scholarly journals The variable subunit associated with protein phosphatase 2A0 defines a novel multimember family of regulatory subunits

1996 ◽  
Vol 317 (1) ◽  
pp. 187-194 ◽  
Author(s):  
Stanislaw ZOLNIEROWICZ ◽  
Christine VAN HOOF ◽  
Nataša ANDJELKOVIĆ ◽  
Peter CRON ◽  
Ilse STEVENS ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Phosphatase ◽  
Amino Acid Sequences ◽  
Regulatory Subunits ◽  
Consensus Sequences ◽  
Reverse Transcription Pcr ◽  
Genes Encoding ◽  
Phosphatase 2A ◽  
Molecular Masses ◽  
Specific Mrnas

Two protein phosphatase 2A (PP2A) holoenzymes were isolated from rabbit skeletal muscle containing, in addition to the catalytic and PR65 regulatory subunits, proteins of apparent molecular masses of 61 and 56 kDa respectively. Both holoenzymes displayed low basal phosphorylase phosphatase activity, which could be stimulated by protamine to an extent similar to that of previously characterized PP2A holoenzymes. Protein microsequencing of tryptic peptides derived from the 61 kDa protein, termed PR61, yielded 117 residues of amino acid sequence. Molecular cloning by enrichment of specific mRNAs, followed by reverse transcription–PCR and cDNA library screening, revealed that this protein exists in multiple isoforms encoded by at least three genes, one of which gives rise to several splicing variants. Comparisons of these sequences with the available databases identified one more human gene and predicted another based on a rabbit cDNA-derived sequence, thus bringing the number of genes encoding PR61 family members to five. Peptide sequences derived from PR61 corresponded to the deduced amino acid sequences of either α or β isoforms, indicating that the purified PP2A preparation was a mixture of at least two trimers. In contrast, the 56 kDa subunit (termed PR56) seems to correspond to the ϵ isoform of PR61. Several regulatory subunits of PP2A belonging to the PR61 family contain consensus sequences for nuclear localization and might therefore target PP2A to nuclear substrates.

scholarly journals Characterization of Novel Carbazole Catabolism Genes from Gram-Positive Carbazole Degrader Nocardioides aromaticivorans IC177

2006 ◽  
Vol 72 (5) ◽  
pp. 3321-3329 ◽  
Author(s):  
Kengo Inoue ◽  
Hiroshi Habe ◽  
Hisakazu Yamane ◽  
Hideaki Nojiri
Keyword(s):  
Amino Acid ◽  
Gene Clusters ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Class Iii ◽  
Rt Pcr ◽  
Gram Positive ◽  
Mononuclear Iron ◽  
Reverse Transcription Pcr ◽  
Genes Encoding

ABSTRACT Nocardioides aromaticivorans IC177 is a gram-positive carbazole degrader. The genes encoding carbazole degradation (car genes) were cloned into a cosmid clone and sequenced partially to reveal 19 open reading frames. The car genes were clustered into the carAaCBaBbAcAd and carDFE gene clusters, encoding the enzymes responsible for the degradation of carbazole to anthranilate and 2-hydroxypenta-2,4-dienoate and of 2-hydroxypenta-2,4-dienoate to pyruvic acid and acetyl coenzyme A, respectively. The conserved amino acid motifs proposed to bind the Rieske-type [2Fe-2S] cluster and mononuclear iron, the Rieske-type [2Fe-2S] cluster, and flavin adenine dinucleotide were found in the deduced amino acid sequences of carAa, carAc, and carAd, respectively, which showed similarities with CarAa from Sphingomonas sp. strain KA1 (49% identity), CarAc from Pseudomonas resinovorans CA10 (31% identity), and AhdA4 from Sphingomonas sp. strain P2 (37% identity), respectively. Escherichia coli cells expressing CarAaAcAd exhibited major carbazole 1,9a-dioxygenase (CARDO) activity. These data showed that the IC177 CARDO is classified into class IIB, while gram-negative CARDOs are classified into class III or IIA, indicating that the respective CARDOs have diverse types of electron transfer components and high similarities of the terminal oxygenase. Reverse transcription-PCR (RT-PCR) experiments showed that the carAaCBaBbAcAd and carDFE gene clusters are operonic. The results of quantitative RT-PCR experiments indicated that transcription of both operons is induced by carbazole or its metabolite, whereas anthranilate is not an inducer. Biotransformation analysis showed that the IC177 CARDO exhibits significant activities for naphthalene, carbazole, and dibenzo-p-dioxin but less activity for dibenzofuran and biphenyl.

