scholarly journals Cloning and expression of a cDNA encoding human inositol 1,4,5-trisphosphate 3-kinase C

2000 ◽  
Vol 352 (2) ◽  
pp. 343-351 ◽  
Author(s):  
Valérie DEWASTE ◽  
Valérie POUILLON ◽  
Colette MOREAU ◽  
Stephen SHEARS ◽  
Kazunaga TAKAZAWA ◽  
...  
Keyword(s):  
Northern Blot Analysis ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Inhibitory Effect ◽  
Enzymic Activity ◽  
Cloning And Expression ◽  
Human Tissues ◽  
Calculated Molecular Mass ◽  
Kinase C ◽  
Inositol 1,4,5 Trisphosphate

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] 3-kinase catalyses the phosphorylation of Ins(1,4,5)P3 to Ins(1,3,4,5)P4. cDNAs encoding two isoenzymes of Ins(1,4,5)P3 3-kinase (3-kinases A and B) have been described previously. In the present study, we report the cloning of a full-length 2052bp cDNA encoding a third human isoenzyme of the Ins(1,4,5)P3 3-kinase family, referred to as isoform C. This novel enzyme has a calculated molecular mass of 75.207kDa and a Km for Ins(1,4,5)P3 of 6µM. Northern-blot analysis showed the presence of a transcript of approx. 3.9kb in various human tissues. Inositol trisphosphate 3-kinase C demonstrates enzymic activity when expressed in DH5αF′ bacteria or COS-7 cells. Calcium alone decreases the Ins(1,4,5)P3 3-kinase activity of the 3-kinase C isoenzyme in transfected COS-7 cells. This inhibitory effect is reversed in the presence of calmodulin. The recombinant bacterial 3-kinase C can be adsorbed on calmodulin–Sepharose in the presence of calcium. The present data show that Ins(1,4,5)P3 3-kinase C: (i) shares a conserved catalytic domain of about 275 amino acids with the two other mammalian isoforms, (ii) could be purified on a calmodulin–Sepharose column and (iii) could be distinguished from the A and B isoenzymes by the effects of calcium and of calmodulin.

scholarly journals Structural identification of the myo-inositol 1,4,5-trisphosphate-binding domain in rat brain inositol 1,4,5-trisphosphate 3-kinase

1997 ◽  
Vol 326 (1) ◽  
pp. 221-225 ◽  
Author(s):  
Shinji TOGASHI ◽  
Kazunaga TAKAZAWA ◽  
Toyoshi ENDO ◽  
Christophe ERNEUX ◽  
Toshimasa ONAYA
Keyword(s):  
Amino Acids ◽  
Rat Brain ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Apparent Homogeneity ◽  
Inositol 1,4,5 Trisphosphate ◽  
Charged Amino Acid Residue

A series of key amino acids involved in Ins(1,4,5)P3 (InsP3) binding and catalytic activity of rat brain InsP3 3-kinase has been identified. The catalytic domain is at the C-terminal end and restricted to a maximum of 275 amino acids [Takazawa and Erneux (1991) Biochem. J. 280, 125–129]. In this study, newly prepared 5′-deletion and site-directed mutants have been compared both for InsP3 binding and InsP3 3-kinase activity. When the protein was expressed from L259 to R459, the activity was lost but InsP3 binding was conserved. Another deletion mutant that had lost only four amino acids after L259 had lost InsP3 binding, and this finding suggests that these residues (i.e. L259DCK262) are involved in InsP3 binding. To further support the data, we have produced two mutants by site-directed mutagenesis on residues C261 and K262. The two new enzymes were designated M4 (C261S) and M5 (K262A). M4 showed similar Vmax and Km values for InsP3 and ATP to wild-type enzyme. In contrast, M5 was totally inactive but had kept the ability to bind to calmodulin–Sepharose. C-terminal deletion mutants that had lost five, seven or nine amino acids showed a large decrease in InsP3 binding and InsP3 3-kinase activity. One mutant that had lost five amino acids (M2) was purified to apparent homogeneity: Km values for both substrates appeared unchanged but Vmax was decreased approx. 40-fold compared with the wild-type enzyme. The results indicate that (1) a positively charged amino acid residue K262 is essential for InsP3 binding and (2) amino acids at the C-terminal end of the protein are necessary to act as a catalyst in the InsP3 3-kinase reaction.

