Prior Dephosphorylation Research Articles

Direct uptake of sphingosine-1-phosphate independent of phospholipid phosphatases

Sphingosine-1-phosphate (S1P) is a lipid mediator that is relatively abundant in plasma and plays an important role in the vascular and immune systems. To date, the only known mechanism for removing S1P from plasma has been dephosphorylation by phospholipid phosphatases (PLPPs) on the surface of cells in contact with the plasma. However, there remains a possibility that PLPP-independent dephosphorylation or direct S1P uptake into cells could occur. To examine these possibilities, here we generated triple KO (TKO) HAP1 cells that lacked all PLPPs (PLPP1–3) present in mammals. In the TKO cells, the intracellular metabolism of externally added deuterium-labeled S1P to ceramide was reduced to 17% compared with the WT cells, indicating that most extracellular S1P is dephosphorylated by PLPPs and then taken up into cells. However, this result also reveals the existence of a PLPP-independent S1P uptake pathway. Tracer experiments using [32P]S1P showed the existence of a direct S1P uptake pathway that functions without prior dephosphorylation. Overexpression of sphingolipid transporter 2 (SPNS2) or of major facilitator superfamily domain containing 2B (MFSD2B), both known S1P efflux transporters, in TKO cells increased the direct uptake of S1P, whereas KO of MFSD2B in TKO cells reduced this uptake. These results suggest that these are channel-type transporters and capable of not only exporting but also importing S1P. Furthermore, we observed that erythroid cells expressing MFSD2B, exhibited high S1P uptake activity. Our findings describing direct S1P uptake may contribute to the elucidation of the molecular mechanisms that regulate plasma S1P concentration.

A Tail of Kinase Regulation: How C‐termini Modulate CK1 Substrate Phosphorylation

CK1 enzymes signal in a variety of important cellular pathways, including DNA damage repair, mitotic checkpoint signaling, circadian rhythm, Wnt signaling, endocytosis, and neurodegenerative disease progression. Like other multifunctional kinases, CK1 must be regulated in space and time in order to target specific subsets of its substrates in each of the pathways it participates in. However, CK1 is generally regarded as a “rogue” kinase, which is constitutively active, ubiquitous throughout cells and tissues, and unregulated except by autoinhibition.CK1 enzymes are known to autophosphorylate their C‐terminal non‐catalytic tails, which are proposed to inhibit their activity by acting as pseudosubstrates. This model would require a phosphorylation‐dependent intramolecular interaction between the C‐terminus and the kinase domain, but we are unaware of any evidence demonstrating such an event. Furthermore, this proposed mechanism of autoinhibition has not been tested in vivo in any organism.We have identified six serine and threonine autophosphorylation sites on the C‐terminus of Schizosaccharomyces pombe Hhp1, one of two soluble CK1 enzymes in this organism, and are testing candidate sites on Hhp2 and the human homologues CK1□/□. When these sites are specifically phosphorylated, the Hhp1 C‐terminus binds the kinase domain via a low‐affinity electrostatic interaction. At concentrations above the Kd of this interaction, the phosphorylated C‐terminus inhibits Hhp1 kinase activity, while mutations that abolish phosphorylation increase the activity of the full‐length kinase.Structural studies have identified two conserved basic patches on the CK1 kinase domain that are hypothesized to interact with the phosphorylated C‐terminus; however, we show that the tail instead interacts with the substrate binding pocket. The physiological substrate Sid4 has a higher affinity than the tail for the Hhp1 kinase domain, and the model substrate casein can out‐compete the phosphorylated C‐terminus for binding to the kinase domain. Our data support a new model of CK1 activation in which the presence of high‐affinity substrates is sufficient to displace the low‐affinity tail and relieve autoinhibition without the need for prior dephosphorylation of CK1.Because the C‐termini of CK1 family members are responsible for most of the sequence variation between isoforms, we hypothesize that these different tails may differ in affinity for the conserved kinase domain, leading to displacement by different cohorts of substrates. Thus, this mechanism may also explain variations in substrate specificities between CK1 enzymes. In support of this model, truncating the tail of Hhp1 or Hhp2 eliminates substrate‐specific differences in catalytic efficiency. We are currently using phosphorylation site mutants of hhp1 to confirm that these changes in catalytic efficiency are dependent on autophosphorylation and to investigate the significance of autophosphorylation in vivo.Support or Funding InformationSNC was supported by the Integrated Biological Systems Training in Oncology Program (2T32CA119925). This work was supported by NIH GM112989 to KLG.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The δ2 glutamate receptor gates long-term depression by coordinating interactions between two AMPA receptor phosphorylation sites

