Creating a common language: defining individualized, personalized and precision prevention in public health.

Background: Because of the limited success of population-based prevention methods and due to developments in genomic screening, public health professionals and health policy makers are increasingly interested in more individualized prevention strategies. However, the terminology applied in this field is still ambiguous and thus has the potential to create misunderstandings. Methods: A narrative literature review was conducted to identify how individualized, personalized and precision prevention are used in research papers and documents. Based on the findings a set of definitions were created that distinguish between these activities in a meaningful way. Results: Definitions were found only for precision prevention, not for individualized or personalized prevention. The definitions of individualized, personalized and precision medicine were therefore used to create the definitions for their prevention counterparts. By these definitions, individualized prevention consists of all types of prevention that are individual-based; personalized prevention also consists of at least one form of -omic screening; and precision prevention further includes psychological, behavioral and socioeconomic data for each patient. Conclusions: By defining these three key terms for different types of individual-based prevention both researchers and health policy makers can differentiate and use them in their proper context.

The phosphorylation state of threonine-220, a uniquely phosphatase-sensitive protein kinase A site in microtubule-associated protein MAP2c, regulates microtubule binding and stability.

Phosphorylation of microtubule-associated protein 2 (MAP2) has a profound effect on microtubule stability and organization. In this work a consensus protein kinase A (PKA) phosphorylation site, T(220), of juvenile MAP2c is characterized. As confirmed by mass spectrometry, this site can be phosphorylated by PKA but shows less than average reactivity among the 3.5 +/- 0.5 phosphate residues incorporated into the protein. In contrast, T(220) is uniquely sensitive to dephosphorylation: three major Ser/Thr protein phosphatases, in the order of efficiency PP2B > PP2A(c) > PP1(c), remove this phosphate group first. MAP2c specifically dephosphorylated at this site binds and stabilizes microtubules stronger than either fully phosphorylated or nonphosphorylated MAP2c. Phosphorylation of this site also affects proteolytic sensitivity of MAP2c, which might represent a further level of control in this system. Thus, the phosphorylation state of T(220) may be a primary determinant of microtubule function.

Functional analysis of the Neurospora crassa PZL-1 protein phosphatase by expression in budding and fission yeast.

The gene pzl-1 from the filamentous fungus Neurospora crassa encodes a putative Ser/Thr protein phosphatase that is reminiscent of the Ppz1/Ppz2 and Pzh1 phosphatases from Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The entire PZL-1 protein, as well as its carboxyl-terminal domain, have been expressed in Escherichia coli as active protein phosphatases. To characterize its cellular role, PZL-1 was also expressed in Sz. pombe and in S. cerevisiae. Expression of PZL-1 in S. cerevisiae from the PPZ1 promoter was able to rescue the altered sensitivity to caffeine and lithium ions of a ppz1 strain. Furthermore, high copy number expression of PZL-1 alleviated the lytic phenotype of a S. cerevisiae slt2/mpk1 mitogen-activated protein (MAP) kinase mutant, similarly to that described for PPZ1, and mimicked the effects of high levels of Ppz1 on cell growth. Expression of PZL-1 in fission yeast from a weak version of the nmt1 promoter fully rescued the growth defect of a pzh1Delta strain in high potassium, but only partially complemented the sodium-hypertolerant phenotype. Strong overexpression of the N. crassa phosphatase in Sz. pombe affected cell growth and morphology. Therefore, PZL-1 appears to fulfil every known function carried out by its S. cerevisiae counterpart, despite the marked divergence in sequence within their NH(2)-terminal moieties.

Protein phosphatase 2A holoenzyme and its subunits from Medicago sativa.

We detected an about 200 kDa holoenzyme of protein phosphatase 2A (PP2A) in the crude extract of Medicago sativa microcallus cells by gel permeation chromatography. By polymerase chain reaction (PCR) we isolated two M. sativa cDNA fragments corresponding to the catalytic (C) subunit, and one each coding for the A and the B regulatory subunits of PP2A. The C subunit sequences were different from that published previously, indicating the existence of at least three different isoforms in M. sativa. Using the PCR fragments as probes, we obtained two distinct full-length clones for both the A and B subunits from an alfalfa cDNA library. Our results demonstrate that the components of the PP2A holoenzyme, namely the catalytic and regulatory subunits, are present in alfalfa in several isoforms and that their sequences are highly similar to their plant, yeast and animal counterparts. The distinct regulatory subunit genes are constitutively expressed during the cell cycle. Interestingly, two A-B subunit pairs had parallel mRNA steady-state levels in different plant tissues suggesting that not all of the possible isoform combinations are present in all tissues. The expression of the MsPP2A Bbeta subunit form was induced by abscisic acid indicating a specific function for this protein in the stress response.

