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Published online 11 August 2003. doi:10.1083/jcb.200302051
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© The Rockefeller University Press, 0021-9525/2003/8/693 $5.00
The Journal of Cell Biology, Volume 162, Number 4, 693-701

Article

Phosphatidylinositol phosphate 5-kinase Iß recruits AP-2 to the plasma membrane and regulates rates of constitutive endocytosis



David Padrón1, Ying Jie Wang2, Masaya Yamamoto2, Helen Yin2 and Michael G. Roth1

1 Department of Biochemistry, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390
2 Department of Physiology, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390

Address correspondence to Michael G. Roth, The University of Texas Southwestern, 5323 Harry Hines Blvd., Dallas, TX 75930-9038. Tel.: (214) 648-3276. Fax: (214) 648-8856. email: michael.roth{at}utsouthwestern.edu

Overexpression of phosphatidylinositol phosphate 5-kinase (PIP5KI) isoforms {alpha}, ß, or {gamma} in CV-1 cells increased phosphatidylinositol 4,5-bisphosphate (PIP2) levels by 35, 180, and 0%, respectively. Endocytosis of transferrin receptors, association of AP-2 proteins with membranes, and the number of clathrin-coated pits at the plasma membrane increased when PIP2 increased. When expression of PIP5KIß was inhibited with small interference RNA in HeLa cells, expression of PIP5KI{alpha} was also reduced slightly, but PIP5KI{gamma} expression was increased. PIP2 levels and internalization of transferrin receptors dropped 50% in these cells; thus, PIP5KI{gamma} could not compensate for loss of PIP5KIß. When expression of PIP5KI{alpha} was reduced, expression of both PIP5KIß and PIP5KI{gamma} increased and PIP2 levels did not change. A similar increase of PIP5KI{alpha} and PIP5KIß occurred when PIP5KI{gamma} was inhibited. These results indicate that constitutive endocytosis in CV-1 and HeLa cells requires (and may be regulated by) PIP2 produced primarily by PIP5KIß.

Key Words: phosphatidylinositol kinase; endocytosis; clathrin; transferrin receptor; phosphatidylinositol 4,5-bisphosphate

Abbreviations used in this paper: HA, hemagglutinin; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP5KI, phosphatidylinositol phosphate 5-kinase; siRNA, small interference RNA.


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