 alpha message in vitro and overexpression in COS-1 cells. Insertion of the 5'-UTR into the beta-galactosidase leader sequence dramatically inhibits translation of the beta-galactosidase message in vitro and in vivo, confirming that this sequence functions as a potent translation regulatory sequence. Cells transfected or microinjected with a PR65 alpha expression vector lacking the 5'-UTR, express high levels of PR65 alpha, accumulating in both nucleus and cytoplasm. PR65 alpha overexpressing rat embryo fibroblasts (REF-52 cells) become multinucleated. These data and previous results (Mayer-Jaekel, R. E., Ohkura, H., Gomes, R., Sunkel, C. E., Baumgartner, S., Hemmings, B. A., and Glover, D. M. (1993) Cell 72, 621-633) suggest that PP2A participates in the regulation of both mitosis and cytokinesis.

Role of protein phosphatase 2A in Drosophila development.

The molecular cloning of protein phosphatase 2A subunits from Drosophila has provided insights into the role this enzyme plays in developmental processes and in cell cycle regulation. The trimeric holoenzyme containing the catalytic, 65-kDa and 55-kDa regulatory subunits appears to be preferentially expressed in proliferative organs such as the gonads, in the developing nervous system and in early syncytial embryos. Analysis of mutant flies affected in the expression of the 55-kDa regulatory subunit suggests that the holoenzyme containing this subunit plays a pivotal role in cell cycle regulation and cell fate determination. The severity of the mutant phenotype correlates with a decrease in 55-kDa subunit protein levels and reduced protein phosphatase activity towards p34cdc2 phosphorylated proteins. The data support the idea that the 'variable' subunits of protein phosphatase 2A holoenzymes play a critical role in directing substrate specificity.

Drosophila mutants in the 55 kDa regulatory subunit of protein phosphatase 2A show strongly reduced ability to dephosphorylate substrates of p34cdc2.

The 55 kDa regulatory subunit of Drosophila protein phosphatase 2A is located in the cytoplasm at all cell cycle stages, by the criterion of immunofluorescence. We are unable to detect significant change in protein phosphatase activity during the nuclear division cycle of syncytial embryos. However, cell cycle function of the enzyme is suggested by the mitotic defects exhibited by two Drosophila mutants, aar1 and twinsP, defective in the gene encoding the 55 kDa subunit. The reduced levels of the 55 kDa subunit correlate with the loss of protein phosphatase 2A-like, okadaic acid-sensitive phosphatase activity of brain extracts against caldesmon and histone H1 phosphorylated by p34cdc2/cyclin B kinase, but not against phosphorylase a. Thus the mitotic defects of aar1 and twinsP are likely to result from the lack of dephosphorylation of specific substrates by protein phosphatase 2A.

Protein phosphatase 2A--a 'ménage à trois'.

Protein phosphorylation is probably the major regulatory mechanism employed by eukaryotic cells. Much work has been devoted to the role of protein kinases and their modulation by hormones, growth factors and neurotransmitters. It is now appreciated that protein phosphatases are also key players in actively regulating many cellular processes. In this article we discuss recent advances in our understanding of the function of protein phosphatase 2A, one of the major serine/threonine-specific protein phosphatases.

Analysis of subunit isoforms in protein phosphatase 2A holoenzymes from rabbit and Xenopus.