Inhibition of serine/threonine-specific protein phosphatases causes premature activation of cdc2MsF kinase at G2/M transition and early mitotic microtubule organisation in alfalfa.

Reversible phosphorylation of serine/threonine residues of cell cycle-regulatory proteins is one of the key molecular mechanisms controlling eukaryotic cell division. In plants, the protein kinase partners (i.e. p34cdc2/CDC28-related kinases) have been extensively studied, while the role of counter-acting protein phosphatases is less well understood. We used endothall (ET) as a cell-permeable inhibitor of serine/threonine-specific protein phosphatases to alter cytological and biochemical characteristics of cell division in cultured alfalfa cells. A high concentration of ET (10 and 50 microM) inhibited both protein phosphatases 1 and 2 (PP1 and PP2A), while a low concentration (1 microM) of ET-treatment primarily reduced the PP2A activity. High concentrations of the inhibitor increased the frequency of hypercondensed early and late prophase chromosomes that could not enter metaphase. In contrast, a low concentration of ET did not interfere with chromosomal events but caused significant alterations in the organisation of microtubules. Exposure of cells to 1 microM ET resulted in disturbance of preprophase band formation, increase in the number of nuclei with prophase microtubule assembly, premature polarisation of the spindle, and abnormal phragmoplast maturation. Under the same conditions, the ET-treated cells exhibited an early increase in cdc2MsF kinase activity. These results suggest that PP2A contributes to the control of mitotic kinase activities and microtubule organisation. Normal chromosome condensation and mitotic progression are dependent on both PP1 and PP2A activities. The presented data support the functional role of protein phosphatases in the co-ordination of chromosomal and microtubule events in dividing plant cells.

A mutation within the catalytic domain of COT1 kinase confers changes in the presence of two COT1 isoforms and in Ser/Thr protein kinase and phosphatase activities in Neurospora crassa.

Neurospora crassa grows by forming spreading colonies. cot-1 belongs to a class of N. crassa colonial temperature-sensitive (cot) mutants and encodes a Ser/Thr protein kinase. We have mapped the cot-1 mutation to a single base change resulting in a His to Arg substitution at amino acid 351, which resides within the catalytic domain. Antibodies raised against COT1 detected and immunoprecipitated a predominant 73-kDa polypeptide in N. crassa extracts, whose abundance was constant under all growth conditions tested. An additional, lower MW COT1 isoform (67-kDa) present in the wild-type was not detected in cot-1 grown at the restrictive temperature. Similarly, this isoform was not detected in cot-3 or cot-5 strains, when grown at restrictive temperatures. Reduced levels of Ser/Thr kinase activity and an increase in type 1 and type 2B phosphatase (calcineurin) activities were measured in a cot-1 background. Apparent changes in the phosphorylation state of the p150(Glued) subunit of the dynactin cytoskeletal motor component (encoded by ro-3, a suppressor of cot-1) and evidence of in vitro physical interactions between COT1 and calcineurin indicate a functional linkage among COT1 kinase, type 2B phosphatase, and dynactin.

Structural and functional characterization of the Drosophila glycogen phosphorylase gene.

We identified a P element insertional mutant of the Drosophila glycogen phosphorylase (DGPH) gene. Glycogen phosphorylase protein concentration and enzyme activity are decreased while glycogen content is increased in flies homozygous for the mutant allele. The DGPH gene has been cloned and sequenced; its open reading frame codes for a protein of 844 amino acids with a predicted molecular mass of 97 kDa. Comparison of the conceptual amino acid sequence of the Drosophila glycogen phosphorylase with glycogen phosphorylase sequences from other organisms shows a high degree of homology to mammalian enzymes. All the residues of the allosteric effector binding sites, the active site, and the site of phosphorylation are exactly conserved, but some of the residues of the glycogen storage site are not.

Protein phosphatase 1 catalytic subunit isoforms from alfalfa: biochemical characterization and cDNA cloning.

