Regulation of phosphoinositide-metabolizing enzymes by clathrin coat proteins

Clathrin plays key roles in endocytic and endo-lysosomal membrane dynamics by facilitating the formation of coated vesicles at the plasma membrane and at the trans-Golgi network (TGN)/endosomal boundary. Assembly of the clathrin lattice critically depends on adaptor proteins and accessory proteins, which connect the clathrin scaffold to the membrane and to transmembrane cargo including receptors, transporters, channels, and SNARE proteins. The recruitment of adaptor proteins to membrane surfaces is triggered by coincidence-detection mechanisms involving phosphoinositides (PIs), cargo proteins, and in many cases small GTPases. To tightly regulate coat formation, there is extensive cross-talk between PI-metabolizing enzymes and adaptor proteins. One of the best studied examples is the endocytic clathrin adaptor complex AP-2, which binds plasma membrane-enriched PI(4,5)P(2). In neurons, PI(4,5)P(2) is synthesized from PI(4)P primarily by the γ-isoform of the type I phosphatidylinositol 4-phosphate 5-kinase family (PIPKIγ), whose enzymatic activity is regulated by direct binding to, amongst others, the small GTPase Arf6 and AP-2. Cargo-bound AP-2 potently stimulates PIPK1γ activity and thereby drives AP-2-membrane interactions. This feed-forward loop is thought to facilitate membrane translocation of additional AP-2 molecules and concomitantly clathrin, but also of endocytic accessory proteins, many of which directly associate with PI(4,5)P(2). It is likely that similar mechanisms support the formation of coated vesicles at the trans-Golgi network (TGN) and on endosomes, involving PI(4)P and PI(3)P respectively, but detailed knowledge is lacking to date. To explore how coat proteins regulate kinase activity, assays are needed to sensitively detect subtle changes in PI synthesis and to discriminate between the various PI species. Here we describe a sensitive and specific radioactivity-based assay to measure PI kinase activity.