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PubMed1991

Characterization of trypanosome protein phosphatase 1 and 2A catalytic subunits.

Oligonucleotides corresponding to highly conserved regions of mammalian protein phosphatase catalytic subunits were used in the polymerase chain reaction (PCR) to generate an amplification product from genomic DNA of Trypanosoma brucei rhodesiense. The PCR product was used to screen a T. b. rhodesiense cDNA library for cDNA clones encoding putative protein phosphatase catalytic subunits. Two cDNA clones, (TPP1A and TPP1B) representing two distinct type 1 catalytic subunit isotypes, encode 39-kDa proteins of 346 amino acids that show 66% and 40% identity, respectively, to mammalian protein phosphatase 1 and 2A catalytic subunits. Both cDNAs are derived from 2.3-kb mRNAs, and Northern blot analysis has provided indirect evidence that these mRNAs are part of the same transcription unit as mRNAs for RNA polymerase II largest subunit. Another cDNA, TPP2, represents the type 2A class of phosphatases and codes for a 34.5-kDa protein of 303 amino acids. The deduced amino acid sequence has 39% and 55% identity, respectively, to the catalytic subunits of mammalian protein phosphatase 1 and 2A. Southern and Northern blot analyses are consistent with TPP2 being encoded by a single copy gene from which is derived a mRNA of 2.5 kb. This finding constitutes the first example in eukaryotes in which a single gene encodes the type 2A class of protein phosphatases. Sera from mice immunized with TPP1A fusion protein reacted with the catalytic subunits of mammalian types 1, 2A and 2B protein phosphatases. However, antisera to TPP2 fusion protein was specific for the type 2A catalytic subunit and recognized a polypeptide of 35 kDa in a Western blot of crude trypanosomal lysate.

Amino Acid Sequence↗

PubMed1989

The importance of nucleoside triphosphate inhibition of liver glycogen synthase phosphatase activity.

The liver glycogen particle contains constitutive glycogen-synthase phosphatase activity which is inhibited by ATP-Mg in a concentration-dependent manner within the physiological range (I0.5 = 0.1 mM). Therefore, we determined whether other nucleoside triphosphate-magnesium complexes also inhibit synthase phosphatase activity. UTP-Mg, CTP-Mg and GTP-Mg were all found to be inhibitory. The maximum inhibition was 85-90% which was greater than that for ATP-Mg. The I0.5 for UTP-Mg was comparable to that of ATP-Mg but it was greater for CTP-Mg and for GTP-Mg. At in vivo physiological concentrations, both UTP and ATP are possible inhibitors of synthase phosphatase activity. In the presence of a saturating concentration of ATP-Mg, added UTP-Mg increased the inhibition suggesting the presence of at least two distinct nucleotide binding sites. Substitution of calcium for magnesium in an ATP complex had no effect on the I0.5, but increased the maximum inhibition. The present studies also suggest that in the multistep conversion of synthase D to synthase I, ATP-Mg inhibition occurs early in the sequence. Addition of glycogen, a known inhibitor of synthase phosphatase activity, to a reaction mixture containing 3 mM ATP-Mg did not further inhibit synthase phosphatase activity when added at concentrations up to 22 mg/ml. The latter data suggest that the presence of a nucleoside triphosphate may desensitize the phosphatase to glycogen inhibition. ATP-Mg and, to a lesser extent, UTP-Mg and CTP-Mg all stimulated phosphorylase phosphatase activity but GTP-Mg did not.

Adenosine Triphosphate↗

PubMed2004

Prostatic acid phosphatase degrades lysophosphatidic acid in seminal plasma.