scholarly journals Cloning and expression in Escherichia coli of a rat brain cDNA encoding a Ca2+/calmodulin-sensitive inositol 1,4,5-trisphosphate 3-kinase

1990 ◽  
Vol 272 (1) ◽  
pp. 107-112 ◽  
Author(s):  
K Takazawa ◽  
J Vandekerckhove ◽  
J E Dumont ◽  
C Erneux
Keyword(s):  
Escherichia Coli ◽  
Protein Kinase ◽  
Rat Brain ◽  
Kinase Activity ◽  
Limited Proteolysis ◽  
Cloning And Expression ◽  
Terminal Part ◽  
Dependent Protein Kinase ◽  
Inositol 1,4,5 Trisphosphate ◽  
Dependent Protein

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). InsP3 3-kinase activity was stimulated by Ca2+ in the presence of calmodulin (CaM) and the protein was associated with two silver-stained bands which migrated with an apparent Mr of approx. 50,000 on SDS/polyacrylamide gels. Upon limited proteolysis with trypsin, the native InsP3 3-kinase was converted into polypeptides of Mr 44,000 and 36,000. Both tryptic fragments displayed InsP3 3-kinase activity that was Ca2+/CaM-sensitive. A cDNA clone, C5, that encodes the C-terminal part of the InsP3 3-kinase, was isolated by immunoscreening of a rat brain cDNA library. The 5′ end of this clone was used in turn to probe the same library, yielding a clone (CP16) containing the entire coding sequence of InsP3 3-kinase. The encoding protein of 459 amino acids (calculated Mr 50,868) has several putative phosphorylation sites for cyclic AMP-dependent protein kinase, protein kinase C and CaM-dependent protein kinase II. When clone C5 was expressed in Escherichia coli, the truncated fusion protein showed Ca2+/CaM-sensitive InsP3 3-kinase activity. Our data demonstrate that the N-terminal part of the protein is not essential for either enzymic or CaM-regulatory properties.

scholarly journals Dual requirement for a newly identified phosphorylation site in p70s6k.

1997 ◽  
Vol 17 (9) ◽  
pp. 5648-5655 ◽  
Author(s):  
B A Moser ◽  
P B Dennis ◽  
N Pullen ◽  
R B Pearson ◽  
N A Williamson ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Mutational Analysis ◽  
Phosphorylation Site ◽  
Serum Stimulation ◽  
Kinase C ◽  
Homologous Site ◽  
Phosphopeptide Analysis

The activation of p70s6k is associated with multiple phosphorylations at two sets of sites. The first set, S411, S418, T421, and S424, reside within the autoinhibitory domain, and each contains a hydrophobic residue at -2 and a proline at +1. The second set of sites, T229 (in the catalytic domain) and T389 and S404 (in the linker region), are rapamycin sensitive and flanked by bulky aromatic residues. Here we describe the identification and mutational analysis of three new phosphorylation sites, T367, S371, and T447, all of which have a recognition motif similar to that of the first set of sites. A mutation of T367 or T447 to either alanine or glutamic acid had no apparent effect on p70s6k activity, whereas similar mutations of S371 abolished kinase activity. Of these three sites and their surrounding motifs, only S371 is conserved in p70s6k homologs from Drosophila melanogaster, Arabidopsis thaliana, and Saccharomyces cerevisiae, as well as many members of the protein kinase C family. Serum stimulation increased S371 phosphorylation; unlike the situation for specific members of the protein kinase C family, where the homologous site is regulated by autophosphorylation, S371 phosphorylation is regulated by an external mechanism. Phosphopeptide analysis of S371 mutants further revealed that the loss of activity in these variants was paralleled by a block in serum-induced T389 phosphorylation, a phosphorylation site previously shown to be essential for kinase activity. Nevertheless, the substitution of an acidic residue at T389, which mimics phosphorylation at this site, did not rescue mutant p70s6k activity, indicating that S371 phosphorylation plays an independent role in regulating intrinsic kinase activity.

