Intracellular control of inositol phosphates by their metabolizing enzymes.

Journal ArticleReviewSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Inositol 1,4,5-trisphosphate 3-kinase distribution in the rat brain. High levels in the hippocampal CA1 pyramidal and cerebellar Purkinje cells suggest its involvement in some memory processes.

The distribution of inositol 1,4,5-trisphosphate (InsP3) 3-kinase was studied in the adult rat brain, using polyclonal antibodies raised against the purified 50,000-Da rat brain enzyme by immunohistochemistry and Western blot, in addition to enzymatic assay. Immunohistochemically, the enzyme was detected in neurons, where it was localized in the dendrites and at the periphery of the cell bodies. Using selective toxin lesions, the highest enzyme levels were found in the dendrites of hippocampal CA1 pyramidal cells and in neurons in the dorsal portion of the lateral septum, regions both involved in long-term potentiation; and in the dendrites of Purkinje cell subpopulations in the cerebellum, a region involved in long-term depression. High levels were found in neurons in the cortex; in the anterior olfactory nucleus; in the striatum (caudate, putamen, olfactory tubercle, Calleja islets and accumbens); in the central nucleus of the amygdala; in the hippocampal dentate gyrus and in the subiculum. The enzyme was not detected in other brain regions. By Western blot, a 50,000-Da immunoreactive band was present in the cortex, caudate-putamen and cerebellum. This band was most highly stained in the hippocampus. InsP3 3-kinase activity, stimulated by calcium/calmodulin, corresponded to 6172-2638 pmol of InsP4 produced/min/mg protein in the hippocampus followed by frontal and parietotemporal cortex and cerebellum. This activity was below 400 in the brainstem and spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Interaction of calmodulin with a putative calmodulin-binding domain of inositol 1,4,5-triphosphate 3-kinase. Effects of synthetic peptides and site-directed mutagenesis of Trp165.

Recombinant rat brain inositol 1,4,5-triphosphate [Ins(1,4,5)P3] 3-kinase was expressed in Escherichia coli as a beta-galactosidase fusion product. It could be adsorbed onto calmodulin-Sepharose and eluted in Ca(2+)-free medium as a 48-kDa protein. Purification could be achieved in a single step. Molecular evidence for a calmodulin-binding domain on Ins(1,4,5)P3 3-kinase can be shown by the following approaches. (a) Inhibition of Ca2+/calmodulin stimulation by a synthetic peptide based on a candidate calmodulin-binding domain. The inhibition was mimicked by a well-characterized peptide derived from the sequence of smooth muscle myosin light-chain kinase calmodulin-binding site. (b) The construction of two mutants by site-directed mutagenesis of Trp165 to Gly or Arg. Both mutants displayed kinase activity but were no longer Ca2+/calmodulin sensitive, supporting, therefore, the role of Trp165 in calmodulin binding.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Human brain inositol 1,4,5-trisphosphate 3-kinase cDNA sequence.

Journal ArticleSCOPUS: no.jinfo:eu-repo/semantics/publishe

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

SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Inositol 1,4,5-trisphosphate 3-kinase mRNA: high levels in the rat hippocampal CA1 pyramidal and dentate gyrus granule cells and in cerebellar Purkinje cells

The distribution of inositol 1,4,5-trisphosphate (InsP3) 3-kinase mRNA in the rat brain is reported using oligonucleotides based on a cDNA clone sequence that encodes rat brain InsP3 3-kinase and the in situ hybridization technique. Moderate levels were found in CA2-4 pyramidal neurons, in the cortex, and in the striatum. The cerebellar granule cells, thalamus, hypothalamus, brainstem, spinal cord, and white matter tracts were almost negative. The levels of InsP3 3-kinase mRNA were highest in the hippocampal CA1 pyramidal neurons, granule cells of the dentate gyrus, and cerebellar Purkinje cells. These results contrast with the lower concentration of the InsP3 receptor already reported in the hippocampus versus the Purkinje cells and suggest a special role for inositol 1,3,4,5-tetrakisphosphate in Ammon's horn.Journal ArticleResearch Support, Non-U.S. Gov'tFLWNAinfo:eu-repo/semantics/publishe

Molecular cloning and expression of a human brain inositol 1,4,5-trisphosphate 3-kinase.

A human hippocampus cDNA library was screened by hybridization with a rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase cDNA. Sequencing of three overlapping clones identified a 1383 bp open reading frame encoding a 461 amino acid protein with a calculated molecular weight of 50988. The coding amino acid sequence showed an overall 93% similarity with the sequence of rat brain InsP3 3-kinase. The cDNA insert of one isolated partial clone (i.e. hh 26) was in frame with the beta galactosidase fragment fused to it as a Bluescript plasmid; it displayed InsP3 3-kinase activity when expressed in Escherichia coli (E. coli). Biochemical characterization of human brain InsP3 3-kinase by SDS polyacrylamide gel electrophoresis and regeneration of enzyme activity reveals three active fractions with apparent Mr of 58,000-64,000, 45,000-50,000 and 37,000-39,000.Comparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

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

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.Journal Articleinfo:eu-repo/semantics/publishe

Inositol 1,4,5-trisphosphate 3-kinase activity in FRTL-5 cells: regulation of the enzyme activity by TSH.

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase phosphorylates the Ca(2+)-mobilizing second messenger InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). InsP3 5-phosphatase dephosphorylates InsP3 to inositol 1,4-bisphosphate (InsP2). We compared the effects of TSH added to a culture of FRTL-5 thyroid cells on the activity of InsP3 5-phosphatase and InsP3 3-kinase. InsP3 3-kinase activity was decreased at a physiological concentration of TSH. Inhibition of this activity started after 3 h of incubation with TSH and was maximal after 24 h. In contrast, InsP3 5-phosphatase activity was not affected by TSH under the same conditions. The inhibitory effect of TSH on InsP3 3-kinase was characterized as follows: a) inhibition of activity was mimicked by both dibutyryl cyclic AMP and forskolin; b) activity obtained by mixing lysates of TSH-stimulated and non-stimulated cells was the sum of each activity measured separately; c) inhibition persisted after a crude lysate of TSH-stimulated cells had been subjected to SDS/polyacrylamide gel electrophoresis and the extraction of InsP3 3-kinase activity. The data suggest that TSH reduced the activity of InsP3 3-kinase in FRTL-5 cells either by a phosphorylation/dephosphorylation mechanism, or by affecting expression of the enzyme.Journal Articleinfo:eu-repo/semantics/publishe