• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of biochemjBJ Latest papers and much more!
Biochem J. Aug 15, 1997; 326(Pt 1): 221–225.
PMCID: PMC1218658

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

Abstract

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.

Full Text

The Full Text of this article is available as a PDF (331K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Berridge MJ, Irvine RF. Inositol phosphates and cell signalling. Nature. 1989 Sep 21;341(6239):197–205. [PubMed]
  • Irvine RF, Letcher AJ, Heslop JP, Berridge MJ. The inositol tris/tetrakisphosphate pathway--demonstration of Ins(1,4,5)P3 3-kinase activity in animal tissues. Nature. 1986 Apr 17;320(6063):631–634. [PubMed]
  • Hill TD, Dean NM, Boynton AL. Inositol 1,3,4,5-tetrakisphosphate induces Ca2+ sequestration in rat liver cells. Science. 1988 Nov 25;242(4882):1176–1178. [PubMed]
  • Morris AP, Gallacher DV, Irvine RF, Petersen OH. Synergism of inositol trisphosphate and tetrakisphosphate in activating Ca2+-dependent K+ channels. Nature. 1987 Dec 17;330(6149):653–655. [PubMed]
  • Irvine RF, Moor RM. Micro-injection of inositol 1,3,4,5-tetrakisphosphate activates sea urchin eggs by a mechanism dependent on external Ca2+. Biochem J. 1986 Dec 15;240(3):917–920. [PMC free article] [PubMed]
  • Joseph SK, Hansen CA, Williamson JR. Inositol tetrakisphosphate mobilizes calcium from cerebellum microsomes. Mol Pharmacol. 1989 Sep;36(3):391–397. [PubMed]
  • Ely JA, Hunyady L, Baukal AJ, Catt KJ. Inositol 1,3,4,5-tetrakisphosphate stimulates calcium release from bovine adrenal microsomes by a mechanism independent of the inositol 1,4,5-trisphosphate receptor. Biochem J. 1990 Jun 1;268(2):333–338. [PMC free article] [PubMed]
  • Gawler DJ, Potter BV, Nahorski SR. Inositol 1,3,4,5-tetrakisphosphate-induced release of intracellular Ca2+ in SH-SY5Y neuroblastoma cells. Biochem J. 1990 Dec 1;272(2):519–524. [PMC free article] [PubMed]
  • Reiser G, Schäfer R, Donié F, Hülser E, Nehls-Sahabandu M, Mayr GW. A high-affinity inositol 1,3,4,5-tetrakisphosphate receptor protein from brain is specifically labelled by a newly synthesized photoaffinity analogue, N-(4-azidosalicyl)aminoethanol(1)-1-phospho-D-myo-inositol 3,4,5-trisphosphate. Biochem J. 1991 Dec 1;280(Pt 2):533–539. [PMC free article] [PubMed]
  • Theibert AB, Estevez VA, Ferris CD, Danoff SK, Barrow RK, Prestwich GD, Snyder SH. Inositol 1,3,4,5-tetrakisphosphate and inositol hexakisphosphate receptor proteins: isolation and characterization from rat brain. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3165–3169. [PMC free article] [PubMed]
  • Cullen PJ, Hsuan JJ, Truong O, Letcher AJ, Jackson TR, Dawson AP, Irvine RF. Identification of a specific Ins(1,3,4,5)P4-binding protein as a member of the GAP1 family. Nature. 1995 Aug 10;376(6540):527–530. [PubMed]
  • Biden TJ, Wollheim CB. Ca2+ regulates the inositol tris/tetrakisphosphate pathway in intact and broken preparations of insulin-secreting RINm5F cells. J Biol Chem. 1986 Sep 15;261(26):11931–11934. [PubMed]
  • Zilberman Y, Howe LR, Moore JP, Hesketh TR, Metcalfe JC. Calcium regulates inositol 1,3,4,5-tetrakisphosphate production in lysed thymocytes and in intact cells stimulated with concanavalin A. EMBO J. 1987 Apr;6(4):957–962. [PMC free article] [PubMed]
  • Biden TJ, Altin JG, Karjalainen A, Bygrave FL. Stimulation of hepatic inositol 1,4,5-trisphosphate kinase activity by Ca2+-dependent and -independent mechanisms. Biochem J. 1988 Dec 15;256(3):697–701. [PMC free article] [PubMed]
  • Biden TJ, Comte M, Cox JA, Wollheim CB. Calcium-calmodulin stimulates inositol 1,4,5-trisphosphate kinase activity from insulin-secreting RINm5F cells. J Biol Chem. 1987 Jul 15;262(20):9437–9440. [PubMed]
  • Morris AJ, Downes CP, Harden TK, Michell RH. Turkey erythrocytes possess a membrane-associated inositol 1,4,5-trisphosphate 3-kinase that is activated by Ca2+ in the presence of calmodulin. Biochem J. 1987 Dec 1;248(2):489–493. [PMC free article] [PubMed]
  • Ryu SH, Lee SY, Lee KY, Rhee SG. Catalytic properties of inositol trisphosphate kinase: activation by Ca2+ and calmodulin. FASEB J. 1987 Nov;1(5):388–393. [PubMed]
  • Yamaguchi K, Hirata M, Kuriyama H. Purification and characterization of inositol 1,4,5-trisphosphate 3-kinase from pig aortic smooth muscle. Biochem J. 1988 Apr 1;251(1):129–134. [PMC free article] [PubMed]
