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

Abstract

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.

Inositol (1,4,5) trisphosphate 3 kinase B controls positive selection of T cells and modulates Erk activity

“…2C). This residual activity could reflect low-level thymic expression of other Itpk isoforms (6,11,12,24). Our data suggest that lack of full-length Itpkb protein expression and concomitant reduction of Itpk activity in thymocytes underlies the defect in T cell development in Ms. T-less mice.…”

Human Inositol 1,4,5-Trisphosphate 3-Kinase Isoform B (IP3KB) Is a Nucleocytoplasmic Shuttling Protein Specifically Enriched at Cortical Actin Filaments and at Invaginations of the Nuclear Envelope

“…Ins(1,3,4,5)P 4 , inositol 1,3,4,5-tetrakisphosphate; HsIP3KB, human IP3KB; IP3KB, inositol 1,4,5-trisphosphate 3-kinase isoform B; MTD, multitargeting domain; NLS, nuclear localization signal; NES, nuclear export signal; LMB, leptomycin B; EGFP, enhanced GFP; TRITC, tetramethylrhodamine-5-isothiocyanate; ROI, region of interest.…”

“…In mammalian cells, multiple intracellular localizations of IP3KB, namely to the plasma membrane, cytoskeleton, and endoplasmic reticulum, have been observed (19). Most likely, these intracellular targeting specificities of IP3KB are of regulatory importance for all processes mediated by IP3KB and its product Ins(1,3,4,5)P 4 . Therefore, the molecular basis of these targeting activities has been studied intensively, revealing an actin binding and nuclear targeting domain (7) as well as an endoplasmic reticulum membrane anchoring domain (20).…”

“…2 -specific IP3Ks remove the Ca 2ϩ -releasing second messenger Ins(1,4,5)P 3 by phosphorylating it to Ins(1,3,4,5)P 4 (1) supporting the formation of higher phosphorylated inositols in the cell (2). So far, three isoforms of IP3K, termed A (3), B (4), and C (5), have been identified in mammals.…”

Specific expression and function of inositol 1,4,5-trisphosphate 3-kinase C (ITPKC) in wild type and knock-out mice

“…Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), the well-known calcium mobilization messenger, can be phosphorylated into inositol 1,3,4,3,4,5)P4) by Ins(1,4,5)P3 3-kinases (Itpk) isoforms a, b and c, or by Ipk2/Ipmk, the inositol phosphate multikinase (Choi et al, 1990;Takazawa et al, 1990Takazawa et al, , 1991Dewaste et al, 2000;Saiardi et al, 2000; reviewed by York, 2006). The a, b and c isoforms of Ins(1,4,5)P3 3-kinases (or ITPKA, ITPKB and ITPKC) share a well conserved carboxy-terminal catalytic domain, an ATP binding motif, an inositol binding motif, and a calmodulin binding domain (reviewed by Erneux et al, 2016).…”