Stimulation of phosphatidylinositol kinase type I-mediated phosphatidylinositol (4,5)-bisphosphate synthesis by AP-2μ–cargo complexes

Abstract

Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is an important factor for a variety of cellular functions ranging from cell signaling to actin cytoskeletal dynamics and endocytic membrane traffic. Here, we have identified the clathrin adaptor complex AP-2 as a regulator of phosphatidylinositol 4-phosphate 5-kinase (PIPK)-mediated PI(4,5)P 2 synthesis. AP-2 directly interacts with the kinase core domain of type I PIPK isozymes via its 2-subunit in vitro and in native protein extracts. Endocytic cargo protein binding to 2 leads to a potent stimulation of PIPK activity. These data thus identify a positive feedback loop consisting of endocytic cargo proteins, AP-2 , and PIPK type I which may provide a specific pool of PI(4,5)P 2 dedicated to clathrin͞AP-2-dependent receptor internalization.phosphoinositides ͉ sorting motifs ͉ clathrin ͉ endocytosis P hosphoinositides have pleiotropic functions in cell physiology including the regulation of signal transduction, membrane traffic (1), and the organization of the actin cytoskeleton (2). Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P 2 ], a phosphoinositide concentrated at the plasma membrane, is required for the generation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP 3 ), remodelling of the actin cytoskeleton (2, 3), clathrin-dependent (4) and -independent pathways of cell entry (5), the exo-endocytic cycling of synaptic and neurosecretory vesicles (6-8), and serves as a substrate for the synthesis of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ]. Biochemical, genetic, and cell biological data suggest that PI(4,5)P 2 plays an essential role in clathrin-dependent endocytosis of plasma membrane proteins including nutrient (4) and growth factor receptors, postsynaptic ion channels, as well as synaptic vesicle proteins (9-11). Endocytic adaptor proteins like the heterotetrameric AP-2 complex (via its ␣ and 2 subunits) (12, 13), AP180͞CALM, epsin (14), Dab2, and HIP1͞1R as well as the large GTPase dynamin (15) all bind directly to PI(4,5)P 2 . In addition to serving as a membrane attachment site for endocytic proteins, PI(4,5)P 2 also plays an active role by allosterically eliciting a conformational change within AP-2 that is required for its interaction with sorting signals of transmembrane proteins (16) and to stably associate with the plasmalemma (17). Impairment of PI(4,5)P 2 hydrolysis by deletion of the phosphoinositide-phosphatase synaptojanin (18-20) results in the accumulation of clathrin-coated pits and vesicles, suggesting that the membrane concentration of PI(4,5)P 2 controls the stability of endocytic clathrin coats.Three isoforms of type I phosphatidylinositol 4-phosphate 5-kinase (PIPK) have been identified in mammals (␣, ␤, ␥), which generate PI(4,5)P 2 by phosporylating PI(4)P at the D-5 position of the inositol ring (21,22). Although isoform-specific functions of type I PIPKs have been shown to contribute to distinct cellular processes such as formation of focal adhesions (23,24), at present it is largely unclear how the s...

Two synaptojanin 1 isoforms are recruited to clathrin-coated pits at different stages

“…Turnover of PI(4,5)P 2 may be needed for the normal growth of a CCP by facilitating dynamic remodeling of the coat. This possibility is consistent with a recent report that type I PIP kinases (PI4P 5-kinases), which are the major PI(4,5)P 2 synthesizing enzymes, interact with AP-2, even though these enzymes are apparently not concentrated at CCPs (42,43).…”

Conserved role for Gga proteins in phosphatidylinositol 4-kinase localization to the trans -Golgi network

“…Specifically, physical and functional interactions have been described between AP-3 and PI4KIIα, AP-2 and PI4P5KIγ, and AP-1 and PI4KIIβ (37)(38)(39). Our data now demonstrate that this property extends to GGA2 and PI4KIIIβ.…”

A phosphoinositide conversion mechanism for exit from endosomes

“…Clathrin coated pit (CCP) nucleation, although dependent on the general PM PI(4,5)P 2 content (Zoncu, Perera et al 2007), is driven by local synthesis of PI(4,5)P 2 by PIP5K type I. The most prominent PI(4,5)P 2 -specific clathrin adaptor is the tetrameric adaptor protein AP-2 and cargo-AP-2-complexes stimulate PIP5K activity (Krauss, Kukhtina et al 2006). While PI(4,5)P 2 -synthesis is restricted to CCP initiation, PI(4,5)P 2 5-phosphatases and 4-phosphatases such as synaptojanin (Perera, Zoncu et al 2006) or SHIP2 (Nakatsu, Perera et al 2010) are recruited during CCP maturation.…”