Long-term depression (LTD) commonly affects learning and memory in various brain regions. Although cerebellar LTD absolutely requires the δ2 glutamate receptor (GluD2) that is expressed in Purkinje cells, LTD in other brain regions does not; why and how cerebellar LTD is regulated by GluD2 remains unelucidated. Here, we show that the activity-dependent phosphorylation of serine 880 (S880) in GluA2 AMPA receptor subunit, which is an essential step for AMPA receptor endocytosis during LTD induction, was impaired in GluD2-null cerebellum. In contrast, the basal phosphorylation levels of tyrosine 876 (Y876) in GluA2 were increased in GluD2-null cerebellum. An in vitro phosphorylation assay revealed that Y876 phosphorylation inhibited subsequent S880 phosphorylation. Conversely, Y876 dephosphorylation was sufficient to restore S880 phosphorylation and LTD induction in GluD2-null Purkinje cells. Furthermore, megakaryocyte protein tyrosine phosphatase (PTPMEG), which binds to the C terminus of GluD2, directly dephosphorylated Y876. These data indicate that GluD2 gates LTD by coordinating interactions between the two phosphorylation sites of the GluA2.

Dephosphorylation-Induced Ubiquitination and Degradation of FMRP in Dendrites: A Role in Immediate Early mGluR-Stimulated Translation

Fragile X syndrome is caused by the loss of fragile X mental retardation protein (FMRP), which represses and reversibly regulates the translation of a subset of mRNAs in dendrites. Protein synthesis can be rapidly stimulated by mGluR-induced and protein phosphatase 2a (PP2A)-mediated dephosphorylation of FMRP, which is coupled to the dissociation of FMRP and target mRNAs from miRNA-induced silencing complexes. Here, we report the rapid ubiquitination and ubiquitin proteasome system (UPS)-mediated degradation of FMRP in dendrites upon DHPG (3,5-dihydroxyphenylglycine) stimulation in cultured rat neurons. Using inhibitors to PP2A and FMRP phosphomutants, degradation of FMRP was observed to depend on its prior dephosphorylation. Translational induction of an FMRP target, postsynaptic density-95 mRNA, required both PP2A and UPS. Thus, control of FMRP levels at the synapse by dephosphorylation-induced and UPS-mediated degradation provides a mode to regulate protein synthesis.

Cytoplasmic Regulators of β-Globin mRNA Are Structurally Modified During Erythroid Terminal Differentiation