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological activities and is detected in various biological fluids, including human seminal plasma. Due to its cell proliferation stimulatory and anti-apoptotic activities, LPA has been implicated in the progression of some cancers such as ovarian cancer and prostate cancer. Here, we show that prostatic acid phosphatase, which is a non-specific phosphatase and which has been implicated in the progression of prostate cancer, inactivates LPA in human seminal plasma. Human seminal plasma contains both an LPA-synthetic enzyme, lysoPLD, which converts lysophospholipids to LPA and is responsible for LPA production in serum, and its major substrate, lysophosphatidylcholine. In serum, LPA accumulated during incubation at 37 degrees C. However, in seminal plasma, LPA did not accumulate. This discrepancy is explained by the presence of a strong LPA-degrading activity. Incubation of LPA with seminal plasma resulted in the disappearance of LPA and an accompanying accumulation of monoglyceride showing that LPA is degraded by phosphatase activity present in the seminal plasma. When seminal plasma was incubated in the presence of a phosphatase inhibitor, sodium orthovanadate, LPA accumulated, indicating that LPA is produced and degraded in the fluid. Biochemical characterization of the LPA-phosphatase activity identified two phosphatase activities in human seminal plasma. By Western blotting analysis in combination with several column chromatographies, the major activity was revealed to be identical to prostatic acid phosphatase. The present study demonstrates active LPA metabolism in seminal plasma and indicates the possible role of LPA signaling in male sexual organs including prostate cancer.

Acid Phosphatase↗

PubMed2005

Crystal structures of protein phosphatase-1 bound to motuporin and dihydromicrocystin-LA: elucidation of the mechanism of enzyme inhibition by cyanobacterial toxins.

The microcystins and nodularins are tumour promoting hepatotoxins that are responsible for global adverse human health effects and wildlife fatalities in countries where drinking water supplies contain cyanobacteria. The toxins function by inhibiting broad specificity Ser/Thr protein phosphatases in the host cells, thereby disrupting signal transduction pathways. A previous crystal structure of a microcystin bound to the catalytic subunit of protein phosphatase-1 (PP-1c) showed distinct changes in the active site region when compared with protein phosphatase-1 structures bound to other toxins. We have elucidated the crystal structures of the cyanotoxins, motuporin (nodularin-V) and dihydromicrocystin-LA bound to human protein phosphatase-1c (gamma isoform). The atomic structures of these complexes reveal the structural basis for inhibition of protein phosphatases by these toxins. Comparisons of the structures of the cyanobacterial toxin:phosphatase complexes explain the biochemical mechanism by which microcystins but not nodularins permanently modify their protein phosphatase targets by covalent addition to an active site cysteine residue.

Bacterial Toxins↗

PubMed2004

A zinc-binding dual-specificity YVH1 phosphatase in the malaria parasite, Plasmodium falciparum, and its interaction with the nuclear protein, pescadillo.

Biochemical evidence revealed protein tyrosine kinase and phosphatase activities in the human malarial parasite Plasmodium falciparum, a member of the Apicomplexa. A novel cDNA sequence of a dual-specificity phosphatase was identified in both sexual and asexual stages of P. falciparum, and named PfYVH1, since the predicted primary structure of the 278-amino acid polypeptide showed significant similarity to the human and yeast YVH1 phosphatases. The N-terminal half of PfYVH1 contained a conserved tyrosine phosphatase catalytic domain within a dual-specificity phosphatase domain. The C-terminal region, consisting of one histidine and eight cysteines, represented a zinc-binding domain with a potentially unconventional architecture. Recombinant PfYVH1 contained 2mol of zinc per mol protein and dephosphorylated both phosphoserine and phosphotyrosine residues. Mutation of specific Cys residues in the putative zinc finger region abolished zinc binding and drastically reduced phosphatase activity, suggesting an allosteric role of zinc in catalysis. PfYVH1 was expressed in essentially all erythrocytic stages of the parasite, and shuttled between the nucleus and the cytoplasm in a stage-specific manner. A Plasmodium ortholog of the nuclear pescadillo protein (PfPES) was also characterized and shown to interact with PfYVH1, thus implicating PfYVH1 in the regulation of parasitic development. PfYVH1 represents the first dual-specificity zinc-finger phosphatase characterized in the protozoan kingdom.

Amino Acid Sequence↗

PubMed2004

Regulation of ErbB4 phosphorylation and cleavage by a novel histidine acid phosphatase.