The catalytic subunit of protein phosphatase 1 (PP1c) was purified from an alfalfa (Medicago sativa) microcallus cell culture. The preparation was inhibited by rabbit muscle inhibitor-2 and okadaic acid and had a molecular mass of 35 kDa. Five distinct cDNAs termed MsPP1alpha, -beta, -gamma, -delta, and -epsilon were cloned from a M. sativa somatic embryo library. MsPP1alpha was identical to a cDNA reported earlier [A. Páy, M. Pirck, L. Bögre, H. Hirt, and E. Heberle-Bors Mol. Gen. Genet. 244, 176-182, 1994], while the others represented novel isoforms encoded by separate genes. The predicted amino acid sequences of MsPP1alpha, -beta, -gamma, -delta, and -epsilon were highly similar to each other and to other known PP1c sequences. The GST-MsPP1ss fusion protein expressed in Escherichia coli was catalytically active and was inhibited by inhibitor-2 and okadaic acid. Affinity-purified polyclonal MsPP1antipeptide antibody detected a protein of 36 kDa in crude cell extracts. These results proved that the cDNA clone encoded an active PP1c which was very similar to the purified enzyme. The mRNA and protein concentrations of PP1c as well as the specific activity of protein phosphatase 1 did not change during the cell cycle in a synchronized alfalfa cell culture. On the other hand, the isoforms exhibited different steady-state mRNA levels in different plant organs suggesting tissue-specific functions.

Protein phosphatase 2A is involved in hyphal growth of Neurospora crassa.

Cantharidin and calyculin A, natural toxins that are inhibitors of protein phosphatases 1 and 2A (PPI and PP2A, respectively). inhibit Neurospora crassa hyphal growth. When N. crassa was grown in the presence of either drug, abnormalities were observed at hyphal tips. In addition, both drugs induced an increase in hyphal branching. Cantharidin inhibited N. crassa hyphal growth in a temperature-dependent manner, as the effect of the drug was more pronounced at 34 degrees C than at 25 degrees C. In addition to the drug-mediated inhibition of phosphatase activity, a genetic approach was used to determine the phenotypic consequences of reduced PP2A activity. Two strains with subnormal PP2A activity were constructed. The first, in which the original pph-1 gene (encoding the PP2A catalytic subunit) was replaced with an ectopically integrated copy of pph-1, exhibited lower levels of pph-1 transcript, lower PP2A activity and increased sensitivity to cantharidin. Similarly, in a second strain, in which the pph-1 gene was cloned in an antisense orientation downstream of the inducible isocitrate lyase promoter, lower levels of pph-1 transcript, as well as of PP2A activity, and a reduction in hyphal growth were observed. The results of this study indicate that PP2A, and probably other Ser/Thr phosphatases, are involved in the regulation of hyphal growth in N. crassa.

pzl-1 encodes a novel protein phosphatase-Z-like Ser/Thr protein phosphatase in Neurospora crassa.

The gene and cDNA of a novel protein phosphatase were cloned from Neurospora crassa. The pzl-1 gene encompasses three introns and is localized to the left arm of chromosome I between cyt-21 and Fsr-12. It encodes a protein of 58.3 kDa containing a Ser/Pro rich N-terminal segment, and a C-terminal domain that is similar to the catalytic subunit of type 1 protein phosphatases. The first 51 amino acid residues, including a potential N-myristoylation site, as well as the C-terminal domain (about 300 residues) have a high level of sequence identity with yeast PPZ phosphatases. However, residues 52-208 do not share high similarity with other proteins. The mRNA of pzl-1 was detected in all phases of asexual development of the filamentous fungus.

Cloning of a novel testis specific protein serine/threonine phosphatase, PPN 58A, from Drosophila melanogaster.

A gene encoding a novel member of the PPP family of protein serine/threonine phosphatases, termed PPN 58A, was cloned from Drosophila melanogaster. The deduced amino acid sequence of PPN 58A exhibits 59-62% identity to D. melanogaster PP1 isoforms, 51% identity to D. melanogaster PPY 55A and < or = 40% identity to other members of the PPP family. The single copy gene PPN 58A maps to chromosome 2 locus 58A. Analysis of PPN 58A mRNA reveals that, like PPY 55A, PPN 58A is a testis specific enzyme.

The catalytic subunits of Ser/Thr protein phosphatases from Caenorhabditis elegans.

The catalytic activities of protein phosphatase 1, 2A, 2B, and 2C were detected in crude extracts of Caenorhabditis elegans with different phosphoprotein substrates and specific inhibitors or activators. The enzymological properties of protein phosphatase 2B as well as those of the catalytic subunits of protein phosphatase 1 and protein phosphatase 2A were determined after partial purification. Gene fragments encoding the catalytic subunits of the protein phosphatase 1-2A-2B superfamily were amplified by polymerase chain reaction and were identified by DNA sequencing. Besides the homologs of protein phosphatase 1, 2B, and X, five protein phosphatase 1-type sequences and four novel protein phosphatase sequences were found. Our data, together with the results of the C. elegans genome project, suggest that this nematode contains an extensive family of Ser/Thr specific protein phosphatases including several up to now biochemically uncharacterized members.

