The Class II Phosphoinositide 3-Kinase PI3K-C2α Is Concentrated in the Trans-Golgi Network and Present in Clathrin-coated Vesicles*
- From the ‡Ludwig Institute for Cancer Research, University College, London W1P 8BT, United Kingdom, the‖Department of Biochemistry and Molecular Biology, University College, London WC1E 6BT, United Kingdom, and the ¶Department of Pharmacology and the Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Abstract
In recent years, a large family of phosphoinositide 3-kinase (PI3K) isozymes has been characterized and cloned. Several of these PI3K enzymes have overlapping tissue distributions and it remains unclear if and how their 3-phosphoinositide products elicit differential, intracellular effects. One possibility is that the PI3K enzymes display a restricted distribution within the cell to produce their 3-phospholipid products in specific, subcellular compartments. In the present study we characterize the subcellular distribution of the novel class II PI3K isozyme PI3K-C2α in several mammalian cell types. Differential centrifugation of COS-1 and U937 cells together with Western blot analysis demonstrated that PI3K-C2α is constitutively associated with phospholipid membranes. Centrifugation of rat brain homogenates and Western blotting revealed that in contrast to the class IA PI3K enzymes, PI3K-C2α could be co-purified with a population of clathrin-coated vesicles (CCVs). Furthermore, a PI3K activity refractory to wortmannin treatment was detected in CCV preparations consistent with the presence of the PI3K-C2α isozyme. These biochemical observations were supported by immunofluorescence analysis that revealed PI3K-C2α to have a punctate distribution and an enrichment of immunoreactivity within a perinuclear site consistent with its presence in the endoplasmic reticulum or Golgi apparatus. Dual label immunofluorescence demonstrated that in this region, the distribution of PI3K-C2α closely paralleled that of γ-adaptin, a component of the AP-1 adaptor that is present in the trans-Golgi and the trans-Golgi network (TGN) resident protein TGN-46. Neither the phospholipid association nor the subcellular localization of PI3K-C2α was dependent upon either its COOH-terminal PX or C2 domains. Mutants lacking these domains demonstrated a similar distribution to the wild type enzyme when expressed as recombinant proteins. Treatment of cells with brefeldin A disrupted the perinuclear staining pattern of both PI3K-C2α and the AP-1 complex demonstrating that the localization of both molecules at the TGN is dependent upon ADP-ribosylation factor GTPase activity.
Footnotes
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↵* This work was supported by National Institutes of Health Grant GM-49217 (to J. H. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom all correspondence should be addressed: Renal Section, Imperial College School of Medicine, Du Cane Road, London W12 ONN, United Kingdom. Tel.: 0181-383-2357; Fax: 0181-383-2062; E-mail jdomin@ic.ac.uk.
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↵2 Arcaro, A., Zvelebil, M. J., Wallasch, C., Ullrich, A., Waterfield, M. D., and Domin, J. (2000) Mol. Cell. Biol., in press.
- Abbreviations:
- PI3K
-
phosphoinositide 3-kinase
- PtdIns
-
phosphatidylinositol
- PtdIns(4)P
-
phosphatidylinositol 4-phosphate
- PtdIns(4
-
5)P2, phosphatidylinositol 4,5-bisphosphate
- PtdIns(3
-
4,5)P2, phosphatidylinositol 3,4,5-bisphosphate
- PX
-
phox homology, AP, adaptor protein
- CCV
-
clathrin-coated vesicle
- FITC
-
fluorescein isothiocyanate
- LDM
-
low density microsome
- M6PR
-
mannose 6-phosphate receptor
- PBS
-
phosphate-buffered saline
- PM
-
plasma membrane
- PAGE
-
polyacylamide gel electrophoresis
- TGN
-
trans-Golgi network
- TRITC
-
tetramethylrhodamine B isothiocyanate
-
- Received August 17, 1999.
- Revision received January 10, 2000.
- The American Society for Biochemistry and Molecular Biology, Inc.