Signaling by a variety of ligands including epidermal growth factor, betacellulin and neuregulin is mediated by the ErbB family of receptor tyrosine kinases. Studies on the prostate have shown that ErbB2 phosphorylation and signaling can be regulated by prostatic acid phosphatase, a histidine acid phosphatase which can dephosphorylate phospho-tyrosine residues in the ErbB2 receptor. Here we report that the histidine acid phosphatase ACPT (testicular acid phosphatase), which is highly homologous to the prostatic acid phosphatase, can dephosphorylate the ErbB4 receptor, which is known to play important roles in neuronal differentiation and synaptogenesis. ACPT and ErbB4 are both expressed in the brain where they are enriched at post-synaptic sites, and furthermore they can be co-immunoprecipitated from brain. We demonstrate that ACPT can inhibit basal and neuregulin-induced tyrosine phosphorylation of ErbB4. We also show that ACPT-dependent dephosphorylation can regulate the proteolytic cleavage of ErbB4, and this process can be reversed by applying the tyrosine phosphatase inhibitor, pervanadate. Furthermore, neuregulin-dependent differentiation of PC12 cells expressing ErbB4 is prevented by co-expressing ACPT. These results indicate that ACPT acts as a tyrosine phosphatase to modulate signals mediated by ErbB4 that are important for neuronal development and synaptic plasticity.

Acid Phosphatase↗

PubMed2002

Phosphatase regulation of gene expression during development of the palate.

In mammalian cells, including those of the embryonic palate, the level of phosphorylation of cellular proteins at any given time reflects the activities of protein kinases and protein phosphatases. Both protein phosphatase-1 (PP-1) and PP-2A inhibit cAMP-mediated increases in transcription by dephosphorylating CREB at ser-133. Western blot analysis indicated that protein phosphatase 1 (PP-1) was expressed constitutively in palatal tissue during its development. Expression of PP-2A was regulated developmentally with maximal expression on gestational day (gd) 14. Densitometric scanning revealed a 30% increase in expression from gd 13 to gd 14. Virtually all phosphatase activity in the tissue extracts could be inhibited by 5 microM okadaic acid, demonstrating that PP-1 and PP-2A account for all detectable ser/thr protein phosphatase activity present in the developing palate. Moreover, no significant differences in PP-1 and PP-2A activities were observed during the period of palate development. Treatment of primary cultures of murine embryonic palate mesenchymal (MEPM) cells with forskolin (20 microM) to elevate intracellular cAMP levels, resulted in a time-dependent increase in CREB ser-133 phosphorylation and a corresponding time dependent decrease in PP-1 and PP-2A levels. Moreover, treatment of MEPM cells with okadaic acid resulted in a dramatic increase in basal CREB ser-133 phosphorylation. This suggests that PP-1 activity may contribute to transcriptional regulation of CREB and that PP-1 and PP-2A are regulated differentially by cAMP. Treatment of MEPM cells with TGF beta 1 (1 ng/ml) under conditions of TGF beta-induced CREB phosphorylation resulted in no effect on the expression of either PP-1 or PP-2A proteins and no significant alterations in total basal protein phosphatase activity. These results demonstrate that transcriptional regulation of CREB in embryonic palatal issue is dependent on the coordinate activity of specific kinases and phosphatases.

Animals↗

PubMed2000

A Theileria parva type 1 protein phosphatase activity.

The protozoan parasite Theileria (spp. parva and annulata) infects bovine leukocytes and provokes a leukaemia-like disease in vivo. In this study, we have detected a type 1 serine/threonine phosphatase activity with phosphorylase a as a substrate, in protein extracts of parasites purified from infected B lymphocytes. In contrast to this type 1 activity, dose response experiments with okadaic acid (OA), a well characterised inhibitor of type 1 and 2A protein phosphatases, indicated that type 2A is the predominant activity detected in host B cells. Furthermore, consistent with polycation-specific activation of the type 2A phosphatase, protamine failed to activate the parasite-associated phosphorylase a phosphatase activity. Moreover, inhibition of phosphorylase a dephosphorylation by phospho-DARPP-32, a specific type 1 inhibitor, clearly demonstrated that a type 1 phosphatase is specifically associated with the parasite, while the type 2A is predominantly expressed in the host lymphocyte. Since an antibody against bovine catalytic protein phosphatase 1 (PP1) subunit only recognised the PP1 in B cells, but not in parasite extracts, we conclude that in parasites the PP1 activity is of parasitic origin. Intriguingly, since type 1 OA-sensitive phosphatase activity has been recently described in Plasmodium falciparum, we can conclude that these medically important parasites produce their one PP1.

Animals↗

PubMed1997

Colorimetric protein phosphatase inhibition assay of laboratory strains and natural blooms of cyanobacteria: comparisons with high-performance liquid chromatographic analysis for microcystins.

