Biomedical subjects

J D York

Publications and source records attributed to J D York.

12 recordsLinked to original sources

PubMed1995

Human DNA-activated protein kinase (DNA-PK) is homologous to phosphatidylinositol kinases.

DNA-activated protein kinase (DNA-PK) is a serine/threonine protein kinase that interacts with a DNA end-binding heterodimeric protein, Ku, and is activated by double-stranded DNA. Genomic clones that contain the DNA-PK gene complement the murine scid defect, indicating that DNA-PK affects double-strand break repair and V(D)J recombination. Here we describe the cDNA sequence of the region that corresponds to about 100 kDa of C-terminal sequence of this large (> p350 kDa) protein. This region contains a kinase domain that has strong homology to phosphatidylinositol kinases.

1-Phosphatidylinositol 4-Kinase↗

PubMed1995

Definition of a metal-dependent/Li(+)-inhibited phosphomonoesterase protein family based upon a conserved three-dimensional core structure.

Inositol polyphosphate 1-phosphatase, inositol monophosphate phosphatase, and fructose 1,6-bisphosphatase share a sequence motif, Asp-Pro-(Ile or Leu)-Asp-(Gly or Ser)-(Thr or Ser), that has been shown by crystallographic and mutagenesis studies to bind metal ions and participate in catalysis. We compared the six alpha-carbon coordinates of this motif from the crystal structures of these three phosphatases and found that they are superimposable with rms deviations ranging from 0.27 to 0.60 A. Remarkably, when these proteins were aligned by this motif a common core structure emerged, defined by five alpha-helices and 11 beta-strands comprising 155 residues having rms deviations ranging from 1.48 to 2.66 A. We used the superimposed structures to align the sequences within the common core, and a distant relationship was observed suggesting a common ancestor. The common core was used to align the sequences of several other proteins that share significant similarity to inositol monophosphate phosphatase, including proteins encoded by fungal qa-X and qutG, bacterial suhB and cysQ (identical to amtA), and yeast met22 (identical to hal2). Evolutionary comparison of the core sequences indicate that five distinct branches exist within this family. These proteins share metal-dependent/Li(+)-sensitive phosphomonoesterase activity, and each predicted tree branch exhibits unique substrate specificity. Thus, these proteins define an ancient structurally conserved family involved in diverse metabolic pathways including inositol signaling, gluconeogenesis, sulfate assimilation, and possibly quinone metabolism. Furthermore, we suggest that this protein family identifies candidate enzymes to account for both the therapeutic and toxic actions of Li+ as it is used in patients treated for manic depressive disease.

Amino Acid Sequence↗

PubMed1994

Crystal structure of inositol polyphosphate 1-phosphatase at 2.3-A resolution.

Bovine inositol polyphosphate 1-phosphatase (1-ptase), M(r) = 44,000, is a Mg(2+)-dependent/Li(+)-sensitive enzyme that catalyzes the hydrolysis of the 1-position phosphate from inositol 1,4-bisphosphate and inositol 1,3,4-trisphosphate. We have determined the crystal structure of recombinant bovine 1-ptase in the presence of Mg2+ by multiple isomorphous replacement. The structure is currently refined to an R value of 0.198 for 15,563 reflections within a resolution range of 8.0-2.3 A. 1-Ptase is monomeric in the crystal, consistent with biochemical data, and folds into an alternatively layered alpha/beta/alpha/beta sandwich. The central core of 1-ptase consists of a six-stranded antiparallel beta sheet perpendicular to two parallel three-turn alpha-helices. The beta sheet is flanked by two antiparallel six-turn alpha-helices aligned parallel to the beta sheet, and the central helices are flanked by a five-stranded largely parallel beta sheet. Two neighboring metal binding sites are located in adjacent acidic pockets formed by the intersection of several secondary structure elements including an unusual kink structure formed by the "DPIDST" sequence motif. The fold of 1-ptase is similar to that of two other metal-dependent/Li(+)-sensitive phosphatases, inositol monophosphate phosphatase and fructose 1,6-bisphosphatase despite minimal amino acid identity. Comparison of the active-site pockets of these proteins will likely provide insight into substrate binding and the mechanisms of metal-dependent catalysis and Li+ inhibition.

