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Phosphatidylinositol 3-kinase is required for the formation of constitutive transport vesicles from the TGN.

Jones SM, Howell KE - J. Cell Biol. (1997)

Bottom Line: Howell. 1993.Cell Biol. 122:775-788).This p62(cplx)-associated PI3-kinase activity is stimulated by activation of the p62(cplx)-associated GTPase, and is specific for phosphatidylinositol (PI) as substrate, and is sensitive to wortmannin at micromolar concentrations.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Structural Biology, University of Colorado School of Medicine, Denver, Colorado 80262, USA.

ABSTRACT
An 85-kD cytosolic complex (p62(cplx)), consisting of a 62-kD phosphoprotein (p62) and a 25-kD GTPase, has been shown to be essential for the cell-free reconstitution of polymeric IgA receptor (pIgA-R)-containing exocytic transport vesicle formation from the TGN (Jones, S.M., J.R. Crosby, J. Salamero, and K.E. Howell. 1993. J. Cell Biol. 122:775-788). Here the p62(cplx) is identified as a regulatory subunit of a novel phosphatidylinositol 3-kinase (PI3-kinase). This p62(cplx)-associated PI3-kinase activity is stimulated by activation of the p62(cplx)-associated GTPase, and is specific for phosphatidylinositol (PI) as substrate, and is sensitive to wortmannin at micromolar concentrations. The direct role of this p62(cplx)-associated PI3-kinase activity in TGN-derived vesicle formation is indicated by the finding that both lipid kinase activity and the formation of pIgA-R-containing exocytic vesicles from the TGN are inhibited by wortmannin with similar dose-response curves and 50% inhibitory concentrations (3.5 microM). These findings indicate that phosphatidylinositol-3-phosphate (PI[3]P) is required for the formation of TGN-derived exocytic transport vesicles, and that the p62(cplx)-associated PI3-kinase and an activated GTPase are the essential molecules that drive production of this PI(3)P.

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The p62cplx-associated PI3-kinase activity and the formation of pIgA-R–containing vesicles from the TGN are sensitive to the same concentrations of wortmannin. PI3-kinase assays  were carried out as described in Materials and Methods at increasing concentrations of wortmannin (0.001–10 μM). The  amount of PI(3)P formed was determined by PhosphorImager  quantitation of the TLC plates. The activity expressed as percent  of control (no wortmannin) is plotted versus wortmannin concentration (A). The cell-free assay was carried out as described in  Materials and Methods at increasing concentrations of wortmannin (0.01–10 μM). The efficiency of formation of pIgA-R–containing vesicles was determined and is plotted versus wortmannin  concentration (B).
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Figure 8: The p62cplx-associated PI3-kinase activity and the formation of pIgA-R–containing vesicles from the TGN are sensitive to the same concentrations of wortmannin. PI3-kinase assays were carried out as described in Materials and Methods at increasing concentrations of wortmannin (0.001–10 μM). The amount of PI(3)P formed was determined by PhosphorImager quantitation of the TLC plates. The activity expressed as percent of control (no wortmannin) is plotted versus wortmannin concentration (A). The cell-free assay was carried out as described in Materials and Methods at increasing concentrations of wortmannin (0.01–10 μM). The efficiency of formation of pIgA-R–containing vesicles was determined and is plotted versus wortmannin concentration (B).

Mentions: The biochemical properties of the p62cplx-associated PI3-kinase activity were determined and compared with those of several other PI3-kinases, including the growth factor– associated p110, Vps34p, and the human Vps34p homologue (Stack et al., 1995a; Volinia et al., 1995) (Figs. 7 and 8; and Table II). The 50% inhibitory concentration (IC50) of the p62cplx-associated PI3-kinase activity with wortmannin was ∼3.5 μM. This value is similar to that determined for Vps34p (∼3 μM) and is three orders of magnitude greater than the IC50s of the p110 PI3-kinase and of the mammalian Vps34p homologue (2–3 nM). The p62cplx-associated PI3-kinase activity was also stimulated by low concentrations of non-ionic detergent (0.1% NP-40), similar to p110 and Vps34p, whereas all these PI3-kinases were inhibited by NP-40 at higher concentrations. p62cplx-associated PI3-kinase and all other PI3-kinase activities were insensitive to high (mM) concentrations of adenosine (data not shown). These properties are used to distinguish PI3-kinases (activation by low concentration of detergent and insensitivity to adenosine) from phosphatidylinositol 4–kinases, which are not detergent activated and are sensitive to adenosine.