Microcystins are cyclic heptapeptide hepatotoxins commonly produced by bloom-forming genera of cyanobacteria. These toxins are potent and specific inhibitors of protein phosphatases 1 and 2A. We have optimised a rapid, simple and sensitive colorimetric protein phosphatase 1 inhibition assay, utilising the activity of protein phosphatase 1 as expressed in a recombinant strain of Escherichia coli, towards the chromogenic substrate, p-nitrophenyl phosphate. A standard curve for the inhibition of protein phosphatase 1 by microcystin-LR was constructed with an IC50 of about 38 ng ml-1 and a limit of detection of 10-20 ng ml-1. Twenty-three laboratory-grown strains and 25 natural bloom samples of cyanobacteria were analysed by high-performance liquid chromatography for microcystins and by the protein phosphatase 1 inhibition assay. Agreement for the microcystin contents of the samples detected by high-performance liquid chromatography and the protein phosphatase 1 inhibition assay showed good correlation (R2 > 0.93, P < 0.0001). The suitability of the colorimetric protein phosphatase 1 inhibition assay as a screen for cyanobacterial microcystins is discussed.

Chromatography, High Pressure Liquid↗

PubMed1998

Characterization and chromosomal localization of PTP-NP-2, a new isoform of protein tyrosine phosphatase-like receptor, expressed on synaptic boutons.

Recently, there have been several reports describing the cloning and characterization of the novel family of protein tyrosine phosphatase-like receptor molecules (known as IA-2 and PTP-NP/PTP-IAR/IA-2beta/phogrin), which may act as autoantigens in diabetes. Here, we report the molecular characterization and chromosomal localization of a new isoform of this family in brain termed PTP-NP-2 (for PTP-NP tyrosine phosphatase isoform), and its function in rat primary hippocampal neurons. PTP-NP-2 has 48% identity to IA-2. The principal difference between PTP-NP-2 and PTP-NP is a 17-amino-acid insert near the N-terminus of PTP-NP that is absent in PTP-NP-2. Genomic DNA analysis indicates that the 17-amino-acid insert is coded by a separate exon, suggesting that both IA-2beta and PTP-NP-2 are isoforms arising by alternate splicing of the same gene. Reverse transcriptase-PCR revealed that both isoforms are present in human SH-SY5Y neuroblastoma cells. PTP-NP-2 mRNA expression is highly restricted, with a 5.5-kb specific transcript in human fetal and adult brain and 5.5 and 3. 8 kb in human adult pancreas. SH-SY5Y neuroblastoma and U87-MG glioblastoma cells showed specific transcripts of 5.5 and 3.8 kb, respectively, indicating the existence of several isoforms of this molecule in the nervous system. The human gene encoding PTP-NP-2 was assigned to human chromosome 7q22-qter using Southern blot analysis of genomic DNAs from rodent/human somatic hybrid cell lines. Confocal microscopy analyses of rat primary hippocampal neurons revealed that PTP-NP-2 is abundantly expressed on synaptic boutons in primary neurons. Wild-type PTP-NP-2 showed no measurable tyrosine phosphatase activity using an in-vitro pNPP assay. Examination of the PTP-NP-2 catalytic consensus sequence revealed that this sequence differed from the typical tyrosine phosphatase-domain consensus sequence by an alanine to aspartate change (amino acid 930). Mutation of aspartate 930 to alanine produced a catalytically active enzyme, suggesting that native PTP-NP and its isoform PTP-NP-2 are catalytically inactive receptor protein tyrosine phosphatase homologues. Taken together, these results indicate that the tyrosine phosphatase PTP-NP-2 is a new isoform of PTP-NP tyrosine phosphatase, is expressed on synaptic boutons and may participate in the regulation of synaptic bouton endocytosis.

Adult↗

PubMed2001

Dephosphorylation of MnDPDP and related compounds by acid and alkaline phosphatase.