Amino Acid Sequence↗

PubMed1994

Inositol polyphosphate 1-phosphatase is present in the nucleus and inhibits DNA synthesis.

Inositol polyphosphate 1-phosphatase, an enzyme of the phosphatidylinositol signaling pathway, hydrolyzes the 1-phosphate from inositol 1,4-bisphosphate and inositol 1,3,4-trisphosphate. We have used indirect immunofluorescence microscopy, Western blot analysis, and enzyme assays to determine the cellular localization of the enzyme. We find that the enzyme is present, but not exclusively, in the nucleus of Madin-Darby bovine kidney cells, and also in COS-7 and HeLa cells that were transiently transfected with a cDNA encoding bovine inositol polyphosphate 1-phosphatase. DNA synthesis, as measured in COS-7 and HeLa cells transiently over-expressing enzyme, was reduced 50% in cells transfected with wild-type enzyme compared with nontransfected cells or cells transfected with an inactive mutant form of the enzyme. These data demonstrate that this response is mediated by one of the substrates or products of inositol polyphosphate 1-phosphatase. We propose that overexpressed inositol polyphosphate 1-phosphatase degrades a stimulatory inositol phosphate(s) and thereby inhibits DNA synthesis.

Amino Acid Sequence↗

PubMed1994

Nuclear phosphatidylinositols decrease during S-phase of the cell cycle in HeLa cells.

In the current study we have measured phosphatidylinositols during the cell cycle. HeLa cells were labeled with [3H]myoinositol to a steady state, synchronized to the G1/S boundary, and the levels of phosphatidylinositol (PtdIns) lipids were measured at various times after release from the block. The levels of total cellular PtdIns, PtdIns(4)P, and PtdIns(4,5)P2 relative to total cellular phospholipid did not vary throughout the cell cycle. We then isolated nuclei from synchronized cells using a non-detergent method and found that the levels of nuclear PtdIns lipids decreased by over 50% at 2 and 4 h after release from the G1/S boundary (S-phase of the cell cycle) and returned to the original levels by 9 h. Separation of individual inositol-containing nuclear lipids showed that PtdIns decreased by 50% while levels of PtdIns(4)P and PtdIns(4,5)P2 decreased by 66%. Levels of the cytoplasmic PtdIns lipids remained constant throughout this period. This experiment indicates that there is specific nuclear. PtdIns turnover that is activated during DNA synthesis.

Cell Cycle↗

PubMed1994

Crystallization and initial X-ray crystallographic characterization of recombinant bovine inositol polyphosphate 1-phosphatase produced in Spodoptera frugiperda cells.

Bovine inositol polyphosphate 1-phosphatase, a monomeric protein with a molecular mass of 44,000 Da, hydrolyzes the 1-position phosphate from inositol 1,3,4-trisphosphate and inositol 1,4-bisphosphate. The low abundance of inositol polyphosphate 1-phosphatase in tissues has precluded structural studies requiring large quantities of enzyme. We used recombinant Baculovirus harboring the cDNA of bovine inositol polyphosphate 1-phosphatase to infect Spodoptera frugiperda (Sf9) insect cells. Recombinant protein (25 mg per 1 x 10(9) cells) was purified to homogeneity. The enzyme produced in Sf9 cells was similar to the native purified protein as determined by immunoblotting catalytic properties, and inhibition by lithium ions. Crystals of the purified recombinant enzyme were grown by vapor diffusion. Precession photography was used to determine the parameters of inositol polyphosphate 1-phosphatase crystals. The tetragonal crystals belong to the space group P4(1) or P4(3), have unit cell dimensions of a = b = 51.6 A, c = 143.3 A, alpha = beta = gamma = 90 degrees, and contain one molecule per asymmetric unit. We have collected a complete diffraction data set extending to 2.3 A and are currently attempting to solve the three-dimensional structure of bovine inositol polyphosphate 1-phosphatase using a multiple isomorphous replacement strategy.