scholarly journals Cloning and characterization of a 92 kDa soluble phosphatidylinositol 4-kinase

1996 ◽  
Vol 320 (2) ◽  
pp. 643-649 ◽  
Author(s):  
Tamotsu NAKAGAWA ◽  
Kaoru GOTO ◽  
Hisatake KONDO
Keyword(s):  
Kinase Activity ◽  
Catalytic Domain ◽  
Adult Brain ◽  
Epitope Tag ◽  
Lipid Kinase ◽  
Calculated Molecular Mass ◽  
Shared Identity ◽  
Brain Cdna Library ◽  
Lipid Kinases ◽  
Phosphatidylinositol 4

A phosphatidylinositol (PtdIns) 4-kinase cDNA cloned from a rat brain cDNA library encoded a protein of 816 amino acids with a calculated molecular mass of 91654 Da. This molecule contained a lipid-kinase-unique domain and a presumed lipid/protein kinase homology domain that are found in other PtdIns 4-kinases and PtdIns 3-kinases. Furthermore, this kinase molecule had 43.3% shared identity with the presumed catalytic domain of yeast PtdIns 4-kinase, PtdInsK1, and the two molecules had a region of similarity that is not conserved in other lipid kinases. By examining PtdIns kinase activity in transfected COS-7 cells using epitope tag immunoprecipitation as well as conventional methods, the product PtdIns phosphate was identified as phosphatidylinositol 4-phosphate (PtdIns4P), but not phosphatidylinositol 3-phosphate (PtdIns3P). The PtdIns 4-kinase activity was recovered predominantly from the soluble fraction and the activity was markedly enhanced in the presence of Triton X-100 and was relatively insensitive to inhibition by adenosine. In addition, the PtdIns 4-kinase activity was completely inhibited in the presence of 10 µM wortmannin. When examined by epitope tag immunocytochemistry, the immunoreactivity for the PtdIns 4-kinase molecule was dominantly aggregated in a cytoplasmic region juxtaposed to the nuclei and was faintly but widely dispersed in the cytoplasm. By in situ hybridization analysis, the mRNA for PtdIns 4-kinase was expressed ubiquitously and was detected in most neurons throughout the grey matter of the brain, with higher expression intensity found in fetal than in adult brain.

scholarly journals Cloning and expression in Escherichia coli of a dog thyroid cDNA encoding a novel inositol 1,4,5-trisphosphate 5-phosphatase

1994 ◽  
Vol 300 (1) ◽  
pp. 85-90 ◽  
Author(s):  
B Verjans ◽  
F De Smedt ◽  
R Lecocq ◽  
V Vanweyenberg ◽  
C Moreau ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Mass ◽  
Bovine Brain ◽  
Cloning And Expression ◽  
Major Protein ◽  
Type I ◽  
Degenerate Primers ◽  
Calculated Molecular Mass ◽  
Major Protein Band ◽  
Inositol 1,4,5 Trisphosphate

In brain and many other tissues, type I inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase is the major isoenzyme hydrolysing the calcium-mobilizing second messenger InsP3. This protein has been purified to apparent homogeneity from a crude soluble fraction of bovine brain, yielding a single major protein band with a molecular mass of 43 kDa after SDS/PAGE. This material was used to determine internal microsequences. A partial DNA sequence has been amplified by PCR by using degenerate primers deduced from two protein sequences (FKAKKYKKV and DENYKSQE). A cDNA clone (BVCT) was isolated by screening a dog thyroid cDNA library. The encoded protein of 412 amino acids has a calculated molecular mass of 47,681 Da. Peptide sequences generated from the bovine brain enzyme were found to be 96% conserved compared with the dog thyroid protein. When clone BVCT was expressed in Escherichia coli, the recombinant protein was shown to hydrolyse both InsP3 and inositol 1,3,4,5-tetrakisphosphate, with apparent Km values of 28 and 3 microM respectively. Enzyme activity was inhibited by EDTA and 2,3-bisphosphoglycerate, both inhibitors of native InsP3 5-phosphatase, but not by EGTA and LiCl, as previously shown for the bovine brain enzyme. Our data show the cloning of type I InsP3 5-phosphatase which, interestingly, does not share any significant sequence identity with the previously cloned type III isoenzyme.