A dimeric and two trimeric forms of protein phosphatase 2A (PP2A) were purified from rabbit and Xenopus tissues and analyzed using antisera specific for the catalytic and regulatory subunits. The dimeric holoenzyme consists of a complex between a 36-kDa catalytic subunit associated with a approximately 65-kDa regulatory subunit. The two trimeric holoenzymes consist of the catalytic subunit complexed with 65- and 55-kDa subunits, or 65- and 72-kDa subunits. Antisera were raised against synthetic peptides specific for the alpha- and beta-isoforms of the 65-kDa (PR65 alpha/beta) and 55-kDa (PR55 alpha/beta) subunits identified by molecular cloning. Anti-peptide antisera to the 36-kDa catalytic subunit of PP2A were prepared against two selected regions: one specific for the alpha-isoform and one to a peptide common to both the alpha- and beta-isoforms. Immunochemical analysis of all three mammalian holoenzymes showed that the catalytic, 55- and 65-kDa subunits are both predominantly of the alpha-isoform, which is consistent with the peptide sequence data. The 65-kDa subunit of PP2A holoenzymes isolated from Xenopus skeletal muscle reacted with both anti-alpha and anti-beta PR65-specific antisera whereas the PP2A holoenzymes isolated from Xenopus oocytes reacted preferentially with the beta-specific antisera, indicating developmental changes in the expression of the 65-kDa subunit isoform. Taken together, these results show that the "core" subunits of the PP2A holoenzymes consist of the catalytic complexed with the 65-kDa subunit and that the association of the third subunit does not appear to be influenced by the isoform of these two core subunits.

The 55 kd regulatory subunit of Drosophila protein phosphatase 2A is required for anaphase.

The gene encoding the Drosophila protein phosphatase 2A 55 kd regulatory subunit (PR55) is located at 85F and directs the synthesis of differentially spliced transcripts. Maternal RNAs are present at very high levels in early embryos and decline around cellularization. Zygotic transcripts are present mainly in the developing embryonic nervous system and gonads. Transcripts are uniformly distributed in third instar larval discs and testes and at lower levels in the proliferative centers of the brain. Mutations in abnormal anaphase resolution (aar) are rescued by the wild-type gene for PR55. aar mutants display intact lagging chromatids that have undergone separation from their sisters, but that remain at the position formerly occupied by the metaphase plate, as well as anaphase figures that show bridging chromatin having two centromeric regions.

Localization of the genes encoding the catalytic subunits of protein phosphatase 2A to human chromosome bands 5q23-->q31 and 8p12-->p11.2, respectively.

The gene for the alpha isoform of the catalytic subunit of human protein phosphatase 2A (PPP2CA) was localized to chromosome 5 using somatic cell hybrids, and then more finely mapped to chromosome region 5q23-->q31 by in situ hybridization using a tritiated cDNA probe. The gene for the beta isoform of the catalytic subunit of this enzyme (PPP2CB) was mapped by the polymerase chain reaction to human chromosome 8 using somatic cell hybrids. Fluorescence in situ hybridization was then used to localize the PPP2CB gene to 8p12-->p11.2, using a mixture of three genomic probes that ranged from 3.5 to 8 kb in size. Finally, Southern blot analysis of somatic cell hybrid DNA suggested that a PPP2CB catalytic subunit pseudogene (PPP2CBP) is on chromosome 16.

The 55 kDa regulatory subunit of protein phosphatase 2A plays a role in the activation of the HPV16 long control region in human cells with a deletion in the short arm of chromosome 11.

Previous results indicated that SV40 small t is essential for SV40-induced transformation of diploid cells but dispensable for the transformation of cells with a deletion on the short arm of chromosome 11 (del-11 cells). From these results we concluded that del-11 cells contain a cellular 'SV40 small t-like' factor, which is able to transactivate the HPV16 long control region (LCR) and to complement SV40 large T in transformation. Since SV40 small t and the regulatory 55 kDa subunit (PR55) of protein phosphatase 2A (PP2A), have been shown to inhibit the enzyme activity of PP2A, the PR55 beta subunit could be the putative 'small t-like' factor. In accordance with this hypothesis, we show that the PR55 beta subunit is highly expressed in del-11 but not in diploid cells and is able to trans-activate the HPV16 LCR in diploid cells. Moreover, inhibition of PP2A by okadaic acid resulted in trans-activation of the HPV16 LCR in diploid cells. Alignment of PR55 and SV40 small t showed a common four amino acid motif DKGG. We present evidence that the integrity of this motif is necessary for the PP2A-mediated ability of SV40 small t to trans-activate the HPV16 LCR.

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

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.