Inositol pyrophosphate mediated pyrophosphorylation of AP3B1 regulates HIV-1 Gag release

Abstract

High-energy inositol pyrophosphates, such as IP7 (diphosphoinositol pentakisphosphate), can directly donate a ␤-phosphate to a prephosphorylated serine residue generating pyrophosphorylated proteins. Here, we show that the ␤ subunit of AP-3, a clathrin-associated protein complex required for HIV-1 release, is a target of IP 7-mediated pyrophosphorylation. We have identified Kif3A, a motor protein of the kinesin superfamily, as an AP3B1-binding partner and demonstrate that Kif3A, like the AP-3 complex, is involved in an intracellular process required for HIV-1 Gag release. Importantly, IP 7-mediated pyrophosphorylation of AP3B1 modulates the interaction with Kif3A and, as a consequence, affects the release of HIV-1 virus-like particles. This study identifies a cellular process that is regulated by IP 7-mediated pyrophosphorylation.phosphorylation ͉ trafficking ͉ kinesin I nositol pyrophosphates such as IP 7 (diphosphoinositol pentakisphosphate or PP-IP 5 ) belong to a class of inositol polyphosphates containing highly energetic pyrophosphate moieties that undergo very rapid turnover (1, 2). Inositol pyrophosphates have been implicated in numerous important cellular events (3, 4), including apoptosis (5, 6) and insulin secretion (7,8), and the use of radiolabeled IP 7 (5␤[ 32 P]IP 7 ) has allowed us to demonstrate that the high-energy pyrophosphate bond can participate in phospho-transfer reactions (9). One of the hallmarks of protein phosphorylation via IP 7 is that the putative targets contain a serine-rich acidic region (9). IP 7 substrates must initially be primed through ATP-dependent protein kinase phosphorylation (10), following which the phospho-serine becomes a substrate of IP 7 -mediated pyrophosphorylation (10). However, the physiological in vivo significance of this posttranslational modification remains unclear. To address the functional significance of protein pyrophosphorylation, we investigate the effect of this posttranslational modification in the functionality of the adaptor protein complex AP-3. ResultsThe ␤ subunit of the adaptor protein complex AP-3 (AP3B1) (Fig. 1A) was initially identified as a potential target for IP 7 phosphorylation by database searching for proteins containing serine-rich acidic regions. Adaptor protein complexes, comprising AP-1 to AP-4, mediate the sorting of transmembrane and cargo proteins to specific membrane compartments within the cell (11, 12). The AP-3 complex in particular is involved in sorting to lysosomes and related organelles (13,14). It contains two large subunits (␤ and ␦), a medium subunit ( 3 ), and a small subunit ( 3 ) (12). The AP3B1 subunit contains three main domains: the N-terminal head domain, the hinge, and the C-terminal ear domain ( Fig. 1 A and Fig. S1 A). The putative target region of IP 7 phosphorylation lies within the hinge domain, which contains three distinct serine-rich acidic stretches that we named regions I, II, and III ( Fig. 1 A and Fig. S1 A). To test whether human AP3B1 is a substrate of IP 7 -mediated phosphorylation, we per...

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