Animals↗

PubMed1993

Cloning, heterologous expression, and chromosomal localization of human inositol polyphosphate 1-phosphatase.

Inositol polyphosphate 1-phosphatase, an enzyme in the phosphatidylinositol signaling pathway, catalyzes the hydrolysis of the 1 position phosphate from inositol 1,3,4-trisphosphate and inositol 1,4-bisphosphate. We used a cDNA that encodes bovine inositol polyphosphate 1-phosphatase as a probe to isolate the human counterpart by low-stringency hybridization. The 1.74-kb human cDNA has 341 bp of 5' untranslated region, 180 bp of 3' untranslated region, poly(A)32, and predicts a protein of 399 amino acids. Human and bovine inositol polyphosphate 1-phosphatases show 84% amino acid sequence identity. Northern blot analysis from a variety of human tissues demonstrates that a 1.9-kb mRNA is ubiquitously expressed with highest levels in pancreas and kidney. Several higher molecular weight mRNAs also are expressed in brain, muscle, heart, and liver. We have confirmed the functional identity of the human cDNA by heterologous expression in NIH 3T3 fibroblasts, COS-7 cells and Escherichia coli. Polymerase chain reaction assay of a panel of human-rodent somatic cell hybrid DNA using human inositol polyphosphate 1-phosphatase-specific DNA primers resulted in amplification of a specific product using chromosome 2 DNA as template. Fluorescence in situ hybridization of metaphase chromosomes localizes the gene to chromosome 2 band q32. The identification of the human inositol polyphosphate 1-phosphatase gene locus provides a target for linkage analysis to identify defects in patients with inherited psychiatric disorders that respond to lithium ions, an inhibitor of the enzyme.

3T3 Cells↗

PubMed1991

Diverse proteins homologous to inositol monophosphatase.

Bovine inositol monophosphatase (IMP) and several homologous proteins were found to share two sequence motifs with bovine inositol polyphosphate 1-phosphatase (IPP). These motifs may correspond to binding sites within IMP and IPP for inositol phosphates or for lithium, since both substances are bound by these proteins. This suggests that the proteins homologous to IMP, which have diverse biological roles but whose function is not clear, may act by enhancing the synthesis or degradation of phosphorylated compounds.

Amino Acid Sequence↗

PubMed1991

Identification, cloning, and expression of a cytosolic megakaryocyte protein-tyrosine-phosphatase with sequence homology to cytoskeletal protein 4.1.

We have isolated a cDNA encoding a third type of protein-tyrosine-phosphatase. We screened human megakaryoblastic cell line (MEG-01) an umbilical vein endothelial cell cDNA libraries to obtain a 3.7-kilobase cDNA designated PTPase MEG. Northern blot analysis of MEG-01 RNA detected a 3.7-kilobase transcript, suggesting that a full-length cDNA has been identified. PTPase MEG cDNA contains an open reading frame of 926 amino acids. The cDNA has a G+C-rich 5' untranslated region of 771 nucleotides that has the potential to form stable stem-loop structures and has two upstream ATG codons. The predicted protein (Mr = 105,910) has no apparent membrane-spanning region and contains a single protein-tyrosine-phosphatase domain (amino acids 659-909) that is 35-40% identical to previously described tyrosine-phosphatase domains. The recombinant phosphatase domain possesses protein-tyrosine-phosphatase activity when expressed in Escherichia coli. The amino-terminal region (amino acids 31-367) is 45% identical to the amino terminus of human erythrocyte protein 4.1, a cytoskeletal protein. The identification of a protein-tyrosine-phosphatase that is related to cytoskeletal proteins implies that cell signaling activities reside not only in transmembrane receptors but in cytoskeletal elements as well.

Amino Acid Sequence↗

PubMed1991

Combinatorial mutagenesis of the reactive site region in plasminogen activator inhibitor I.

