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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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Properties of the  p62cplx-associated PI3-kinase.  PI3-kinase assays were carried out as described in Materials and Methods. Either  Mg2+ (A) or Mn2+ (B) were  used as the cation in the presence of multiple substrates: PI,  PI(4)P, and PI(4,5)P2, as  listed at the top of each lane.  The mobility of PI(3)P is  noted at the right of B.
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Figure 7: Properties of the p62cplx-associated PI3-kinase. PI3-kinase assays were carried out as described in Materials and Methods. Either Mg2+ (A) or Mn2+ (B) were used as the cation in the presence of multiple substrates: PI, PI(4)P, and PI(4,5)P2, as listed at the top of each lane. The mobility of PI(3)P is noted at the right of 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)

Properties of the  p62cplx-associated PI3-kinase.  PI3-kinase assays were carried out as described in Materials and Methods. Either  Mg2+ (A) or Mn2+ (B) were  used as the cation in the presence of multiple substrates: PI,  PI(4)P, and PI(4,5)P2, as  listed at the top of each lane.  The mobility of PI(3)P is  noted at the right of B.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139785&req=5

Figure 7: Properties of the p62cplx-associated PI3-kinase. PI3-kinase assays were carried out as described in Materials and Methods. Either Mg2+ (A) or Mn2+ (B) were used as the cation in the presence of multiple substrates: PI, PI(4)P, and PI(4,5)P2, as listed at the top of each lane. The mobility of PI(3)P is noted at the right of 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