The Class II Phosphoinositide 3-Kinase PI3K-C2α Is Concentrated in the Trans-Golgi Network and Present in Clathrin-coated Vesicles*

By J. Domin, I. Gaidarov, M. E. Smith, J. Keen, M. Waterfield. Journal of Biological Chemistry, 2000

150 citations recorded by the source corpus.

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.

Subfields: Cell Biology, Molecular Biology, Physiology

View Paper: https://www.semanticscholar.org/paper/cfd6d2106813f6ff73435bf5688e82541c1c025a

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In Cell Biology

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Citations in the corpus

• Targeting phosphoinositide 3-kinase: moving towards therapy. (2008, background): “Mainly the PI3KC2α isoform has been reported to associate with clathrin and has been attributed a role in clathrin assembly and clathrinmediated vesicular trafficking [20,21].”
• Rab proteins as membrane organizers (2001, background): “As membrane flows through the early endosome, phosphoinositides other than PtdIns(3)P (for example, PtdIns(3,4)P 2 and PtdIns(3,4,5)P 3”
• The emerging mechanisms of isoform-specific PI3K signalling (2010, background): “Class II PI3Ks seem to be predominantly and constitutively associated with intracellular membranes, with low levels in the cytoso”