Purification and characterization of the catalytic subunit of protein phosphatase 1 from Neurospora crassa.

Protein phosphorylation is a universal regulatory mechanism in eukaryotic cells. The phosphorylation state of proteins is affected by the antagonistic activities of protein kinases and phosphatases. Protein phosphatases (PPs) can be classified as serine/threonine and tyrosine specific phosphatases. Ser/Thr phosphatases are divided into four subclasses (PP1, PP2A, PP2B, PP2C) on the basis of their substrate specificity, metal ion dependence and inhibitor sensitivity. We were able to detect the activities of all four Ser/Thr protein phosphatases in the mycelial extract of Neurospora crassa. The catalytic subunit of PP1 was purified 1500-fold with a yield of 1.3% using ammonium sulfate-ethanol precipitation, DEAE-Sephacel, heparin-Sepharose and MonoQ FPLC chromatography. The protein product was nearly homogenous, as judged by SDS-polyacrylamide gel electrophoresis. The most important properties of the enzyme were the following: /1/ its molecular mass proved to be 35 kD, /2/ it was completely inhibited by inhibitor-2, microcystin and okadaic acid, /3/ it was bound to heparin-Sepharose, and /4/ its specific activity was 2000 mU/mg. These biochemical properties are very similar to those of the homologous enzyme from rabbit muscle and indicate a high level of conservation of PP1 structure during evolution.

Isolation and characterization of the catalytic subunit of protein phosphatase 2A from Neurospora crassa.

The catalytic subunit of protein phosphatase 2A (PP2Ac) was purified from Neurospora crassa extract by (NH4)2SO4-ethanol precipitation followed by DEAE-Sephacel, heparin-Sepharose, and MonoQ chromatography steps about 900-fold to a specific activity of 1200 U/g with a 2% yield. The apparent M(r) of PP2Ac was estimated to be 35 kDa by gel filtration and 33 kDa by SDS polyacrylamide gel electrophoresis. Half maximal inhibition of PP2Ac was achieved at 0.3 nM okadaic acid, 0.1 nM microcystin-LR, 56 nM cantharidin and 280 nM endothall concentrations. The preparation was completely inhibited by 20 mM NaF, was insensitive to rabbit muscle inhibitor-2, and was specific for the alpha-subunit of rabbit muscle phosphorylase kinase. According to its biochemical properties, N. crassa PP2Ac is very similar to its mammalian counterparts. Antipeptide antibodies raised against the N-terminal and C-terminal ends of human PP2Ac did not cross-react with N. crassa PP2Ac, indicating sequence differences outside the catalytic core of the enzyme.

Dephosphorylation of tau protein from Alzheimer's disease patients.

Tau protein-prepared post-mortem from brains of Alzheimer's disease patients was treated with protein phosphatase 1 catalytic subunit, 2A catalytic subunit, and 2B (calcineurin). Dephosphorylation was monitored by immunoblotting with two monoclonal antibodies, TAU-1 and SMI31, which recognize in tau the dephospho- and phospho-states, respectively, of proline-directed protein kinase phosphorylation sites. Out of the three enzymes tested, protein phosphatase 2A was only effective in dephosphorylating tau at these Alzheimer-type epitopes.

Drosophila protein phosphatase V functionally complements a SIT4 mutant in Saccharomyces cerevisiae and its amino-terminal region can confer this complementation to a heterologous phosphatase catalytic domain.

The sequence of a Drosophila melanogaster cDNA encoding a novel 35 kDa protein serine/threonine phosphatase, termed PPV, is presented. PPV is 40-41% identical to Drosophila PP1, 53% identical to Drosophila PP2A and 63% identical to Saccharomyces cerevisiae SIT4. Complementation studies demonstrated that PPV can functionally rescue a temperature sensitive mutant of SIT4, a protein phosphatase required for the G1 to S transition of the cell cycle. When placed under the SIT4 promoter, PPV cDNA is able to replace the SIT4 gene in S. cerevisiae. The amino-terminal domain of PPV fused to another phosphatase catalytic region (PP1) also rescues the temperature sensitive SIT4 mutant and the SIT4 deletion mutant, implicating this region in binding to regulatory subunits and/or altering specificity. In Drosophila, a substantial transient increase in both PPV mRNA and protein occurs in late syncytial and early cellular blastoderm embryos. At the latter stage PPV is localized to the cytoplasm of cells at the cortex. This increase in PPV correlates with introduction of the G2 phase of the cell cycle, elevated zygotic transcription and cellularization, indicating that PPV may play a role in one or more of these processes.