Abstract 1093The high-level accumulation of β globin in mature erythrocytes requires a correspondingly high level of its encoding mRNA in terminally differentiating erythroid progenitors. We recently identified two RNA-binding proteins–AUF1 and YB1–that appear to regulate levels of β-globin mRNA in these cells by assembling a cytoplasm-restricted RNA-protein ’β-complex’ on its 3’UTR. The function of the β-complex was predicted by in vitro analyses mapping it to a cis-acting determinant of β-globin mRNA stability, and subsequently validated by in vivo siRNA studies demonstrating that simultaneous knockdown of AUF1 and YB1 ablated the β-complex and coordinately reduced the accumulation of β-globin mRNA in K562 cells. Although both AUF1 and YB1 are ubiquitously expressed, studies in cultured cells and in Epo-induced CD34+ primary cells indicated that their β-globin mRNA-specific regulatory properties are restricted to erythroid cells during later stages of terminal differentiation. Based upon these observations, we reasoned that AUF1 and YB1 undergo erythroid and differentiation stage-restricted alterations that permit their assembly into the mRNA-regulatory β-complex.Our analyses of AUF1 focused on three structural isoforms, observed in K562 cells, resulting from alternative pre-mRNA processing events that retain or exclude exon 7. GST-AUF1 fusion isoforms that retain exon 7 fail to bind the β-globin 3’UTR in vitro, while related isoforms that exclude exon 7 bind the 3’UTR with high efficiency. These results, which implicate the importance of exon 7 exclusion to AUF1 function, were subsequently validated in intact K562 cells using an AUF1 isotype-specific siRNA knockdown strategy. In these in vivo experiments, a reduction in exon 7-excluded AUF1 effected a two-fold decrease in steady-state β-globin mRNA, while similar reductions in exon 7-retained AUF1 isoforms had no measurable effect. The isotype-specific mRNA-binding characteristics of AUF1 may be particularly important during terminal differentiation: Epo-induced CD34+ cells display an increase in exon 7-excluded AUF1, paralleling their capacity to assemble a regulatory β-complex in vitro. Among several possible mechanisms, we asked whether the isoform-specific function of AUF1 might relate to the unusually high number (20) of phosphorylation-capable residues encoded by exon 7. In vitro analyses were fully consistent with this possibility, demonstrating that the β-globin mRNA-binding activity of exon 7-retained AUF1 could be restored by prior dephosphorylation. These experiments suggest that post-transcriptional regulation of β-globin mRNA during erythroid differentiation is likely to be effected by alternative splicing of AUF1 pre-mRNA that eliminates phosphorylation-active exon 7 amino-acids in the translated protein.Based upon these results, we reasoned that related post-translational processes might similarly regulate the β-globin mRNA-binding specificity of YB1. Our analyses focused on a specific residue (Ser102) that is a known target for regulatory phosphorylation and can be experimentally identified using a Ser102 phospho-specific YB1 antibody. In in vitro studies with K562 cytoplasmic extract, which contains both phospho- and dephospho- forms of YB1, we observed that only dephospho-YB1 adheres to the β-globin 3’UTR; likewise, in in vivo studies of CD34+ cells we noted a substantial increase in the ratio of dephospho:phospho-YB1 following Epo induction. Both experiments indicate the likely importance of this post-translational process to the function of YB1 during terminal differentiation. Confirmatory studies are currently being conducted in vivo using an epitope-tagged YB1 containing a position 102 Ser->Ala substitution.Collectively, our analyses indicate that the β-globin mRNA-binding specificities of AUF1 and YB1–and, hence, their corresponding regulatory activities–are determined by post-transcriptional and -translational events. This work suggests mechanisms through which erythroid progenitors can maintain dynamic regulatory control during an interval when transcriptional processes are beginning to silence, and identifies new pathways that can be therapeutically targeted in patients with congenital disorders of β-globin gene expression. Disclosures:No relevant conflicts of interest to declare.

The 1.4 Å crystal structure of the large and cold-active Vibrio sp. alkaline phosphatase

Alkaline phosphatase (AP) from the cold-adapted Vibrio strain G15-21 is among the AP variants with the highest known kcat value. Here the structure of the enzyme at 1.4 Å resolution is reported and compared to APs from E. coli, human placenta, shrimp and the Antarctic bacterium strain TAB5. The Vibrio AP is a dimer although its monomers are without the long N-terminal helix that embraces the other subunit in many other APs. The long insertion loop, previously noted as a special feature of the Vibrio AP, serves a similar function. The surface does not have the high negative charge density as observed in shrimp AP, but a positively charged patch is observed around the active site that may be favourable for substrate binding. The dimer interface has a similar number of non-covalent interactions as other APs and the “crown"-domain is the largest observed in known APs. Part of it slopes over the catalytic site suggesting that the substrates may be small molecules. The catalytic serines are refined with multiple conformations in both monomers. One of the ligands to the catalytic zinc ion in binding site M1 is directly connected to the crown-domain and is closest to the dimer interface. Subtle movements in metal ligands may help in the release of the product and/or facilitate prior dephosphorylation of the covalent intermediate. Intersubunit interactions may be a major factor for promoting active site geometries that lead to the high catalytic activity of Vibrio AP at low temperatures.

