Type Igamma661 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)P(2) is synthesized by type I phosphatidylinositol phosphate kinases (PIPKI). We have discovered a direct interaction between the mu2-subunit of the AP2 complex and PIPKIgamma661 via a yeast two-hybrid screen. This interaction was confirmed using both the mu2-subunit in glutathione S-transferase pulldowns and via coimmunoprecipitation of endogenous PIPKIgamma661 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 PIPKIgamma661. Because AP2 regulates endocytosis of transferrin receptor from the plasma membrane, we also examined a role for PIPKIgamma661 using a flow cytometry endocytosis assay. We observed that stable expression of wild type PIPKIgamma661 in Madin-Darby canine kidney cells enhanced transferrin uptake, whereas stable expression of kinase-dead PIPKIgamma661 had an inhibitory effect. Neither condition affected the overall cellular level of PI(4,5)P(2). RNA interference-based knockdown of PIPKIgamma661 in HeLa cells also had an inhibitory effect on transferrin endocytosis using the same assay system. Collectively, this evidence implies an important role for PIPKIgamma661 in the AP2-mediated endocytosis of transferrin.
Bairstow
Shawn F
SF
Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
Ling
Kun
K
Su
Xiaojing
X
Firestone
Ari J
AJ
Carbonara
Chateen
C
Anderson
Richard A
RA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004285:Dogs; D004705:Endocytosis; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D011990:Receptors, Transferrin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D050656:Transcription Factor AP-2 / Q000737:chemistry / Q000378:metabolism*; D014168:Transferrin / Q000378:metabolism
D017931:DNA Primers; D015778:Minor Histocompatibility Antigens; D018129:Phosphatidylinositol Phosphates; D021122:Protein Subunits; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; D050656:Transcription Factor AP-2; D014168:Transferrin; C060974:phosphatidylinositol 3,4,5-triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C402182:phosphatidylinositol phosphate 4-kinase