Type Iγ661 phosphatidylinositol phosphate kinase directly interacts with AP2 and regulates endocytosis

Clathrin-coated vesicles mediate sorting and intracellular transport of membrane-bound proteins. The formation of these coats is initiated by the assembly of adaptor proteins (AP), which specifically bind to membrane cargo proteins via recognition of endocytic sorting motifs. The lipid signaling molecule phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2) is critical for this process, as it serves as both a targeting and regulatory factor. PI( 4,5) P2 is synthesized by type I phosphatidylinositol phosphate kinases (PIPKI). We have discovered a direct interaction between the mu 2-subunit of the AP2 complex and PIPKI gamma 661 via a yeast two-hybrid screen. This interaction was confirmed using both the mu 2-subunit in glutathione S-transferase pulldowns and via coimmunoprecipitation of endogenous PIPKI gamma 661 with the AP2 complex from HEK293 cells. The interaction is mediated, in vivo, by a tyrosine-based motif in the 26-amino acid tail of PIPKI gamma 661. Because AP2 regulates endocytosis of transferrin receptor from the plasma membrane, we also examined a role for PIPKI gamma 661 using a flow cytometry endocytosis assay. We observed that stable expression of wild type PIPKI gamma 661 in Madin-Darby canine kidney cells enhanced transferrin uptake, whereas stable expression of kinase-dead PIPKI gamma 661 had an inhibitory effect. Neither condition affected the overall cellular level of PI(4,5) P2. RNA interference-based knockdown of PIPKI gamma 661 in HeLa cells also had an inhibitory effect on transferrin endocytosis using the same assay system. Collectively, this evidence implies an important role for PIPKI gamma 661 in the AP2-mediated endocytosis of transferrin.