A Target of Phosphatidylinositol 3,4,5‐Trisphosphate with a Zinc Finger Motif Similar to that of the ADP‐Ribosylation‐Factor GTPase‐Activating Protein and Two Pleckstrin Homology Domains

Abstract

We have purified a protein that binds phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] using beads bearing a PtdIns(3,4,5)P3 analogue. This protein, with a molecular mass of 43 kDa, was termed PtdIns(3,4,5)P3-binding protein. The partial amino acid sequences were determined and a full-length cDNA encoding the protein was isolated from bovine brain cDNA library. The clone harbored an open reading frame of 373 amino acids which contained one zinc finger motif similar to that of ADP-ribosylation-factor GTPase-activating protein and two pleckstrin homology domains. The entire sequence was 83 % similar to centaurin a, another PtdIns(3,4,5)P3-binding protein. The protein bound PtdIns(3,4,5)P3 with a higher affinity than it did inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3-phosphate suggesting that the binding to PtdIns(3,4,5)P3 was specific. The binding activity was weaker in the mutants with a point mutation in the conserved sequences in each pleckstrin homology domain. Introduction of both mutations abolished the activity. These results suggest that this new binding protein hinds PtdIns(3,4,5)P3 through two pleckstrin domains present in the molecule.Keywords: phosphatidylinositol 3,4,5-trisphosphate; inositolphospholipid 3-kinase; signal transduction.

Recombinant p42IP4, a brain‐specific 42‐kDa high‐affinity Ins(1,3,4,5)P4 receptor protein, specifically interacts with lipid membranes containing Ptd‐Ins(3,4,5)P3

“…(1,3,4,5,6)P 5 3.2 ^1.7 nm (3) Glycero-PhosphoIns(3,4,5)P 3 6.0 ^2.0 nm (7) DiC8-Ptd-Ins(3,4,5)P 3 3.5 ^1.5 nm (5) DiC8-Ptd-Ins(3,4)P 2 1.5 ^0.4 mm (4) DiC8-Ptd-Ins(4,5)P 2 . 10 mm (2) DiC16-Ptd-Ins(3,4,5)P 3 : IC 50 /slope 121 ^69 nm/2.9^0.9 (6) p42 IP4 has two PH domains and possibly both PH domains contribute to ligand binding [19]. This could mean that p42 IP4 binds the ligand in a horse-shoe-like manner, as proposed for the structure of p110g [39].…”

“…Porcine and rat p42 IP4 have 374 amino acid residues. PIP3BP reported by Tanaka et al [19] is the corresponding bovine homologue with one amino-acid deletion. Sequence homology analysis reveals that p42 IP4 has a zinc finger domain at its N-terminus followed by two PH-domains.…”

“…Some of the PH domain-containing proteins that bind PtdIns(3,4,5)P 3 also bind inositols, phosphorylated at position D3. For p42 IP4 which binds Ins(1,3,4,5)P 4 with a K d in the nm range [17], the role of the PH domain in determining ligand specificity is not yet clear; this is also the case for centaurin-a and PIP3BP [19,20]. The structure of the PH domain of Btk [9] which binds Ins(1,3,4,5)P 4 and Ptd-InsP 3 , was resolved by X-ray crystallography [23].…”

“…p42 IP4 is expressed mainly in brain [17,18]; these proteins displaying two PH domains comprise the porcine p42 IP4 [17], the rat p42 IP4 [18], and the bovine Ptd-Ins(3,4,5)P 3 -binding protein (PIP3BP) [19]. The homologous protein rat centaurin-a [20] differs from the p42 IP4 proteins by disruption of the N-terminal PH domain and by a 45 amino acid extension at the C-terminus [18].…”

“…p42 IP4 is expressed mainly in brain [17,18]; these proteins displaying two PH domains comprise the porcine p42 IP4 [17], the rat p42 IP4 [18], and the bovine Ptd-Ins(3,4,5)P 3 -binding protein (PIP3BP) [19] GST, glutathione-S-transferase; PI3-kinase, phosphoinositide-3-kinase; Ins, inositol; Ptd-Ins, phosphatidylinositol; PH, pleckstrin homology; PLC, phospholipase C; SH2 domain, src homology-2 domain; Btk, Bruton's tyrosine kinase; diC16, dipalmitoyl-l-a; diC8, dioctanly-l-a; GroPIns(3, 4,5)P 3, glycerophosphoinositol 3,4,5-trisphosphate; PIP3BP, Ptd-Ins(3,4,5)P 3 -binding protein; Sf9, Spodoptera frugiperda. Note: Theodor Hanck and Rolf Stricker contributed equally to this work.…”