Regulation of AMP Deaminase by Phosphoinositides

Abstract

AMP deaminase (AMPD) converts AMP to IMP and is a diverse and highly regulated enzyme that is a key component of the adenylate catabolic pathway. In this report, we identify the high affinity interaction between AMPD and phosphoinositides as a mechanism for regulation of this enzyme. We demonstrate that endogenous rat brain AMPD and the human AMPD3 recombinant enzymes specifically bind inositide-based affinity probes and to mixed lipid micelles that contain phosphatidylinositol 4,5-bisphosphate. Moreover, we show that phosphoinositides specifically inhibit AMPD catalytic activity. Phosphatidylinositol 4,5-bisphosphate is the most potent inhibitor, effecting pure noncompetitive inhibition of the wild type human AMPD3 recombinant enzyme with a K i of 110 nM. AMPD activity can be released from membrane fractions by in vitro treatment with neomycin, a phosphoinositide-binding drug. In addition, in vivo modulation of phosphoinositide levels leads to a change in the soluble and membrane-associated pools of AMPD activity. The predicted human AMPD3 sequence contains pleckstrin homology domains and (R/ K)X n (R/K)XKK sequences, both of which are characterized phosphoinositide-binding motifs. The interaction between AMPD and phosphoinositides may mediate membrane localization of the enzyme and function to modulate catalytic activity in vivo.Phosphoinositides and inositol polyphosphates (referred to collectively as inositides) are components of many pathways in eukaryotic cells, functioning in second messenger cascades, acting as regulators of many proteins, and operating as membrane localization signals (1-3). Numerous protein and lipid kinases, adaptor proteins, ion channels, phospholipases, modulators of small GTPases, and actin-binding proteins are regulated by inositides (1-3). To identify novel targets for inositides, our laboratories and others have used purification schemes employing affinity resins that contain tethered inositol polyphosphate head groups (3-9). These affinity purifications were successful in the identification of inositide binding in the clathrin adaptor/assembly protein AP-2 (6), centaurin ␣ (7), a centaurin ␣ orthologue (8), and a phospholipase C (PLC) 1 -related protein (9). In addition to AP-2 and centaurin ␣, we isolated several other proteins from rat brain, one of which was approximately 80 kDa (5). Published reports show that inositol polyphosphates modulate the activity of the enzyme AMP deaminase (EC 3.5.4.6) (AMPD) (10, 11), an enzyme family whose endogenous, purified subunit molecular masses are between 66 and 88 kDa. Therefore, we hypothesized that AMPD could be the 80-kDa protein isolated using the inositide affinity resin.AMPD is a diverse and highly regulated enzyme located at a branchpoint in the adenine nucleotide catabolic pathway and is important in regulating nucleotide pools. AMPD is also a component of the purine nucleotide cycle, an energy-generating pathway reportedly operative in many animal tissues (reviewed in Ref. 12). The AMPD1 gene encodes human isoform M and rat iso...

Regulation of Inositol Phospholipid Binding and Signaling through Syndecan-4

“…It has been suggested that inositol polyphosphates may compete with phosphoinositides in the regulation of phospholipase C␦ (40), AP-2, Bruton's tyrosine kinase, synaptotagmin II (20), and AMP deaminase (reviewed in Ref. 41). Levels of InsP 6 in some cells may reach 15-60 M (37), and it is the most abundant inositol polyphosphate in most vertebrate cells.…”

N-terminal Sequence and Distal Histidine Residues Are Responsible for pH-regulated Cytoplasmic Membrane Binding of Human AMP Deaminase Isoform E

“…A positively charged surface within the PH domain has been shown to represent the ligand binding site to phosphoinositides (45). In fact, the C-terminal region of isoform E has two putative PH domains (31), and these stretches of sequence contain 11 of the 29 histidine residues found in the AMPD3 polypeptide. Data presented in this study have shown that N-carbethoxylation of as many as 18 histidine residues in each isoform E subunit is accompanied by the near elimination of its membrane binding capacity.…”

“…Negative charge potentials of intracellular membranes are due, in part, to the presence of a variety of anionic phospholipids. Among these, PtdIns(4,5)P2 is a component of the cytoplasmic membrane that can interact with isoform E. PtdIns(4,5)P2 is a pure noncompetitive inhibitor of this enzyme with a K i of 110 nM (31), which may also explain the associated catalytic inhibition of membrane-bound isoform E. While other membrane-derived sources of phosphate groups could bind and inhibit this enzyme, low micromolar concentrations of neomycin are able to simultaneously relieve catalytic inhibition and disrupt the protein-lipid interaction involving isoform E. Neomycin is an aminoglycoside antibiotic with six primary amine groups that binds strongly to PtdIns(4,5)P2 in intracellular membranes (36,38,39). Although neomycin does not distinguish between PtdIns(4,5)P2 and PtdIns(3,4)P2 (39), the former is the major bisphosphate inositol in mammalian cells and therefore a more likely in vivo target for isoform E.…”

“…Neomycin-induced Dissociation of AMPD3 Enzymes from Erythrocyte Ghosts-Neomycin was evaluated as an antagonist of the associations between AMPD3 enzymes and EG under the premise that these interactions may be related to their affinity for PtdIns(4,5)P2, an integral membrane phospholipid that is also a potent noncompetitive inhibitor of catalytic activity (31). Neomycin is an aminoglycoside antibiotic containing six primary amine groups that bind to PtdIns(4,5)P2 with micromolar affinity (36 -39).…”

“…Knowledge of the physical basis for these protein-lipid interactions and the effects they have on catalytic behavior of AMPD3 enzymes have been advanced by two recent observations (31). 1) Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), an integral membrane phospholipid, is a potent noncompetitive inhibitor of human recombinant isoform E and its primary proteolytic product, ⌬M90.…”