EDD Mediates DNA Damage-induced Activation of CHK2

EDD, the human orthologue of Drosophila melanogaster "hyperplastic discs," is overexpressed or mutated in a number of common human cancers. Although EDD has been implicated in DNA damage signaling, a definitive role has yet to be demonstrated. Here we report a novel interaction between EDD and the DNA damage checkpoint kinase CHK2. EDD and CHK2 associate through a phospho-dependent interaction involving the CHK2 Forkhead-associated domain and a region of EDD spanning a number of putative Forkhead-associated domain-binding threonines. Using RNA interference, we demonstrate a critical role for EDD upstream of CHK2 in the DNA damage signaling pathway. EDD is necessary for the efficient activating phosphorylation of CHK2 in response to DNA damage following exposure to ionizing radiation or the radiomimetic, phleomycin. Cells depleted of EDD display impaired CHK2 kinase activity and an inability to respond to DNA damage. These results identify EDD as a novel mediator in DNA damage signal transduction via CHK2 and emphasize the potential importance of EDD in cancer.

Phosphorylation of the Head Domain of Neurofilament Protein (NF-M): A FACTOR REGULATING TOPOGRAPHIC PHOSPHORYLATION OF NF-M TAIL DOMAIN KSP SITES IN NEURONS

In neurons the phosphorylation of neurofilament (NF) proteins NF-M and NF-H is topographically regulated. Although kinases and NF subunits are synthesized in cell bodies, extensive phosphorylation of the KSP repeats in tail domains of NF-M and NF-H occurs primarily in axons. The nature of this regulation, however, is not understood. As obligate heteropolymers, NF assembly requires interactions between the core NF-L with NF-M or NF-H subunits, a process inhibited by NF head domain phosphorylation. Phosphorylation of head domains at protein kinase A (PKA)-specific sites seems to occur transiently in cell bodies after NF subunit synthesis. We have proposed that transient phosphorylation of head domains prevents NF assembly in the soma and inhibits tail domain phosphorylation; i.e. assembly and KSP phosphorylation in axons depends on prior dephosphorylation of head domain sites. Deregulation of this process leads to pathological accumulations of phosphorylated NFs in the soma as seen in some neurodegenerative disorders. To test this hypothesis, we studied the effect of PKA phosphorylation of the NF-M head domain on phosphorylation of tail domain KSP sites. In rat cortical neurons we showed that head domain phosphorylation of endogenous NF-M by forskolin-activated PKA inhibits NF-M tail domain phosphorylation. To demonstrate the site specificity of PKA phosphorylation and its effect on tail domain phosphorylation, we transfected NIH3T3 cells with NF-M mutated at PKA-specific head domain serine residues. Epidermal growth factor stimulation of cells with mutant NF-M in the presence of forskolin exhibited no inhibition of NF-tail domain phosphorylation compared with the wild type NF-M-transfected cells. This is consistent with our hypothesis that transient phosphorylation of NF-M head domains inhibits tail domain phosphorylation and suggests this as one of several mechanisms underlying topographic regulation.

Phosphorylation-dependent interactions between enzymes of plant metabolism and 14-3-3 proteins.