The enzymatic dephosphorylation of the magnetic resonance imaging contrast agent Teslascan was studied in in vitro experiments with acid phosphatase (prostatic, from human semen) and alkaline phosphatase (from human placenta). The active component, MnDPDP (manganese (II)-N,N'-dipyridoxylethylenediamine-N,N'-diacetate-5,5'-bis(phosphate), was dephosphorylated by both enzymes to the monophosphate MnDPMP and the totally dephosphorylated compound MnPLED. The corresponding zinc compound, ZnDPDP (which is a result of in vivo metabolism), was also dephosphorylated by both enzymes to ZnDPMP and ZnPLED. In separate experiments, both enzymes dephosphorylated MnDPMP and ZnDPMP. With the same amount of enzyme units, alkaline phosphatase was almost four times more active than acid phosphatase in dephosphorylating MnDPDP and ZnDPDP with only minor differences whether the substrate contained Mn or Zn. A similar difference in enzymatic activity was seen with the monophosphates, MnDPMP and ZnDPMP. This, taken together with the approximately 50 times higher activity of alkaline phosphatase than acid phosphatase in serum shows that alkaline phosphatase is responsible for most of the dephosphorylation of MnDPDP and its metabolites in vivo.

Acid Phosphatase↗

PubMed2002

Protein Ser/Thr phosphatases with kelch-like repeat domains.

This report describes the presence in plants of protein Ser/Thr phosphatases of the PPP family, homologous to PfPPalpha phosphatase from Plasmodium falciparum. Like PfPPalpha, they possess large N-terminal domains and catalytic domains that are more closely related to the protein phosphatase 1 group. The N-terminal domains of PfPPalpha and its plant homologues contain tandem kelch-like repeats, not previously identified in any protein phosphatases, suggesting that the N-terminal domains may form beta-propeller structures mediating protein-protein interactions. We therefore suggest that this novel phosphatase group be designated as PPKLs for protein phosphatases with kelch-like repeat domains. Four PPKL isoforms are encoded in the Arabidopsis thaliana genome, of which at least three are expressed. PPKLs appear to be ubiquitous in Viridiplantae. The existence of a protein phosphatase group shared by Viridiplantae and Apicomplexa, but not other eukaryotes, is in line with the theory of the origin of Apicomplexa by endosymbiosis of nonphotosynthetic eukaryotes with red algae.

Amino Acid Sequence↗

PubMed2003

Parallel purification of three catalytic subunits of the protein serine/threonine phosphatase 2A family (PP2A(C), PP4(C), and PP6(C)) and analysis of the interaction of PP2A(C) with alpha4 protein.

The protein serine/threonine phosphatase (PP) type 2A family consists of three members: PP2A, PP4, and PP6. Specific rabbit and sheep antibodies corresponding to each catalytic subunit, as well as a rabbit antibody recognizing all three subunits, were utilized to examine the expression of these enzymes in select rat tissue extracts. PP2A, PP4, and PP6 catalytic subunits (PP2A(C), PP4(C), and PP6(C), respectively) were detected in all rat tissue extracts examined and exhibited some differences in their levels of expression. The expression of alpha4, an interacting protein for PP2A family members that may function downstream of the target of rapamycin (Tor), was also examined using specific alpha4 sheep antibodies. Like the phosphatase catalytic subunits, alpha4 was ubiquitously expressed with particularly high levels in the brain and thymus. All three PP2A family members, but not alpha4, bound to the phosphatase affinity resin microcystin-Sepharose. The phosphatase catalytic subunits were purified to apparent homogeneity (PP2A(C) and PP4(C)) or near homogeneity (PP6(C)) from bovine testes soluble extracts following ethanol precipitation and protein extraction. In contrast to PP2A(C), PP4(C) and PP6(C) exhibited relatively low phosphatase activity towards several substrates. Purified PP2A(C) and native PP2A in cellular extracts bound to GST-alpha4, and co-immunoprecipitated with endogenous alpha4 and ectopically expressed myc-tagged alpha4. The interaction of PP2A(C) with alpha4 was unaffected by rapamycin treatment of mammalian cells; however, protein serine/threonine phosphatase inhibitors such as okadaic acid and microcystin-LR disrupted the alpha4/PP2A complex. Together, these findings increase our understanding of the biochemistry of alpha4/phosphatase complexes and suggest that the alpha4 binding site within PP2A may include the phosphatase catalytic domain.

Adaptor Proteins, Signal Transducing↗

PubMed1989

Interplay of phosphorylation and dephosphorylation in vision: protein phosphatases of bovine rod outer segments.

