Abstract
A segment of inositol 1,4,5-trisphosphate 3-kinase responsible for inositol 1,4,5-trisphosphate (InsP(3)) binding was characterized and confirmed by three different approaches employing the fully active expressed catalytic domain of the enzyme. Part of this moiety was protected from limited tryptic proteolysis by InsP(3). Sequencing of two fragments of 16 and 21 kDa, generated in the absence or presence of InsP(3), respectively, identified segment Glu-271 to Arg-305 as being protected. 15 monoclonal antibodies, all binding to epitopes within this region, inhibited enzyme activity and interfered with inositol phosphate binding. Detailed enzyme kinetic parameters of 32 site-directed mutants revealed residues Arg-276 and Lys-303 in this segment and Arg-322, located nearby, as directly involved in and five other closely neighbored residues, all located within a segment of 73 amino acids, as also influencing InsP(3) binding. Part of this region is similar in sequence to an InsP(3) binding segment in InsP(3) receptors. Combined with the finding that mutants influencing only ATP binding all lie outside this region, these data indicate that an InsP(3) binding core domain is inserted between two segments acting together in ATP binding and phosphate transfer.
Highlights
The second messenger inositol 1,4,5-trisphosphate (InsP3)1 is phosphorylated by a 3-kinase to inositol 1,3,4,5-tetrakisphosphate (InsP4)
An InsP3 binding segment has been located in the catalytic domain of type II inositol polyphosphate 5-phosphatases, with two short, highly conserved amino acid motifs (WXGDXNXR and PXWCDRXL) identified by mutational analysis as being involved in catalysis and/or substrate binding
For phospholipase C␦1 the structural requirements for InsP3 binding are already fairly well understood because the pleckstrin homology domain of this enzyme has been identified as the InsP3-binding element, and a crystal structure of this domain bound to InsP3 has been obtained [15]
Summary
InsP3, inositol 1,4,5-trisphosphate; InsP4, inositol 1,3,4,5-tetrakisphosphate; IP3K, inositol 1,4,5-trisphosphate 3-kinase; IP3R, inositol 1,4,5-trisphosphate receptor; GST, glutathione S-transferase; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; CaM, calmodulin; IP3K147-C, COOH-terminal fragment (amino acids 147– 452) of chicken IP3K. IP3K is known as a highly selective InsP3-binding protein [16] with high affinity for this substrate, as illustrated by Km values ranging from 0.7 to 3.1 M [17,18,19,20,21] depending on the. This domain shows the highest degree of amino acid conservation (30% identities) between isoforms and between species Within this region Lys-197 and Asp-414 have been found to be involved in ATP binding in rat isoform A by site-directed mutagenesis [29]. The enzymatic properties of recombinantly expressed mutant proteins clearly indicate the direct involvement in InsP3 binding of three residues (Arg-276 and Lys-303) within or close to this region (Arg-322) and a participation in binding of five neighbored residues, all lying within a 73-amino acid segment, centered in the catalytic domain
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