Reactive oxygen species mediate phorbol ester-regulated tyrosine phosphorylation and phospholipase A2 activation: potentiation by vanadate.

We have previously shown that vanadate potentiates the activating effect of phorbol ester (TPA) on cellular phospholipase A2 (PLA2) in a pathway dependent on the formation of reactive oxygen species (ROS). Here we evaluate the chain of enzymes (protein kinases and phosphatases) that participate in this process. Treatment of macrophages with vanadate plus TPA led to activation of protein kinase C (PKC) and NADPH oxidase (O2- generation in intact cells), massive cellular protein tyrosine phosphorylation, suppression of protein tyrosine phosphatase (PTP) activity and a sustained activation of protein tyrosine kinase (PTK) and myelin basic protein kinase activity (the latter three enzyme activities were assessed in cell lysates). Inhibition of ROS formation by diphenyleneiodonium (DPI) prevented PTP inhibition, PTK activation and protein tyrosine phosphorylation by vanadate plus TPA. Vanadate plus H2O2 mimicked the effect of vanadate plus TPA on PKC activation, cellular protein tyrosine phosphorylation, PTP and PTK, but their effects were resistant to DPI. Suppression of PKC activity (down-regulation; selective inhibitors) prevented the above-mentioned effects of vanadate plus TPA, but not of vanadate plus H2O2. Collectively, the results show that ROS formation induced by TPA in association with vanadate is essential in the modulation of protein tyrosine phosphorylation and PLA2 activity.

Mutations in the protein phosphatase 1 gene at 87B can differentially affect suppression of position-effect variegation and mitosis in Drosophila melanogaster.

The suppressor of position effect variegation (PEV) locus Su-var(3)6 maps to 87B5-10. The breakpoints of deficiencies that define this interval have been placed on a 250-kb molecular map of the region. The locus is allelic to the ck19 complementation group previously shown to encode a type 1 serine-threonine protein phosphatase (PP1) catalytic subunit. When introduced into flies by P element-mediated transformation, a 5.8-kb genomic fragment carrying this gene overcomes the suppressor phenotype of Su-var(3)6(01) and recessive lethality of all mutations of the locus. Four of the mutant alleles at the locus show a broad correlation between high levels of suppression of PEV, a high frequency of aberrant mitosis and low PP1 activity in larval extracts. However, some alleles with low PP1 activity show weak suppression of PEV with a high frequency of abnormal mitosis, whereas others show strong suppression of PEV with normal mitosis. The basis for these discussed.

Dephosphorylation of distinct sites on microtubule-associated protein MAP1B by protein phosphatases 1, 2A and 2B.

Rat brain microtubule-associated protein MAP1B has been tested as a substrate for Ser/Thr protein phosphatases (PP). The dephosphorylation reactions were followed by specific antibodies recognizing phosphorylated and phosphorylatable epitopes. One set of phosphorylation sites on MAP1B are referred to as mode I sites, and their phosphorylation is presumably catalyzed by proline-directed protein kinases. These mode I sites are efficiently dephosphorylated by PP2B and 2A but not by PP1. Another set of phosphorylation sites on MAP1B are named mode II sites, and their phosphorylation is possibly due to casein kinase II. These mode II sites are dephosphorylated by PP2A and PP1, the PP2B being ineffective. The selectivity of phosphatases for different sites within the same protein indicates the complexity of the dephosphorylation reactions regulating the functionality of MAP1B in neurons.

Protein phosphatase 1-deficient mutant Drosophila is affected in habituation and associative learning.

The learning and memory of Drosophila melanogaster strains carrying the Su-var(3)6(01) mutation, which is known to affect the structural gene of a protein phosphatase 1 isoenzyme, PP1(87B), were studied in various behavioral paradigms. Three lines of Drosophila comprising the Su-var(3)6(01) mutation in different genetic backgrounds were shown to have diminished protein phosphatase 1 activity and behavioral anomalies. Associative olfactory learning and visual conditioning were impaired. Olfactory acuity for the odorants used and response to electric shock were largely unchanged in the mutant lines. The motility and flight activity of the mutants were reduced. Habituation of the landing response, a nonassociative learning process, was more pronounced in heterozygotes of the mutants than in the wild-type control strains. Taken together with earlier data, the results indicate that protein phosphatase PP1(87B), while affecting several cellular processes, is also part of the biochemical machinery of various forms of neuromodulation in Drosophila.