  • Takazawa K, Passareiro H, Dumont JE, Erneux C. Ca2+/calmodulin-sensitive inositol 1,4,5-trisphosphate 3-kinase in rat and bovine brain tissues. Biochem Biophys Res Commun. 1988 Jun 16;153(2):632–641. [PubMed]
  • Takazawa K, Passareiro H, Dumont JE, Erneux C. Purification of bovine brain inositol 1,4,5-trisphosphate 3-kinase. Identification of the enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Biochem J. 1989 Jul 15;261(2):483–488. [PMC free article] [PubMed]
  • Lee SY, Sim SS, Kim JW, Moon KH, Kim JH, Rhee SG. Purification and properties of D-myo-inositol 1,4,5-trisphosphate 3-kinase from rat brain. Susceptibility to calpain. J Biol Chem. 1990 Jun 5;265(16):9434–9440. [PubMed]
  • Takazawa K, Lemos M, Delvaux A, Lejeune C, Dumont JE, Erneux C. Rat brain inositol 1,4,5-trisphosphate 3-kinase. Ca2(+)-sensitivity, purification and antibody production. Biochem J. 1990 May 15;268(1):213–217. [PMC free article] [PubMed]
  • Takazawa K, Vandekerckhove J, Dumont JE, Erneux C. Cloning and expression in Escherichia coli of a rat brain cDNA encoding a Ca2+/calmodulin-sensitive inositol 1,4,5-trisphosphate 3-kinase. Biochem J. 1990 Nov 15;272(1):107–112. [PMC free article] [PubMed]
  • Choi KY, Kim HK, Lee SY, Moon KH, Sim SS, Kim JW, Chung HK, Rhee SG. Molecular cloning and expression of a complementary DNA for inositol 1,4,5-trisphosphate 3-kinase. Science. 1990 Apr 6;248(4951):64–66. [PubMed]
  • Takazawa K, Erneux C. Identification of residues essential for catalysis and binding of calmodulin in rat brain inositol 1,4,5-trisphosphate 3-kinase. Biochem J. 1991 Nov 15;280(Pt 1):125–129. [PMC free article] [PubMed]
  • Erneux C, Moreau C, Vandermeers A, Takazawa K. 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. Eur J Biochem. 1993 Jun 1;214(2):497–501. [PubMed]
  • Communi D, Takazawa K, Erneux C. Lys-197 and Asp-414 are critical residues for binding of ATP/Mg2+ by rat brain inositol 1,4,5-trisphosphate 3-kinase. Biochem J. 1993 May 1;291(Pt 3):811–816. [PMC free article] [PubMed]
  • Sayers JR, Schmidt W, Eckstein F. 5'-3' exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis. Nucleic Acids Res. 1988 Feb 11;16(3):791–802. [PMC free article] [PubMed]
  • Chadwick CC, Saito A, Fleischer S. Isolation and characterization of the inositol trisphosphate receptor from smooth muscle. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2132–2136. [PMC free article] [PubMed]
  • Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. [PubMed]
  • Takazawa K, Perret J, Dumont JE, Erneux C. Molecular cloning and expression of a new putative inositol 1,4,5-trisphosphate 3-kinase isoenzyme. Biochem J. 1991 Sep 15;278(Pt 3):883–886. [PMC free article] [PubMed]
  • Chou PY, Fasman GD. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. [PubMed]
  • Fukuda M, Aruga J, Niinobe M, Aimoto S, Mikoshiba K. Inositol-1,3,4,5-tetrakisphosphate binding to C2B domain of IP4BP/synaptotagmin II. J Biol Chem. 1994 Nov 18;269(46):29206–29211. [PubMed]
  • Ross TS, Jefferson AB, Mitchell CA, Majerus PW. Cloning and expression of human 75-kDa inositol polyphosphate-5-phosphatase. J Biol Chem. 1991 Oct 25;266(30):20283–20289. [PubMed]
  • Verjans B, De Smedt F, Lecocq R, Vanweyenberg V, Moreau C, Erneux C. Cloning and expression in Escherichia coli of a dog thyroid cDNA encoding a novel inositol 1,4,5-trisphosphate 5-phosphatase. Biochem J. 1994 May 15;300(Pt 1):85–90. [PMC free article] [PubMed]
  • Furuichi T, Yoshikawa S, Miyawaki A, Wada K, Maeda N, Mikoshiba K. Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P400. Nature. 1989 Nov 2;342(6245):32–38. [PubMed]
  • Mignery GA, Newton CL, Archer BT, 3rd, Südhof TC. Structure and expression of the rat inositol 1,4,5-trisphosphate receptor. J Biol Chem. 1990 Jul 25;265(21):12679–12685. [PubMed]
  • Mignery GA, Südhof TC. The ligand binding site and transduction mechanism in the inositol-1,4,5-triphosphate receptor. EMBO J. 1990 Dec;9(12):3893–3898. [PMC free article] [PubMed]
  • Miyawaki A, Furuichi T, Ryou Y, Yoshikawa S, Nakagawa T, Saitoh T, Mikoshiba K. Structure-function relationships of the mouse inositol 1,4,5-trisphosphate receptor. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4911–4915. [PMC free article] [PubMed]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Compound
    Compound
    PubChem Compound links
  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...