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J. Biol. Chem., Vol. 281, Issue 29, 20632-20642, July 21, 2006

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Type I661 Phosphatidylinositol Phosphate Kinase Directly Interacts with AP2 and Regulates Endocytosis^*

From the Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706

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)P₂) 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 µ2-subunit of the AP2 complex and PIPKI661 via a yeast two-hybrid screen. This interaction was confirmed using both the µ2-subunit in glutathione S-transferase pulldowns and via coimmunoprecipitation of endogenous PIPKI661 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 PIPKI661. Because AP2 regulates endocytosis of transferrin receptor from the plasma membrane, we also examined a role for PIPKI661 using a flow cytometry endocytosis assay. We observed that stable expression of wild type PIPKI661 in Madin-Darby canine kidney cells enhanced transferrin uptake, whereas stable expression of kinase-dead PIPKI661 had an inhibitory effect. Neither condition affected the overall cellular level of PI(4,5)P₂. RNA interference-based knockdown of PIPKI661 in HeLa cells also had an inhibitory effect on transferrin endocytosis using the same assay system. Collectively, this evidence implies an important role for PIPKI661 in the AP2-mediated endocytosis of transferrin.

Received for publication, February 15, 2006 , and in revised form, May 10, 2006.

^* This work was supported in part by National Institutes of Health Biotechnology Training Program Grant GM08349 (to S. F. B.), American Heart Association Grant 133-EY51 (to K. L.), and National Institutes of Health Grants GM57549 and CA104708 (to R. A. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Graduate of the Biomolecular Chemistry Predoctoral Training Program. Current address: Baxter Healthcare Corp., Rt. 120 and Wilson Rd., Round Lake, IL 60073.

² To whom correspondence should be addressed: 3750 Medical Science Center, 1300 University Ave., University of Wisconsin Medical School, Madison, WI 53706. Tel.: 608-262-3753; Fax: 608-262-1257; E-mail: raanders{at}wisc.edu.

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