Far-Western overlays of soluble extracts of cauliflower revealed many proteins that bound to digoxygenin (DIG)-labelled 14-3-3 proteins. Binding to DIG-14-3-3s was prevented by prior dephosphorylation of the extract proteins or by competition with 14-3-3-binding phosphopeptides, indicating that the 14-3-3 proteins bind to phosphorylated sites. The proteins that bound to the DIG-14-3-3s were also immunoprecipitated from extracts with anti-14-3-3 antibodies, demonstrating that they were bound to endogenous plant 14-3-3 proteins. 14-3-3-binding proteins were purified from cauliflower extracts, in sufficient quantity for amino acid sequence analysis, by affinity chromatography on immobilised 14-3-3 proteins and specific elution with a 14-3-3-binding phosphopeptide. Purified 14-3-3-binding proteins included sucrose-phosphate synthase, trehalose-6-phosphate synthase, glutamine synthetases, a protein (LIM17) that has been implicated in early floral development, an approximately 20 kDa protein whose mRNA is induced by NaCl, and a calcium-dependent protein kinase that was capable of phosphorylating and rendering nitrate reductase (NR) sensitive to inhibition by 14-3-3 proteins. In contrast to the phosphorylated NR-14-3-3 complex which is activated by dissociation with 14-3-3-binding phosphopeptides, the total sugar-phosphate synthase activity in plant extracts was inhibited by up to 40% by a 14-3-3-binding phosphopeptide and the phosphopeptide-inhibited activity was reactivated by adding excess 14-3-3 proteins. Thus, 14-3-3 proteins are implicated in regulating several aspects of primary N and C metabolism. The procedures described here will be valuable for determining how the phosphorylation and 14-3-3-binding status of defined target proteins change in response to extracellular stimuli.

Phosphorylation of CD45 by Casein Kinase 2: MODULATION OF ACTIVITY AND MUTATIONAL ANALYSIS

CD45 is a receptor-type protein-tyrosine phosphatase (PTP) that is required for antigen-specific stimulation and proliferation in lymphocytes. This study was designed to determine the nature of specific kinases in lymphocytes that phosphorylate CD45 and to determine the effect of phosphorylation on CD45 PTP activity. A major cytoplasmic lymphocyte kinase that phosphorylated CD45 was identified as casein kinase 2 (CK2) by use of an in-gel kinase assay in combination with immunoprecipitation, immunodepletion, and specific inhibition. Mutational analysis of CK2 consensus sites showed that the target for CK2 was in an acidic insert of 19 amino acids in the D2 domain, and Ser to Ala mutations at amino acids 965, 968, 969, and 973 abrogated CK2 phosphorylation of CD45. CK2 phosphorylation increased CD45 activity 3-fold toward phosphorylated myelin basic protein, and this increase was reversible by PP2A treatment. Mutation of Ser to Glu at the CK2 sites had the same effect as phosphorylation and also tripled the Vmax of CD45. CD45 isolated in vivo was highly phosphorylated and could not be phosphorylated by CK2 without prior dephosphorylation with phosphatase PP2A. We conclude that CK2 is a major lymphocyte kinase that is responsible for in vivo phosphorylation of CD45, and phosphorylation at specific CK2 sites regulates CD45 PTP activity.

The comparative efficiencies of the Ser( P)-, Thr( P)- and Tyr( P)-residues as specificity determinants for casein kinase-1

The β-casein derived phosphopeptide, Glu-Glu-Ser( P)-Glu-Glu-Ser-Ile-Thr-NHMe and two derivatives in which the Ser( P)-residue is replaced by the Thr( P)- and Tyr( P)-residue have been compared for their susceptibility to phosphorylation by casein kinase-1. While both the Ser( P)- and Thr( P)-peptides are good substrates with similar kinetic constants, the Tyr( P)-peptide is a substrate as poor as the unphosphorylated derivative EEEEESIT, exhibiting a 21-fold higher K m and 6-fold lower V max values. While prior dephosphorylation of the Ser( P)-peptide caused a marked loss in its phosphoacceptor capacity, prior dephosphorylation of the Tyr( P)-peptide caused no significant change in its poor phosphoacceptor capacity. Thus the order of efficiency of phosphoaminoacids as specificity determinants for casein kinase-1 was found to be Ser( P)=Thr( P)⪢Tyr( P) and this order is markedly different from Tyr( P)>Ser( P)⪢Thr( P) which was previously established for casein kinase-2 [Meggio et al. (1991) FEBS Lett. 279, 307–309].

