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

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-C2␣ in several mammalian cell types. Differential centrifugation of COS-1 and U937 cells together with Western blot analysis demonstrated that PI3K-C2␣ 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-C2␣ could be copurified 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-C2␣ isozyme. These biochemical observations were supported by immunofluorescence analysis that revealed PI3K-C2␣ 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-C2␣ closely paralleled that of ␥-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-C2␣ 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-C2␣ and the AP-1 complex demonstrating that the localization of both molecules at the TGN is dependent upon ADP-ribosylation factor GTPase activity.Phosphoinositide 3-kinase (PI3K) 1 was originally isolated from bovine brain as a heterodimeric complex consisting of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit. Since then, additional purification strategies and reverse transcriptase-polymerase chain reaction-based approaches have identified seven other mammalian PI3K catalytic subunits. On

Endothelial PI3K-C2α, a class II PI3K, has an essential role in angiogenesis and vascular barrier function

“…In contrast to class I PI3Ks that generate PtdIns(3,4,5)P 3 , the major product of class II and III PI3Ks is PtdIns(3)P, which is mainly accumulated in endosomes [16][17][18][20][21][22][23][24][25] .…”

“…The actions of C2α and class I PI3Ks in EC are distinct in that, different from C2α, class I PI3Ks exert a great impact on Akt stimulation and cell proliferation and are not involved in vesicular trafficking including VE-cadherin transfer or VE-cadherin assembly at the cell-cell junction [10][11][12][13] . Therefore, these observations reveal novel biological activities of C2α and underscore broader roles for PI3K family members in vascular physiology and pathophysiology.In contrast to class I PI3Ks that generate PtdIns(3,4,5)P 3 , the major product of class II and III PI3Ks is PtdIns(3)P, which is mainly accumulated in endosomes [16][17][18][20][21][22][23][24][25] .Consistent with this, C2α in EC was localized in endosomes and TGN, which are organelles responsible for processing, sorting and packaging proteins into distinct carriers for transport to their final destinations. Another class II PI3K-C2β and class III Vps34 are also localized primarily in the clathrin-coated or endocytic compartment 38,39 .…”

“…Class II PI3Ks comprise three members, PI3K-C2α (C2α), PI3K-C2β (C2β) and PI3K-C2γ (C2γ), and mainly produce PtdIns(3)P in vivo 16,17 . Among the three class II PI3Ks, C2α is distinct from C2β, C2γ and other PI3K 4 members in that it has unique structures including a clathrin-binding site in the Nterminal stretch and relative resistance to PI3K inhibitors 16,18 . C2α is expressed in selected cell populations including epithelium, vascular endothelium, and smooth muscle 19,20 .…”

“…C2α is expressed in selected cell populations including epithelium, vascular endothelium, and smooth muscle 19,20 . C2α is enriched in clathrin-coated endocytic vesicles, other endosomes and the trans-Golgi network (TGN), and is suggested to regulate intracellular vesicular trafficking 16,18,21,22 . In vitro studies 21,23,24 showed that various extracellular stimuli including cytokines, insulin and integrin ligation modestly stimulated C2α.…”

The Type Iα Inositol Polyphosphate 4-Phosphatase Generates and Terminates Phosphoinositide 3-Kinase Signals on Endosomes and the Plasma Membrane

“…Therefore, under serumstimulated conditions resulting in increased PtdIns(3,4)P 2 synthesis, the 4-phosphatase may generate a PtdIns(3)P pool on early endosomes, via the hydrolysis of its cognate substrate PtdIns(3,4)P 2 . Furthermore, given that wortmannin inhibits hVPS34-mediated synthesis of PtdIns(3)P on early endosomes, the PtdIns(3,4)P 2 pool hydrolyzed by the 4-phosphatase in wortmannin-treated cells may be synthesized by a wortmannin-resistant PI3-kinase, such as the class II C2␣ PI 3-kinase (Domin et al, 2000).…”