Antiproliferative plant and synthetic polyphenolics are specific inhibitors of vertebrate inositol-1,4,5-trisphosphate 3-kinases and inositol polyphosphate multikinase. VOLUME 280 (2005) PAGES 13229-13240

Abstract

In this paper we analyzed putative amino acids involved in inhibitor binding and identified Lys268 and Lys272 as inhibitor binding sites. However, verification of these results showed that not these amino acids but Lys336 is critical for inhibitor binding. The failure was corrected (as shown in bold) in the updated sections that follow. PAGE 13229: In the Abstract, the sentence in lines 25-28 should read: “Mutagenesis studies revealed that both the calmodulin binding and the ATP binding domains in IP3K are involved in inhibitor binding.” PAGE 13235: In the right column, under the subheading “Search for Inhibitor Binding Sites,” lines 31-47 should read: “The point mutant K336Q revealed a drastically reduced inhibition by THF (Table VII). Substitution of Lys336 led to a 260-fold increase in the IC50. On the other hand, kinetic parameters of the enzyme with respect to both substrates were nearly unchanged (Table VII). Thus, by substituting lysine 336, which is involved in ATP binding and located in the C-lobe (37, 38), we have created inhibitor-resistant IP3K-A with full enzymatic activity and normal substrate affinity. As shown in the sequence alignment of IP3K isoforms and IPMK in Fig. 4B, this region shows high homology between IP3K isoforms but not between IP3K and IPMK. Both the absence of the CaM binding domain (above) and the differences in the ATP binding site (55) may thus contribute to the observed differences in inhibitor selectivity of IPMK as compared with IP3K (see Fig. 1C).TABLE VIIKinetic constants and IC50 values for THF inhibition of mutated or truncated IP3K isoforms or IPMKEnzymeKm InsP3Km ATPSpecific activityInhibitorIC50μmμmunits/mgμmRnIP3K-CCaM/cataFragment comprising CaM binding and catalytic domain (assay without Ca2+-CaM).0.2333THF0.19RnIP3K-CcatbFragment comprising only catalytic domain.1.74216THF0.61GgIP3K-AaFragment comprising CaM binding and catalytic domain (assay without Ca2+-CaM).0.407415THF0.29GgIP3K-AK336QcFragment comprising CaM binding and catalytic domain. Lys336 is substituted by Gln (assay without Ca2+-CaM).0.5410516THF84.3HsIPMK wt0.35172.5THF>100OthersSee Table VHsIPMKΔdDeletion of amino acids 266-371.0.40395.5THF0.52OthersSee Table Va Fragment comprising CaM binding and catalytic domain (assay without Ca2+-CaM).b Fragment comprising only catalytic domain.c Fragment comprising CaM binding and catalytic domain. Lys336 is substituted by Gln (assay without Ca2+-CaM).d Deletion of amino acids 266-371. Open table in a new tab PAGE 13237: Table VII has been updated. Fig. 4B has been corrected as shown, and its legend should read: “Identification of sites of GgIP3K-A involved in inhibitor binding. Partial sequences from the catalytic domains of mammalian IP3K isoforms and HsIPMK comprising the “GFR” motif (underlined) and parts of the ATP binding domain downstream of this motif (55) are aligned. The amino acid in GgIP3K-A (bold) shown by mutagenesis to be involved in inhibitor binding (Lys336) is boxed.” PAGE 13238: In the right column, under the subheading “Structural Basis of Inhibitor Selectivity,” lines 1-16 should read: “The markedly differing inhibitor selectivity of IP3K and IPMK reflects different inhibitor binding sites within these two homologous enzymes (see the structural sketch in Fig. 4A). One important position in the IP3K catalytic domain for inhibitor binding seems to be located within the ATP binding segment, since substitution of Lys336 downstream of the GFR motif (Ref. 56, see Fig. 4) caused a 260-fold decrease of the inhibitory effect. No corresponding Lys residue is present in IPMK (see Fig. 4B). The location of Lys336 in the three-dimensional structure of IP3K-A (37, 38) indicates that it is located in the C-lobe and is involved in ATP binding. An interaction of inhibitor with this side chain thus may induce a major conformational change modifying the nucleotide binding site.”