Two types of protein phosphatases were identified in carefully prepared bovine rod outer segments (ROS). Extraction of the ROS with a medium-salt buffer solubilized protein phosphatase activity that was mainly type 2A, since it was active toward phosphorylase a in the absence of divalent cations, was not retained by heparin-Sepharose, dephosphorylated the alpha-subunit of phosphorylase kinase faster that the beta-subunit, and was unaffected by inhibitor 2. Further extraction of the resulting membranes with a high-salt buffer solubilized additional phosphatase activity which was predominantly type 1, since it was retained by heparin-Sepharose and was blocked by inhibitor 2. The molecular mass of the type 2A phosphatase estimated by gel permeation chromatography on Superose 12 was 100 kDa, suggesting it may be the 2A2 form. Only the ROS type 2A phosphatase dephosphorylated opsin and rhodopsin efficiently. Concordant with this finding, the purified catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle dephosphorylated opsin efficiently, while the type 1 catalytic subunit isolated from this tissue was inactive. Together, the results suggest that the ROS type 2A protein phosphatase plays an important role in regenerating rhodopsin from the various phosphorylated species in vivo. The activity of the enzyme per retina (approximately 85 pmol of Pi released/min) is comparable to that of rhodopsin kinase (100 pmol of phosphate transferred/min).

Animals↗

PubMed1996

VHR and PTP1 protein phosphatases exhibit remarkably different active site specificities toward low molecular weight nonpeptidic substrates.

The dual-specificity protein phosphatases have recently been shown to act as key regulators of mitogenic signaling pathways as well as of the cell cycle process. The are unusual catalysts in that they can utilize protein substrates containing phosphotyrosine as well as phosphoserine/threonine. The dual-specificity phosphatases and the protein tyrosine phosphatases (PTPase) share the active site motif (H/V)C(X)5R(S/T) but display little amino acid sequence identity outside of the active site. Although the dual-specificity phosphatases and the PTPases appear to bring about phosphate monoester hydrolysis through a similar mechanism, there is very limited information about the structural features that control the substrate specificity for the two groups of enzymes. As a first step in the development of selective dual-specificity phosphatase inhibitors, we have examined the active site substrate specificity of the human dual-specificity phosphatase, VHR [for VH1-Related; Ishibashi et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 12170-12174]. Like the tyrosine-specific PTP1, VHR also preferentially catalyzes the hydrolysis of aromatic phosphates. However, we demonstrate herein that relatively modest changes in the substitution patterns on the phosphorylated aromatic nucleus generates dramatic, and differential, swings in substrate selectivity for VHR and PTP1. For example, VHR appears to be significantly more accommodating than PTP1 toward sterically-demanding substrates. Thus, the active site specificity of these two protein phosphatases is decidedly dissimilar. In addition, we have also identified several low molecular weight compounds that are more efficient substrates than the most potent peptidic substrates ever reported for VHR. Finally, we have shown that the Michaelis constants exhibited by these substrates are accurate assessments of enzyme affinity. Consequently, it should be possible to develop phospatase-selective inhibitors based upon the distinct substrate specificities of these enzymes.

Amino Acid Sequence↗

PubMed1997

Protein phosphorylation in rat cardiac microsomes: effects of inhibitors of protein kinase A and of phosphatases.

The phosphorylation of rat cardiac microsomal proteins was investigated with special attention to the effects of okadaic acid (an inhibitor of protein phosphatases), inhibitor 2 of protein phosphatase 1 and inhibitor of cyclic AMP-dependent protein kinase (protein kinase A). The results showed that okadaic acid (5 microM) modestly but reproducibly augmented the protein kinase A-catalyzed phospholamban (PLN) phosphorylation, although exerted little effect on the calcium/calmodulin kinase-catalyzed PLN phosphorylation. Microsomes contained three other substrates (M(r) 23, 19 and 17 kDa) that were phosphorylated by protein kinase A but not by calcium/calmodulin kinase. The protein kinase A-catalyzed phosphorylation of these three substrates was markedly (2-3 fold) increased by 5 microM okadaic acid. Calmodulin was found to antagonize the action of okadaic acid on such phosphorylation. Protein kinase A inhibitor was found to decrease the protein kinase A-catalyzed phosphorylation of microsomal polypeptides. Unexpectedly, inhibitor 2 was also found to markedly decrease protein kinase A-catalyzed phosphorylation of phospholamban as well these other microsomal substrates. These results are consistent with the views that protein phosphatase 1 is capable of dephosphorylating membrane-associated phospholamban when it is phosphorylated by protein kinase A, but not by calcium/calmodulin kinase, and that under certain conditions, calcium/calmodulin-stimulated protein phosphatase (protein phosphatase 2B) is also able to dephosphorylate PLN phosphorylated by protein kinase A. Additionally, the observations show that protein phosphatase 1 is extremely active against the three protein kinase A substrates (M(r) 23, 19 and 17 kDa) that were present in the isolated microsomes and whose state of phosphorylation was particularly affected in the presence of dimethylsulfoxide. Protein phosphatase 2B is also capable of dephosphorylating these three substrates.

