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.
Highlights
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
Endogenous Brain AMPD Binds Inositide Affinity Probes— Using an aminopropyl-InsP4 resin to purify inositide-binding proteins from rat brain, we previously identified a protein at approximately 80 kDa that eluted in fractions containing the clathrin adaptor/assembly protein AP-2 (3, 5, 6, 45)
Membrane Association of AMPD May Involve Phosphoinositides—To investigate whether AMPD may interact with endogenous phosphoinositides within the membrane, we examined the effects of neomycin, a phosphoinositide-binding drug (47), on AMPD activity in membrane fractions
Summary
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. We identify the high affinity interaction between AMPD and phosphoinositides as a mechanism for regulation of this enzyme. 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). 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 ␣. AMPD activity is highly regulated through interactions with other proteins (23–25), phosphorylation (26, 27), and small molecules (10, 11, 28 –32). Whereas hydropathy analysis suggests that AMPD isoforms do not contain putative transmem-
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