scholarly journals Molecular cloning and expression of a new putative inositol 1,4,5-trisphosphate 3-kinase isoenzyme

1991 ◽  
Vol 278 (3) ◽  
pp. 883-886 ◽  
Author(s):  
K Takazawa ◽  
J Perret ◽  
J E Dumont ◽  
C Erneux
Keyword(s):  
Amino Acid ◽  
Kinase Activity ◽  
Cloning And Expression ◽  
Fusion Product ◽  
Molecular Evidence ◽  
Reading Frame ◽  
Acid Protein ◽  
Sds Page ◽  
Inositol 1,4,5 Trisphosphate ◽  
Kinase A

A human hippocampus cDNA library in lambda ZAP II was screened by hybridization with a rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase cDNA. Two clones (hh6 and hh3) were isolated and sequenced. The insert of clone hh6 was shown to correspond to the 3′ end of the coding sequence of 50,000-Mr InsP3 3-kinase (referred to as 3-kinase-A). Sequencing of the clone hh3 insert yielded an open reading frame encoding a 472-amino acid protein with a calculated Mr of 53,451 (referred to as 3-kinase-B). The C-terminal part of 3-kinase-B (residues 187-462) was 68% identical with 3-kinase-A in amino acid sequence. The cDNA of clone hh3 was rescued as a Bluescript plasmid and expressed in Escherichia coli as a beta-galactosidase fusion product. It showed InsP3 3-kinase activity that was stimulated in the presence of Ca2+/calmodulin (more than 7-fold in a crude bacterial lysate from expressed plasmid). Regeneration of InsP3 3-kinase activity after SDS/PAGE identified a major polypeptide (Mr 60,000-65,000). The Km for InsP3 of expressed 3-kinase-B was 1.6 microM. These data provide molecular evidence for the existence of InsP3 3-kinase isoenzymes.

Neomycin reversibly disrupts mitotic progression in stamen hair cells of Tradescantia

1991 ◽  
Vol 98 (2) ◽  
pp. 159-168
Author(s):  
PAUL M. LARSEN ◽  
TUNG-LING L. CHEN ◽  
STEPHEN M. WOLNIAK
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Hair Cells ◽  
Inhibitory Effect ◽  
Mitotic Progression ◽  
Metaphase Arrest ◽  
Nuclear Envelope Breakdown ◽  
Regulatory Cascade ◽  
Kinase C ◽  
Inositol 1,4,5 Trisphosphate

Neomycin has been reported to inhibit polyphosphoinositide cycling by preventing the hydrolysis of phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. Inositol 1,4,5-trisphosphate, through the mobilization of calcium, and 1,2-diacylglycerol, through the activation of protein kinase C, trigger many physiological responses. The addition of 2 mM neomycin to stamen hair cells of Tradescantia virginiana at various points during mitosis arrests cells in prophase, prior to nuclear envelope breakdown, or in metaphase. Arrest in prophase is irreversible. Metaphase arrest can persist for over 2h before the cells attempt to revert to interphase without dividing. Entry into anaphase by the majority of cells in our sample arrested in metaphse occurred after treatment with 1,2-dioctanoylglycerol while 1,3-dioctanoylglycerol was totally ineffective at reversal. Perfusion of 100 μM calcium chloride solution past the cells was sufficient to reverse arrest in approximately half of the cells in the sample. Magnesium could not be substituted for calcium in the reversal. Clindamycin, another member of this class of aminoglycoside antibiotics, with no known inhibitory effect on polyphosphoinositide cycling, is without effect on mitotic progression in stamen hair cells. Our results indirectly implicate one or more episodes of polyphosphoinositide cycling and its resultant protein phosphorylation by protein kinase C in the regulatory cascade that leads to anaphase.

scholarly journals Lys-197 and Asp-414 are critical residues for binding of ATP/Mg2+ by rat brain inositol 1,4,5-trisphosphate 3-kinase

1993 ◽  
Vol 291 (3) ◽  
pp. 811-816 ◽  
Author(s):  
D Communi ◽  
K Takazawa ◽  
C Erneux
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Rat Brain ◽  
Catalytic Domain ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Calmodulin Binding ◽  
Inositol 1,4,5 Trisphosphate

Rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase A was expressed in Escherichia coli in order to identify the amino acid residues involved in substrate ATP/Mg2+ binding. Two amino acid regions that are conserved in the catalytic domain of InsP3 3-kinase isoenzymes A and B had characteristics consistent with two ATP/Mg(2+)-binding motives. Site-directed mutagenesis was performed on residues Lys-197, Lys-207 and Asp-414 to generate three mutant enzymes, referred to as C5 K197I, C5 K207I and C5 D414N. Comparison of the wild-type and mutant proteins with regard to enzymic activity revealed that C5 K197I exhibited 10% of control enzyme activity, C5 D414N was totally inactive and C5 K207I was fully active. The reduced levels of enzyme activity for C5 K197I and C5 D414N were correlated with an altered ability of the mutant enzymes to bind ATP/Mg2+, as determined by ATP-agarose affinity chromatography. Neither Ca2+/calmodulin binding nor InsP3 binding appeared to be affected. Mutant C5 K207I showed the same characteristics as the wild-type enzyme. Taken together, these results strongly indicated (i) that amino acid residues Lys-197 and Asp-414 are necessary for InsP3 3-kinase activity and form part of the ATP/Mg(2+)-binding domain, and (ii) that amino acid residues Lys-197, Lys-207 and Asp-414 are not involved in either InsP3 binding or enzyme stimulation by Ca2+/calmodulin.

scholarly journals Cloning and expression of a cDNA encoding human inositol 1,4,5-trisphosphate 3-kinase C

2000 ◽  
Vol 352 (2) ◽  
pp. 343 ◽  
Author(s):  
Valérie DEWASTE ◽  
Valérie POUILLON ◽  
Colette MOREAU ◽  
Stephen SHEARS ◽  
Kazunaga TAKAZAWA ◽  
...  
Keyword(s):  
Cloning And Expression ◽  
Kinase C ◽  
Inositol 1,4,5 Trisphosphate

scholarly journals Purification of catalytic domain of rat spleen p72syk kinase and its phosphorylation and activation by protein kinase C

1998 ◽  
Vol 331 (2) ◽  
pp. 649-657 ◽  
Author(s):  
Peter BOROWSKI ◽  
Max HEILAND ◽  
Lutz KORNETZKY ◽  
Sven MEDEM ◽  
Rainer LAUFS
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Catalytic Domain ◽  
Assay Method ◽  
Dependent Manner ◽  
Tyrosine Kinase Activity ◽  
Kinase C ◽  
Pkc Isoforms

The catalytic domain of p72syk kinase (CDp72syk) was purified from a 30000 g particulate fraction of rat spleen. The purification procedure employed sequential chromatography on columns of DEAE-Sephacel and Superdex-200, and elution from HA-Ultrogel by chloride. The analysis of the final CDp72syk preparation by SDS/PAGE revealed a major silver-stained 40 kDa protein. The kinase was identified by covalent modification of its ATP-binding site with [14C]5´-fluorosulphonylbenzoyladenosine and by immunoblotting with a polyclonal antibody against the ‘linker ’ region of p72syk. By using poly(Glu4, Tyr1) as a substrate, the specific activity of the enzyme was determined as 18.5 nmol Pi/min per mg. Casein, histones H1 and H2B and myelin basic protein were efficiently phosphorylated by CDp72syk. The kinase exhibited a limited ability to phosphorylate random polymers containing tyrosine residues. CDp72syk autophosphorylation activity was associated with an activation of the kinase towards exogenous substrates. The extent of activation was dependent on the substrates added. CDp72syk was phosphorylated by protein kinase C (PKC) on serine and threonine residues. With a newly developed assay method, we demonstrated that the PKC-mediated phosphorylation had a strong activating effect on the tyrosine kinase activity of CDp72syk. Studies extended to conventional PKC isoforms revealed an isoform-dependent manner (α > βI = βII > γ) of CDp72syk phosphorylation. The different phosphorylation efficiencies of the PKC isoforms closely correlated with the ability to enhance the tyrosine kinase activity.

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