Adenosine Triphosphatases↗

PubMed2002

Tyrosine hydroxylase dephosphorylation by protein phosphatase 2A in bovine adrenal chromaffin cells.

This study was undertaken to characterise the protein phosphatases in bovine adrenal chromaffin cells acting on tyrosine hydroxylase. Cells were pre-labelled with 32Pi and permeabilized with digitonin. The extent of dephosphorylation of Ser-8, Ser-19, Ser-31 and Ser-40 on tyrosine hydroxylase was found to be 30%, 38%, 37% and 71% respectively over 5 min. For Ser-19, Ser-31 and Ser-40 the dephosphorylation was entirely due to protein phosphatase 2A, as the dephosphorylation could be completely blocked by microcystin, but not by the protein phosphatase I inhibitory peptide. Permeabilization did not change the distribution of protein phosphatase 2A or tyrosine hydroxylase, or the activity of PP2A, from that occurring in intact cells. The dephosphorylation of Ser-8 was not altered by any inhibitor, suggesting the involvement of other protein phosphatases. The method developed here can be used to determine the protein phosphatases acting on substrates in conditions closely approximating those in situ, including the endogenous state of substrate phosphorylation and phosphatase location.

Adrenal Medulla↗

PubMed1980

Dephosphorylation and activation of exogenous glycogen synthase by adipose-tissue phosphatase.

Exogenous purified rabbit skeletal-muscle glycogen synthase was used as a substrate for adipose-tissue phosphoprotein phosphatase from fed and starved rats in order to (1) compare the relationship between phosphate released from, and the kinetic changes imparted to, the substrate and (2) ascertain if decreases in adipose-tissue phosphatase activity account for the apparent decreased activation of endogenous glycogen synthase from starved as compared with fed rats. Muscle glycogen synthase was phosphorylated with [gamma-(32)P]ATP and cyclic AMP-dependent protein kinase alone, or in combination with a cyclic AMP-independent protein kinase, to 1.7 or 3mol of phosphate per subunit. Adipose-tissue phosphatase activity determined with phosphorylated skeletal-muscle glycogen synthase as substrate was decreased by 35-60% as a consequence of starvation. This decrease in phosphatase activity had little effect on the capacity of adipose-tissue extracts to activate exogenous glycogen synthase (i.e. to increase the glucose 6-phosphate-independent enzyme activity), although there were marked differences in the activation profiles for the two exogenous substrates. Glycogen synthase phosphorylated to 1.7mol of phosphate per subunit was activated rapidly by adipose-tissue extracts from either fed or starved rats, and activation paralleled enzyme dephosphorylation. Glycogen synthase phosphorylated to 3mol of phosphate per subunit was activated more slowly and after a lag period, since release of the first mol of phosphate did not increase the glucose 6-phosphate-independent activity of the enzyme. These patterns of enzyme activation were similar to those observed for the endogenous adipose-tissue glycogen synthase(s): the glucose 6-phosphate-independent activity of the endogenous enzyme from fed rats increased rapidly during incubation, whereas that of starved rats, like that of the more highly phosphorylated muscle enzyme, increased only very slowly after a lag period. The observations made here suggest that (1) changes in glucose 6-phosphate-independent glycogen synthase activity are at best only a qualitative measure of phosphoprotein phosphatase activity and (2) the decrease in glycogen synthase phosphatase activity during starvation is not sufficient to explain the differential glycogen synthase activation in adipose tissue from fed and starved rats. However, alterations in the phosphorylation state of glycogen synthase combined with decreased activity of phosphoprotein phosphatase, both as a consequence of starvation, could explain the apparent markedly decreased enzyme activation.

Adipose Tissue↗