Plasminogen activator inhibitor (PAI-I) rapidly inactivates tissue plasminogen activator (t-PA) and urokinase (UK) with nearly identical association rate constants. The contributions of Ser344, Ala345, and Arg346 (P3, P2, and P1 residues, respectively) in PAI-I to inhibition of UK and t-PA were evaluated using combinatorial mutagenesis of the human PAI-I cDNA. A bacteriophage lambda expression library potentially encoding the 8000 unique PAI-I species were screened for inhibitory activity against UK using a fibrin indicator gel. 390 plaques demarcated by zones of retarded fibrinolysis were analyzed to determine the DNA sequences of their associated active PAI-1 species. We found 134 unique PAI-1 variants that retained inhibitory activity towards UK; they contained a variety of amino acids in their P3 and P2 positions but only Arg or, infrequently, Lys in their P1 position. Each of the unique active PAI-1 were assayed for inhibitory activity towards UK or t-PA; many substitutions differentially affected the ability of the inhibitor to inactivate UK and t-PA. For example, replacement of Ser344 and Ala344 with Val and Pro, respectively, yielded a PAI-1 variant exhibiting an association rate constant that was unchanged for t-PA but decreased 23-fold for UK, relative to native PAI-1. In general, the PAI-1 variants were more potent inhibitors of t-PA than UK. Hence, t-PA appears more tolerant than UK of structural diversity present in the P3 and P2 positions of the PAI-1 variants.

Amino Acid Sequence↗

PubMed1990

Isolation and heterologous expression of a cDNA encoding bovine inositol polyphosphate 1-phosphatase.

Inositol polyphosphate 1-phosphatase, an enzyme of the phosphatidylinositol signaling pathway, catalyzes the hydrolysis of the 1-position phosphate from inositol 1,3,4-trisphosphate and inositol 1,4-bisphosphate. The protein was isolated from calf brain and digested with trypsin or CNBr, and the amino acid sequence of several peptides was determined. Degenerate oligonucleotide primers were designed from amino acid sequence and used to synthesize an 80-base-pair (bp) fragment by the polymerase chain reaction. This product was used to isolate a 1.6-kbp cDNA with an open reading frame of 400 amino acids, 185 bp of 5' untranslated region, and 171 bp of 3' untranslated region followed by a putative poly(A) tail. The coding region of the cDNA was inserted into an expression vector that was used to obtain the recombinant protein from Escherichia coli cells. The recombinant enzyme (44 kDa) had a specific activity and other properties similar to those of native bovine brain inositol polyphosphate 1-phosphatase. It hydrolyzed both inositol phosphate substrates and was inhibited by lithium ions. The enzyme shows minimal sequence similarity to inositol monophosphate phosphatase, the other enzyme inhibited by lithium ions in the signaling pathway.

Amino Acid Sequence↗

PubMed1989

Isolation, characterization, and cDNA cloning of a vampire bat salivary plasminogen activator.

Vampire bat saliva contains a plasminogen activator that presumably assists these hematophagous animals during feeding. Here, we report that the vampire bat salivary plasminogen activator, Bat-PA, is homologous to tissue-type plasminogen activator (t-PA) but contains neither a kringle 2 domain nor a plasmin-sensitive processing site. Three Bat-PA species corresponding to full-length, finger-, and finger- epidermal growth factor homology domain- forms of t-PA have been isolated. Bat-PA(H), the full-length form, was purified and its activity has been characterized. Bat-PA(H) and t-PA are of similar efficacy when monitored for their abilities to catalyze plasminogen activation in the presence of a fibrin cofactor. Interestingly, Bat-PA activity toward plasminogen is stimulated 45,000-fold in the presence of fibrin I; the corresponding value for t-PA is only 205-fold. Bat-PA(H) is the only Bat-PA species which binds tightly to fibrin, although each of the three species exhibit remarkable stimulation by a fibrin cofactor.

Amino Acid Sequence↗