Effects of the Selective α1-Antagonist, Doxazosin, on Hepatic Lipid Levels and Metabolism in the Golden Hamster

The effect of the selective alpha 1-antagonist, doxazosin, on lipid metabolism was studied in cholesterol-fed golden hamsters. The hamsters were studied after short-term (1 week) and long-term (6-8 weeks) treatment. Doxazosin was added to the food (0.05-0.1%). Doxazosin lowered, within 1 week, plasma cholesterol and triglyceride concentrations by 12 and 19%, respectively. These effects were slightly larger during long-term treatment (cholesterol by 15% and triglycerides by 27%). Lipoprotein lipase (LPL) activity in postheparin plasma or in adipose tissue or heart was affected by doxazosin. The hepatic triglyceride secretion rate was lowered by 40%. Doxazosin treatment partially prevented the accumulation of cholesterol and triglycerides in the liver. Hepatic cholesterol synthesis was decreased by 25-40%. When determined under optimal conditions, i.e., after prior dephosphorylation, the hepatic microsomal HMG-CoA reductase activity was lowered by 10-25% in the doxazosin-treated animals. However, if HMG-CoA reductase was determined under conditions to prevent phosphorylation and dephosphorylation of the enzyme, representing the in situ expressed activity, the activity in the doxazosin-treated animals was 40% lower than in the controls. These results indicate that the plasma lipid-lowering effect of doxazosin is largely due to its interference with hepatic lipid metabolism and that one of the effects is a lowering of hepatic cholesterol synthesis, probably due to an increase in the phosphorylation grade of HMG-CoA reductase.

Altered phosphorylation of tau protein in heat-shocked rats and patients with Alzheimer disease.

Six hours after heat shocking 2- to 3-month-old male and female Sprague-Dawley rats at 42 degrees C for 15 min, we analyzed tau protein immunoreactivity in SDS extracts of cerebrums and peripheral nerves by using immunoblot analysis and immunohistochemistry with the anti-tau monoclonal antibody Tau-1, which recognizes a phosphate-dependent non-phosphorylated epitope, and with 125I-labeled protein A. In the cerebral extracts, we found altered phosphorylation of tau in heat-shocked females, characterized by a marked reduction in the amount of nonphosphorylated tau, a doubling of the ratio of total (phosphorylated plus nonphosphorylated) tau to nonphosphorylated tau, and the appearance of the slowest moving phosphorylated tau polypeptide (68 kDa). Similar, but milder, changes were observed in male rats. These changes progressively increased in females from 3 to 6 h after heat shocking. In contrast, both phosphorylated tau and nonphosphorylated tau were reduced in peripheral nerves after heat shocking. In immunoblots of SDS extracts from Alzheimer disease-affected brain, the two slowest moving phosphorylated tau polypeptides (62 kDa and 66 kDa, respectively) were detected by Tau-1 after dephosphorylation and by Tau-2 (an anti-tau-monoclonal antibody that recognizes a phosphate-independent epitope) without prior dephosphorylation only in regions that contained tau immunoreactivity in histologic preparations. In addition, quantitative immunoblot analysis of cortex and the underlying white matter with Tau-1 and 125I-labeled protein A showed that the amount of phosphorylated tau progressively increased in the Alzheimer disease-affected cerebral cortex, while concurrently a proportionally lesser amount of tau entered the white matter axons. The similar findings for the rat heat-shock model and Alzheimer disease suggest that life stressors may play a role in the etiopathogenesis of Alzheimer disease.

Phosphotyrosine as a specificity determinant for casein kinase-2, a growth related Ser/Thr-specific protein kinase

The motif Ser-Ser-Ser-Glu-Glu is readily phosphorylated by casein kinase-2 (CK-2), a growth-related protein kinase whose consensus sequence is Ser(Thr)-Xaa-Xaa-Glu(Asp) [(1990) Biochim. Biophys. Acta. 1054, 267–283]. Here we show that phosphotyrosine can replace carboxylic acids as specificity determinant for CK-2 phosphorylation, the phosphotyrosyl peptide Ser-Ser-Ser-TyrP-TyrP actually being a substrate more efficient than Ser-Ser-Ser-Glu-Glu itself both in terms of K m (0.69 vs 2.43 mM) and V???. Prior dephosphorylation of phosphotyrosine entirely prevents the subsequent phosphorylation of serine by CK-2. While Ser-Ser-Ser-TyrP-TyrP is better than Ser-Ser-Ser-SerP-SerP, which in turn is better than Ser-Ser-Ser-Glu-Glu, Ser-Ser-Ser-ThrP-ThrP is a less efficient substrate than Ser-Ser-Ser-Glu-Glu. Thus the order of efficiency of phosphoamino acids as specificity determinants for CK-2 appears to be TyrP>SerP>ThrP.