scholarly journals A novel 50 kDa protein forms complexes with protein phosphatase 4 and is located at centrosomal microtubule organizing centres

2000 ◽  
Vol 347 (3) ◽  
pp. 845-855 ◽  
Author(s):  
C. James HASTIE ◽  
Graeme K. CARNEGIE ◽  
Nick MORRICE ◽  
Patricia T. W. COHEN
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Amino Acid ◽  
Protein Phosphatase ◽  
Amino Acid Sequences ◽  
Regulatory Subunit ◽  
Mammalian Skeletal Muscle ◽  
Microtubule Organization ◽  
Microtubule Organizing Centres ◽  
Molecular Masses

Protein phosphatase 4 (PPP4) is a protein serine/threonine phosphatase that has been implicated in microtubule organization at centrosomes. Complexes of PPP4 with high apparent molecular masses (450 and 600 kDa) were purified from mammalian skeletal muscle and testis to near homogeneity. Amino acid sequences derived from a protein component present in both complexes were utilized to identify a human cDNA. The encoded putative PPP4 regulatory subunit (termed PPP4R2), comprising 453 amino acids, had a molecular mass of 50.4 kDa. The interaction of PPP4R2 with PPP4 catalytic subunit (PPP4c) was confirmed by co-sedimentation of PPP4c with PPP4R2 expressed in bacteria and human cells. PPP4c formed a complex of 450 kDa with baculovirus expressed His6-tagged PPP4R2. Immunocytological detection of PPP4R2 at centrosomes suggests that it may target PPP4c to this location. Native 450 kDa and 600 kDa PPP4 complexes are inactive, but can be activated by basic proteins, suggesting that PPP4R2 may also regulate the activity of PPP4c at centrosomal microtubule organising centres.

scholarly journals Correct Regulation of the Septation Initiation Network in Schizosaccharomyces pombe Requires the Activities of par1 and par2

Genetics ◽  
2001 ◽  
Vol 158 (4) ◽  
pp. 1413-1429 ◽  
Author(s):  
Wei Jiang ◽  
Richard L Hallberg
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Protein Phosphatase ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Loss Of Function ◽  
Regulatory Subunits ◽  
Genes Encoding ◽  
Phosphatase 2A ◽  
Septation Initiation Network ◽  
Mutant Cells

Abstract In Schizosaccharomyces pombe, the initiation of cytokinesis is regulated by a septation initiation network (SIN). We previously reported that deletion of par1 and par2, two S. pombe genes encoding B′ regulatory subunits of protein phosphatase 2A, causes a multiseptation phenotype, very similar to that seen in hyperactive SIN mutants. In this study, we examined the genetic interactions between par deletions and mutations in the genes encoding components of SIN and found that deletion of par1 and par2 suppressed the morphological and viability defects caused by overproduction of Byr4p and rescued a loss-of-function allele of spg1. However, par deletions could not suppress any mutations in genes downstream of spg1 in the SIN pathway. We showed further that, in suppressing the lethality of a spg1 loss-of-function allele, the correct localization of Cdc7p to the spindle pole body (SPB), which is normally lost in spg1 mutant cells, was restored. The fact that par mutant cells themselves exhibited a symmetric localization of Cdc7p to SPBs indicated a hyperactivity of SIN in such cells. On the basis of our epistasis analyses and cytological studies, we concluded that par genes normally negatively regulate SIN at or upstream of cdc7, ensuring that multiple rounds of septation do not occur.

scholarly journals Variable Expressions of Staphylococcus aureus Bicomponent Leucotoxins Semiquantified by Competitive Reverse Transcription-PCR

2000 ◽  
Vol 66 (9) ◽  
pp. 3931-3938 ◽  
Author(s):  
St�phane Bronner ◽  
Patricia Stoessel ◽  
Alain Gravet ◽  
Henri Monteil ◽  
Gilles Pr�vost
Keyword(s):  
Staphylococcus Aureus ◽  
Reverse Transcription ◽  
Regulatory System ◽  
Optimization Procedure ◽  
Reverse Transcription Pcr ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Specific Mrnas ◽  
Casamino Acids ◽  
Global Regulatory System

