Developmental expression of protein phosphatase 2A in the kidney.
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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 non-brush 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. (+info)
Leptin receptor activation of SH2 domain containing protein tyrosine phosphatase 2 modulates Ob receptor signal transduction.
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Leptin exerts its weight-reducing effects by binding to its receptor and activating signal transduction in hypothalamic neurons and other cell types. To identify the components of the leptin signal transduction pathway, an approach was developed in which bacterially expressed phosphorylated fragments of Ob receptor b (Ob-Rb) were used as affinity agents. Leptin binding to the Ob-Rb form of the leptin receptor leads to tyrosyl phosphorylation of the cytoplasmic domain of its receptor. Two of the three cytoplasmic tyrosines of Ob-Rb, at positions 985 and 1138, are phosphorylated after leptin treatment. Affinity chromatography using a tyrosine-phosphorylated fragment spanning Tyr 985 of Ob-Rb was used to identify proteins that bind to this site. The SH2 domain containing protein tyrosine phosphatase 2 (SHP-2) was isolated from bovine and mouse hypothalamus by using this method. After cotransfection of Ob-Rb, Janus kinase 2 (JAK2), and SHP-2 into 293T cells, leptin results in direct binding of SHP-2 to the phosphorylated Tyr 985. The bound SHP-2 is itself tyrosine phosphorylated after leptin treatment. SHP-2 is not phosphorylated after leptin treatment when a Y-->F 985 receptor mutant is cotransfected. In the absence of SHP-2 phosphorylation, the level of JAK2 phosphorylation was increased. Tyrosyl phosphorylation of the leptin receptor and signal transducer and activater of transcription 3 (STAT3) are not affected by phosphorylation of SHP-2. These data suggest that activation of SHP-2 by the leptin receptor results in a decreased phosphorylation of JAK2 and may act to attenuate leptin signal transduction. The method used in this report can in principle be used to isolate additional components of the leptin, or other, signal transduction pathway. (+info)
Molecular characterization and immunolocalization of Dictyostelium discoideum protein phosphatase 2A.
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Protein phosphatase 2A (PP2A) was previously purified from Dictyostelium and biochemically characterized. The purified PP2A holoenzyme was composed of a 37 kDa catalytic 'C-subunit', a 65 kDa 'A-subunit' and a 55 kDa 'B-subunit'. We report here the characterization of the genes encoding the Dictyostelium PP2A subunits as well as the immunolocalization of the PP2A subunits in Dictyostelium. The cDNAs encoding the B- and C-subunits were isolated from a Dictyostelium library and the deduced amino acid sequences reveal strong conservation with the mammalian PP2A homologues. Southern blot analysis suggests that each of the PP2A subunit genes is present in a single copy. The PP2A subunits were localized mainly to the cytosol in Dictyostelium cells. However, immunofluorescence confocal microscopy demonstrates that the B-subunit of PP2A is highly enriched in centrosomes, suggesting a potential role for this PP2A regulatory subunit in the centrosomal function. (+info)
Molecular interactions among protein phosphatase 2A, tau, and microtubules. Implications for the regulation of tau phosphorylation and the development of tauopathies.
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Hyperphosphorylated forms of the neuronal microtubule (MT)-associated protein tau are major components of Alzheimer's disease paired helical filaments. Previously, we reported that ABalphaC, the dominant brain isoform of protein phosphatase 2A (PP2A), is localized on MTs, binds directly to tau, and is a major tau phosphatase in cells. We now describe direct interactions among tau, PP2A, and MTs at the submolecular level. Using tau deletion mutants, we found that ABalphaC binds a domain on tau that is indistinguishable from its MT-binding domain. ABalphaC binds directly to MTs through a site that encompasses its catalytic subunit and is distinct from its binding site for tau, and ABalphaC and tau bind to different domains on MTs. Specific PP2A isoforms bind to MTs with distinct affinities in vitro, and these interactions differentially inhibit the ability of PP2A to dephosphorylate various substrates, including tau and tubulin. Finally, tubulin assembly decreases PP2A activity in vitro, suggesting that PP2A activity can be modulated by MT dynamics in vivo. Taken together, these findings indicate how structural interactions among ABalphaC, tau, and MTs might control the phosphorylation state of tau. Disruption of these normal interactions could contribute significantly to development of tauopathies such as Alzheimer's disease. (+info)
Interconversion of Mn(2+)-dependent and -independent protein phosphatase 2A from human erythrocytes: role of Zn(2+) and Fe(2+) in protein phosphatase 2A.