Phosphatidylinositol 3-kinase is required for the formation of constitutive transport vesicles from the TGN.

Jones SM, Howell KE - J. Cell Biol. (1997)

The p62cplx-associated PI3-kinase activity and the formation of pIgA-R–containing vesicles from the TGN are sensitive to the same concentrations of wortmannin. PI3-kinase assays  were carried out as described in Materials and Methods at increasing concentrations of wortmannin (0.001–10 μM). The  amount of PI(3)P formed was determined by PhosphorImager  quantitation of the TLC plates. The activity expressed as percent  of control (no wortmannin) is plotted versus wortmannin concentration (A). The cell-free assay was carried out as described in  Materials and Methods at increasing concentrations of wortmannin (0.01–10 μM). The efficiency of formation of pIgA-R–containing vesicles was determined and is plotted versus wortmannin  concentration (B).
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getmorefigures.php?uid=PMC2139785&req=5

Figure 8: The p62cplx-associated PI3-kinase activity and the formation of pIgA-R–containing vesicles from the TGN are sensitive to the same concentrations of wortmannin. PI3-kinase assays were carried out as described in Materials and Methods at increasing concentrations of wortmannin (0.001–10 μM). The amount of PI(3)P formed was determined by PhosphorImager quantitation of the TLC plates. The activity expressed as percent of control (no wortmannin) is plotted versus wortmannin concentration (A). The cell-free assay was carried out as described in Materials and Methods at increasing concentrations of wortmannin (0.01–10 μM). The efficiency of formation of pIgA-R–containing vesicles was determined and is plotted versus wortmannin concentration (B).
Mentions: The biochemical properties of the p62cplx-associated PI3-kinase activity were determined and compared with those of several other PI3-kinases, including the growth factor– associated p110, Vps34p, and the human Vps34p homologue (Stack et al., 1995a; Volinia et al., 1995) (Figs. 7 and 8; and Table II). The 50% inhibitory concentration (IC50) of the p62cplx-associated PI3-kinase activity with wortmannin was ∼3.5 μM. This value is similar to that determined for Vps34p (∼3 μM) and is three orders of magnitude greater than the IC50s of the p110 PI3-kinase and of the mammalian Vps34p homologue (2–3 nM). The p62cplx-associated PI3-kinase activity was also stimulated by low concentrations of non-ionic detergent (0.1% NP-40), similar to p110 and Vps34p, whereas all these PI3-kinases were inhibited by NP-40 at higher concentrations. p62cplx-associated PI3-kinase and all other PI3-kinase activities were insensitive to high (mM) concentrations of adenosine (data not shown). These properties are used to distinguish PI3-kinases (activation by low concentration of detergent and insensitivity to adenosine) from phosphatidylinositol 4–kinases, which are not detergent activated and are sensitive to adenosine.

Bottom Line: Howell. 1993.Cell Biol. 122:775-788).This p62(cplx)-associated PI3-kinase activity is stimulated by activation of the p62(cplx)-associated GTPase, and is specific for phosphatidylinositol (PI) as substrate, and is sensitive to wortmannin at micromolar concentrations.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Structural Biology, University of Colorado School of Medicine, Denver, Colorado 80262, USA.

ABSTRACT
An 85-kD cytosolic complex (p62(cplx)), consisting of a 62-kD phosphoprotein (p62) and a 25-kD GTPase, has been shown to be essential for the cell-free reconstitution of polymeric IgA receptor (pIgA-R)-containing exocytic transport vesicle formation from the TGN (Jones, S.M., J.R. Crosby, J. Salamero, and K.E. Howell. 1993. J. Cell Biol. 122:775-788). Here the p62(cplx) is identified as a regulatory subunit of a novel phosphatidylinositol 3-kinase (PI3-kinase). This p62(cplx)-associated PI3-kinase activity is stimulated by activation of the p62(cplx)-associated GTPase, and is specific for phosphatidylinositol (PI) as substrate, and is sensitive to wortmannin at micromolar concentrations. The direct role of this p62(cplx)-associated PI3-kinase activity in TGN-derived vesicle formation is indicated by the finding that both lipid kinase activity and the formation of pIgA-R-containing exocytic vesicles from the TGN are inhibited by wortmannin with similar dose-response curves and 50% inhibitory concentrations (3.5 microM). These findings indicate that phosphatidylinositol-3-phosphate (PI[3]P) is required for the formation of TGN-derived exocytic transport vesicles, and that the p62(cplx)-associated PI3-kinase and an activated GTPase are the essential molecules that drive production of this PI(3)P.

Show MeSH
Related in: MedlinePlus