ABSTRACT A competitive reverse transcription-PCR method was developed for the semiquantitation of the expression of genes encoding bicomponent leucotoxins of Staphylococcus aureus, e.g., Panton-Valentine leucocidin (lukPV), gamma-hemolysin (hlgA and hlgCB), and LukE-LukD (lukED). The optimization procedure included RNA preparation; reverse transcription; the use of various amounts of enzymes, antisense primer, and RNA; and the final amplification chain reaction. Reproducible results were obtained, with sensitivity for detection of cDNA within the range of 1 mRNA/104 CFU to 102 mRNA/CFU, depending on the gene. Both specific mRNAs were more significantly expressed at the late-exponential phase of growth. Expression was about 100-fold higher in yeast extract-Casamino Acids-pyruvate medium than in heart infusion medium. Expression of the widely distributed gamma-hemolysin locus in the NTCC 8178 strain was around 10-fold diminished compared with that in the ATCC 49775 strain. Because of the lower level of hlgA expression, the corresponding protein, which is generally not abundant in culture supernatant, should be investigated for its contribution to the leucotoxin-associated virulence. The agr, sar, and agr sar mutant strains revealed a great dependence with regard to leucotoxin expression on the global regulatory system inS. aureus, except that expression of hlgA was not affected in the agr mutant.

Structural and antigenic polymorphism of the 35- to 48-kilodalton merozoite surface antigen (MSA-2) of the malaria parasite Plasmodium falciparum

1991 ◽  
Vol 11 (2) ◽  
pp. 963-971
Author(s):  
B Fenton ◽  
J T Clark ◽  
C M Khan ◽  
J V Robinson ◽  
D Walliker ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Amino Acid ◽  
Surface Antigen ◽  
Dna Sequences ◽  
Amino Acid Sequences ◽  
Merozoite Surface Antigen ◽  
Parasite Plasmodium ◽  
Genes Encoding ◽  
Parasite Plasmodium Falciparum ◽  
Group B

Merozoite surface antigen MSA-2 of the human parasite Plasmodium falciparum is being considered for the development of a malaria vaccine. The antigen is polymorphic, and specific monoclonal antibodies differentiate five serological variants of MSA-2 among 25 parasite isolates. The variants are grouped into two major serogroups, A and B. Genes encoding two different variants from serogroup A have been sequenced, and their DNA together with deduced amino acid sequences were compared with sequences encoded by other alleles. The comparison shows that the serological classification reflects differences in DNA sequences and deduced primary structure of MSA-2 variants and serogroups. Thus, the overall homologies of DNA and amino acid sequences are over 95% among variants in the same serogroup. In contrast, similarities between the group A variants and a group B variant are only 70 and 64% for DNA and amino acid sequences, respectively. We propose that the MSA-2 protein is encoded by two highly divergent groups of alleles, with limited additional polymorphism displayed within each group.

scholarly journals Cloning and Characterization of Two Xyloglucanases from Paenibacillus sp. Strain KM21

2005 ◽  
Vol 71 (12) ◽  
pp. 7670-7678 ◽  
Author(s):  
Katsuro Yaoi ◽  
Tomonori Nakai ◽  
Yoshiro Kameda ◽  
Ayako Hiyoshi ◽  
Yasushi Mitsuishi
Keyword(s):  
Glycoside Hydrolase ◽  
Amino Acid Sequences ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
C Terminus ◽  
Paenibacillus Sp ◽  
Genes Encoding ◽  
Molecular Masses

ABSTRACT Two xyloglucan-specific endo-β-1,4-glucanases (xyloglucanases [XEGs]), XEG5 and XEG74, with molecular masses of 40 kDa and 105 kDa, respectively, were isolated from the gram-positive bacterium Paenibacillus sp. strain KM21, which degrades tamarind seed xyloglucan. The genes encoding these XEGs were cloned and sequenced. Based on their amino acid sequences, the catalytic domains of XEG5 and XEG74 were classified in the glycoside hydrolase families 5 and 74, respectively. XEG5 is the first xyloglucanase belonging to glycoside hydrolase family 5. XEG5 lacks a carbohydrate-binding module, while XEG74 has an X2 module and a family 3 type carbohydrate-binding module at its C terminus. The two XEGs were expressed in Escherichia coli, and recombinant forms of the enzymes were purified and characterized. Both XEGs had endoglucanase active only toward xyloglucan and not toward Avicel, carboxymethylcellulose, barley β-1,3/1,4-glucan, or xylan. XEG5 is a typical endo-type enzyme that randomly cleaves the xyloglucan main chain, while XEG74 has dual endo- and exo-mode activities or processive endo-mode activity. XEG5 digested the xyloglucan oligosaccharide XXXGXXXG to produce XXXG, whereas XEG74 digestion of XXXGXXXG resulted in XXX, XXXG, and GXXXG, suggesting that this enzyme cleaves the glycosidic bond of unbranched Glc residues. Analyses using various oligosaccharide structures revealed that unique structures of xyloglucan oligosaccharides can be prepared with XEG74.

Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling

2001 ◽  
Vol 353 (3) ◽  
pp. 417-439 ◽  
Author(s):  
Veerle JANSSENS ◽  
Jozef GORIS
Keyword(s):  
Protein Phosphatase ◽  
Cellular Localization ◽  
Protein Phosphatase 2A ◽  
Viral Proteins ◽  
Catalytic Subunit ◽  
Post Translational Modification ◽  
Regulatory Subunits ◽  
Regulatory Ability ◽  
Phosphatase 2A ◽  
Cycle Regulation

Protein phosphatase 2A (PP2A) comprises a family of serine/threonine phosphatases, minimally containing a well conserved catalytic subunit, the activity of which is highly regulated. Regulation is accomplished mainly by members of a family of regulatory subunits, which determine the substrate specificity, (sub)cellular localization and catalytic activity of the PP2A holoenzymes. Moreover, the catalytic subunit is subject to two types of post-translational modification, phosphorylation and methylation, which are also thought to be important regulatory devices. The regulatory ability of PTPA (PTPase activator), originally identified as a protein stimulating the phosphotyrosine phosphatase activity of PP2A, will also be discussed, alongside the other regulatory inputs. The use of specific PP2A inhibitors and molecular genetics in yeast, Drosophila and mice has revealed roles for PP2A in cell cycle regulation, cell morphology and development. PP2A also plays a prominent role in the regulation of specific signal transduction cascades, as witnessed by its presence in a number of macromolecular signalling modules, where it is often found in association with other phosphatases and kinases. Additionally, PP2A interacts with a substantial number of other cellular and viral proteins, which are PP2A substrates, target PP2A to different subcellular compartments or affect enzyme activity. Finally, the de-regulation of PP2A in some specific pathologies will be touched upon.

scholarly journals Genes for low-molecular-weight heat shock proteins of soybeans: sequence analysis of a multigene family.

1985 ◽  
Vol 5 (12) ◽  
pp. 3417-3428 ◽  
Author(s):  
R T Nagao ◽  
E Czarnecka ◽  
W B Gurley ◽  
F Schöffl ◽  
J L Key
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Heat Shock ◽  
Dna Sequences ◽  
Regulatory Element ◽  
Amino Acid Sequences ◽  
Striking Similarity ◽  
Low Molecular Weight ◽  
Genes Encoding ◽  
Flanking Regions

Soybeans, Glycine max, synthesize a family of low-molecular-weight heat shock (HS) proteins in response to HS. The DNA sequences of two genes encoding 17.5- and 17.6-kilodalton HS proteins were determined. Nuclease S1 mapping of the corresponding mRNA indicated multiple start termini at the 5' end and multiple stop termini at the 3' end. These two genes were compared with two other soybean HS genes of similar size. A comparison among the 5' flanking regions encompassing the presumptive HS promoter of the soybean HS-protein genes demonstrated this region to be extremely homologous. Analysis of the DNA sequences in the 5' flanking regions of the soybean genes with the corresponding regions of Drosophila melanogaster HS-protein genes revealed striking similarity between plants and animals in the presumptive promoter structure of thermoinducible genes. Sequences related to the Drosophila HS consensus regulatory element were found 57 to 62 base pairs 5' to the start of transcription in addition to secondary HS consensus elements located further upstream. Comparative analysis of the deduced amino acid sequences of four soybean HS proteins illustrated that these proteins were greater than 90% homologous. Comparison of the amino acid sequence for soybean HS proteins with other organisms showed much lower homology (less than 20%). Hydropathy profiles for Drosophila, Xenopus, Caenorhabditis elegans, and G. max HS proteins showed a similarity of major hydrophilic and hydrophobic regions, which suggests conservation of functional domains for these proteins among widely dispersed organisms.

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