Role of acidic phosphoproteins in the partial reconstitution of the active 60 S ribosomal subunit

We have recently shown that rat liver 60 S ribosomal subunits active in protein synthesis can be reconstituted from inactive core particles lacking 30% of the total proteins, mainly L10a, L12, L22, L24, A33 and the acidic phosphoproteins P1–P2, obtained by treatment of 60 S subunits with dimethylmaleic anhydride [(1987) Eur. J. Biochem. 163, 15–20]. In this study, an ethanol extract of the 60 S subunit which contains only P1–P2 was also shown to be effective in reconstitution with the DMMA-core-particies: it strongly stimulated the EF-2-dependent GTP hydrolysis and, to a lesser extent, polyphenylalanine synthesis; like the DMMA wash it shifted the thermal denaturation curve of the DMMA-core particles towards that of control subunits. Prior dephosphorylation of the ethanol extract by alkaline phosphatase inhibited the reconstruction process.

Uptake and utilization of deoxynucleoside 5'-monophosphates by Mycoplasma mycoides subsp. mycoides.

Mycoplasma mycoides subsp. mycoides has been shown to possess an unusual capacity for the uptake and utilization of exogenous deoxyribonucleoside 5'-monophosphates intact without prior dephosphorylation. In this study, it was found that once inside the cell, deoxyribonucleoside 5'-monophosphates were rapidly phosphorylated to the triphosphate level and incorporated into DNA. Catabolism of deoxyribonucleoside 5'-monophosphates was also observed. Competition studies indicated that a single uptake system with a higher affinity for deoxyribonucleotides mediates the uptake of nucleoside 5'-monophosphates.

The pathway of AMP catabolism and its control in isolated rat hepatocytes subjected to anoxia.

In anoxic conditions, the hepatic concentrations of high energy phosphates, most notably ATP, decrease to a marked extent, whereas those of AMP and Pi increase several-fold (Deuticke and Gerlach, 1966; Busch et al., 1968; Hems and Brosnan, 1970; Jackson et al., 1976). The exact pathway of the further catabolism of AMP in this situation has been a subject of controversy: whereas Deuticke and Gerlach (1966) have proposed that the initial degradation of AMP occurs by way of AMP deaminase and is followed by the dephosphorylation of IMP by 5′-nucleotidase, Busch et al. (1968) have asserted that the degradation of AMP involves a prior dephosphorylation by the same enzyme, followed by deamination of adenosine by adenosine deaminase. The present study was undertaken with isolated rat hepatocytes in order to try to solve this controversy. As in our previous work, coformycin was used to determine the pathway of degradation of AMP. In the liver, this inosine analog inhibits selectively adenosine deaminase at concentrations around 0.1 μM, whereas at concentrations around 50 μM, it also inhibits AMP deaminase (Van den Berghe et al., 1980). If the initial degradation of AMP occurs by way of 5′-nucleotidase, the low concentration of coformycin would decrease the formation of the terminal products of adenine nucleotide catabolism. If, however, the initial step of the degradation of AMP is catalyzed by AMP deaminase, the decrease in the production of purine catabolites would be observed only at high concentrations of coformycin. Further details concerning the experiments reported and a description of the methodology can be found in Vincent et al. (1982).

Pathways of pyrimidine deoxyribonucleotide biosynthesis in Mycoplasma mycoides subsp. mycoides.