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Human erythrocyte Mn(2+)-dependent (C'A') and -independent (CA) protein-serine/threonine phosphatase (PP) 2A are composed of 34-kDa catalytic C' and C subunits, in which the metal dependency resides, and 63-kDa regulatory A' and A subunits, respectively. Each catalytic and regulatory subunit gave the same V8- and papain-peptide maps, respectively. Stoichiometric zinc and substoichiometric iron were detected in CA but not in C'A' [Nishito et al. (1999) FEBS Lett. 447, 29-33]. The Mn(2+)-dependent protein-tyrosine phosphatase (PTP) activity of C'A' was about 70-fold higher than that of CA. Pre-incubation of CA with 25 mM NaF changed CA to a Mn(2+)-dependent form with higher PTP activity. The same NaF treatment had no effect on C'A'. Pre-incubation of C'A' with ZnCl(2), zinc-metallothionein, or FeCl(2) activated the Mn(2+)-independent PP activity, but pre-incubation with FeCl(3) did not. Ascorbate in the pre-incubation and assay mixture significantly stimulated the effect of FeCl(2). Pre-incubation of C'A' with 5 microM ZnCl(2) and 15 microM FeCl(2) in the presence of 1 mM ascorbate synergistically stimulated the Mn(2+)-independent PP activity, with concomitant suppression of the Mn(2+)-dependent PP and PTP activities. The PP and PTP activities of CA were unaffected by the same zinc and/or iron treatment. Micromolar concentrations of vanadate strongly inhibited the Mn(2+)-dependent PP activity of C'A' but only slightly inhibited the PP activity of CA. Using the distinct effect of vanadate as an indicator, the interconversion between CA and C'A' with the above mentioned treatments was proved. These results support the notion that Mn(2+)-independent CA is a Zn(2+)- and Fe(2+)-metalloenzyme, whose apoenzyme is Mn(2+)-dependent C'A'. (+info)
Induction of apoptosis by adenovirus E4orf4 protein is specific to transformed cells and requires an interaction with protein phosphatase 2A.
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We previously have shown that adenovirus type 5 E4orf4 protein associates with protein phosphatase 2A (PP2A) and induces apoptosis in transformed cells in a p53-independent manner. Here we show that the interaction between E4orf4 and PP2A is required for induction of apoptosis by the viral protein. This conclusion is supported by a mutation analysis of E4orf4 protein, showing a correlation between the ability to bind PP2A and to induce apoptosis, and by the observation that transfection of an antisense construct of the PP2A-B55 subunit reduces expression of the PP2A-B55 subunit and inhibits induction of apoptosis by E4orf4, but not by p53. The mutant analysis also indicates that even a low level of interaction with PP2A is sufficient to initiate the E4orf4 apoptotic pathway. In addition, E4orf4 inhibits cellular transformation by various oncogenes, and this function is coupled to its ability to induce apoptosis. Furthermore, expression of oncogenes in primary cell cultures sensitizes these cells to induction of apoptosis by E4orf4. Our results suggest that E4orf4 is a potentially useful tool for cancer gene therapy. (+info)
Alternative promoters direct tissue-specific expression of the mouse protein phosphatase 2Cbeta gene.
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Type 2C protein phosphatases (PP2Cs), a class of ubiquitous and evolutionally conserved serine/threonine protein phosphatases, are encoded in at least four distinct genes and implicated in the regulation of various cellular functions. Of these four PP2C genes, the expression of the PP2Cbeta gene has been reported to be tissue-specific and development-dependent. To understand more precisely the regulatory mechanism of this expression, we have isolated and characterized overlapping mouse genomic lambda clones. A comparison of genomic sequences with PP2Cbeta cDNA sequences provided information on the structure and localization of intron/exon boundaries and indicated that PP2Cbeta isoforms with different 5' termini were generated by alternative splicing of its pre-mRNA. The 5'-flanking region of exon 1 had features characteristic of a housekeeping gene: it was GC-rich, lacked TATA boxes and CAAT boxes in the standard positions, and contained potential binding sites for the transcription factor SP1. In the 5'-flanking region of exon 2, several consensus sequences were found, such as a TATA-like sequence and negative regulatory element box-1, -2 and -3. Subsequent analysis by transient transfection assay with a reporter gene showed that these regions act as distinct promoters. Analysis of PP2Cbeta transcripts by reverse transcriptase-PCR showed that exon-1 transcripts were expressed ubiquitously in all of the tissues examined, whereas exon-2 transcripts were predominantly expressed in the testis, intestine and liver. These results suggest that the alternative usage of two promoters within the PP2Cbeta gene regulates tissue-specific expression of PP2Cbeta mRNA. (+info)
Spatial gradients of cellular phospho-proteins.
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If a protein is rapidly phosphorylated and dephosphorylated at separate cellular locations and protein diffusion is slow, then a spatial gradient of the phosphorylated form of the protein may develop within the cell. We have estimated the potential size of such gradients using measured values of protein diffusion coefficients and protein kinase and phosphatase activities. We analysed two different cellular geometries: (1) where the kinases is located on the plasma membrane of a spherical cell and the phospatase is distributed homogenously in the cytoplasm and (2) where the kinase is located on one planar membrane and the phosphatase on a second parallel planar membrane. The estimated gradients of phospho-proteins were potentially very large, which has important implications for cellular signalling. (+info)