By measuring the specific activity of deoxyribonucleotides isolated from DNA after the incorporation of 14C-labeled precursors with and without competition from other nucleotide precursors, we defined the major pathways of pyrimidine deoxyribonucleotide synthesis in Mycoplasma mycoides subsp. mycoides. Uracil, guanine, and thymine are required for the synthesis of nucleotides. Cytidine competed effectively with uracil to provide all of the deoxycytidine nucleotide, as well as most of the deoxyribose-1-phosphate, for the synthesis of thymidylate from thymine via thymidine phosphorylase. Each of dUMP, dCMP, and dTMP competed with cytidine for incorporation into DNA thymidylate. Appreciable incorporation of exogenous deoxyribonucleoside 5'-monophosphates into DNA without prior dephosphorylation was observed. Dephosphorylation also occurred since the added deoxyribonucleotide provided phosphate for the synthesis of the other nucleotides in DNA in competition with the 32Pi in the growth medium. Hydroxyurea inhibited cell growth and decreased the intracellular level of dATP, consistent with the action of a ribonucleoside diphosphate reductase with regulatory properties similar to those of the Escherichia coli enzyme.

Comparison of the liver glycogen synthase from normal and streptozotocin-induced diabetic rats

Glycogen synthase in the liver extracts of short-term (3 days) streptozotocin-induced diabetic rats is poorly activated by the endogenous synthase phosphatase as well as phosphatase(s) from the liver extracts of normal animals. However, synthase in the liver extracts of diabetic rats is readily activated by the 35,000 M r rabbit liver protein phosphatase (H. Brandt, F. L. Capulong, and E. Y. C. Lee ( J. Biol. Chem. 250, 8038–8044 (1975)). The purified synthases from normal and diabetic animals respond differently after incubations with three different phosphatases. Both normal and diabetic synthase are activated by the 35,000 M r protein phosphatase; however, the total activity of diabetic, but not the normal, synthase is significantly increased. Normal, but not the diabetic, synthase is activated by a synthase phosphatase from normal rats; this activation is accompanied by an increase in total synthase activity. Incubation of the diabetic synthase with calf intestinal alkaline phosphatase results in a reduction of the total synthase activity, whereas synthase activity of the normal is not significantly affected. The reduction in total activity of the diabetic synthase by treatment with alkaline phosphatase was prevented by prior dephosphorylation with 35,000 M r rabbit liver protein phosphatase. In addition to their differences in responses to different phosphatases, the normal and diabetic synthases are also different in their molecular weights as determined by sucrose density gradient centrifugation (154,000 ± 17,000 ( n = 6) for the normal and 185,000 ± 15,000 ( n = 8) for the diabetic synthase) and their kinetic properties.

Adenine Nucleotide Degradation by the Obligate Intracellular Bacterium Rickettsia typhi

Adenosine 5'-triphosphate (ATP) was catabolized by whole cells and cell-free extracts of Rickettsia typhi to adenosine 5'-diphosphate (ADP) and then to adenosine 5'-monophosphate (AMP), the end product of ATP catabolism under the experimental conditions used. The only intermediate of the pathway from ATP to AMP which was identified by thin-layer chromatography and quantitated by the (14)C content was ADP, whereas products such as adenine, adenosine, hypoxanthine, inosine, and inosine 5'-monophosphate were not detected. The enzymes which could be theoretically responsible for the catabolism or the anabolism of AMP were not detected by standard assay procedures. Most importantly, 5'-nucleotidase or nonspecific phosphatase and AMP nucleosidase activities were undetectable under a variety of experimental conditions. Although these two enzymes remove AMP from the adenylate pool in other cells, they are apparently nonfunctional in R. typhi. The biosynthesis of ATP was initiated by adenylate kinase because no adenine phosphoribosyltransferase or adenosine kinase could be detected. Furthermore, AMP was transported intact without prior dephosphorylation. These observations suggest that for R. typhi the in vivo activity of adenine nucleotide interconversion was limited to the nucleotides, with AMP being the end product of ATP catabolism, and that the salvage of purine bases and nucleosides was not an essential feature of purine metabolism. These results elucidate the findings of a previous study which showed that in the absence of glutamate as a source of energy, the adenylate energy charge of resting cells of R. typhi is drastically lowered by the high proportion of AMP.