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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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Phosphorylation of p62cplx purified from detergent-solubilized native SGF reveals a 100-kD subunit not present if the  SGF is high pH–washed before solubilization. p62cplx purified  from detergent-solubilized, high pH–washed SGF (A) and native  SGF (B) (10 ng) was incubated in an in vitro phosphorylation reaction containing 10 μCi [γ32P]ATP at increasing concentration  of calcium from 0 to 1.0 μM. The calcium concentrations are  noted above each lane and the molecular weight markers at the  right of B. The mobility of p62 is noted at the left of A and B.  Quantitation of the phosphorylation is plotted in PhosphorImager units (C).
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Figure 3: Phosphorylation of p62cplx purified from detergent-solubilized native SGF reveals a 100-kD subunit not present if the SGF is high pH–washed before solubilization. p62cplx purified from detergent-solubilized, high pH–washed SGF (A) and native SGF (B) (10 ng) was incubated in an in vitro phosphorylation reaction containing 10 μCi [γ32P]ATP at increasing concentration of calcium from 0 to 1.0 μM. The calcium concentrations are noted above each lane and the molecular weight markers at the right of B. The mobility of p62 is noted at the left of A and B. Quantitation of the phosphorylation is plotted in PhosphorImager units (C).

Mentions: Since in the yeast system the PI3-kinase regulatory subunit (Vps 15p) is a protein kinase that has been postulated to phosphorylate the PI3-kinase catalytic subunit (Vps34p) (Stack et al., 1995a) and it has been shown that the mammalian p110γ subunit can autophosphorylate (Vanhaesebroeck et al., 1997), in vitro phosphorylation reactions were performed in an attempt to identify the p62cplx-associated PI3-kinase catalytic subunit. The p62cplx was purified from detergent-solubilized, high pH washed, and native membranes using an immunoaffinity column prepared by covalently attaching an IgG fraction of p62 antisera. Kinase assays demonstrated that a single band at 62 kD was phosphorylated when the p62cplx was purified from detergent-solubilized, high pH–washed SGF and the phosphorylation was stimulated at increasing concentrations of Ca2+ (Fig. 3 A). Moreover, when the p62cplx was purified from detergent-solubilized native SGF, which has PI3-kinase activity (data not shown), an additional ∼100-kD phosphorylated subunit was present and the phosphorylation of this subunit exhibited the same calcium activation profile as the p62 subunit (Fig. 3, B and C). From these data we conclude that the membrane-associated p62cplx contains an additional 100-kD subunit that is detected only when the SGF is not high pH washed before detergent solubilization. Both the 62- and 100-kD subunits are likely to be phosphorylated by the same kinase because their phosphorylation displays the same calcium activation curve.


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

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

Phosphorylation of p62cplx purified from detergent-solubilized native SGF reveals a 100-kD subunit not present if the  SGF is high pH–washed before solubilization. p62cplx purified  from detergent-solubilized, high pH–washed SGF (A) and native  SGF (B) (10 ng) was incubated in an in vitro phosphorylation reaction containing 10 μCi [γ32P]ATP at increasing concentration  of calcium from 0 to 1.0 μM. The calcium concentrations are  noted above each lane and the molecular weight markers at the  right of B. The mobility of p62 is noted at the left of A and B.  Quantitation of the phosphorylation is plotted in PhosphorImager units (C).
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Related In: Results  -  Collection

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

Figure 3: Phosphorylation of p62cplx purified from detergent-solubilized native SGF reveals a 100-kD subunit not present if the SGF is high pH–washed before solubilization. p62cplx purified from detergent-solubilized, high pH–washed SGF (A) and native SGF (B) (10 ng) was incubated in an in vitro phosphorylation reaction containing 10 μCi [γ32P]ATP at increasing concentration of calcium from 0 to 1.0 μM. The calcium concentrations are noted above each lane and the molecular weight markers at the right of B. The mobility of p62 is noted at the left of A and B. Quantitation of the phosphorylation is plotted in PhosphorImager units (C).
Mentions: Since in the yeast system the PI3-kinase regulatory subunit (Vps 15p) is a protein kinase that has been postulated to phosphorylate the PI3-kinase catalytic subunit (Vps34p) (Stack et al., 1995a) and it has been shown that the mammalian p110γ subunit can autophosphorylate (Vanhaesebroeck et al., 1997), in vitro phosphorylation reactions were performed in an attempt to identify the p62cplx-associated PI3-kinase catalytic subunit. The p62cplx was purified from detergent-solubilized, high pH washed, and native membranes using an immunoaffinity column prepared by covalently attaching an IgG fraction of p62 antisera. Kinase assays demonstrated that a single band at 62 kD was phosphorylated when the p62cplx was purified from detergent-solubilized, high pH–washed SGF and the phosphorylation was stimulated at increasing concentrations of Ca2+ (Fig. 3 A). Moreover, when the p62cplx was purified from detergent-solubilized native SGF, which has PI3-kinase activity (data not shown), an additional ∼100-kD phosphorylated subunit was present and the phosphorylation of this subunit exhibited the same calcium activation profile as the p62 subunit (Fig. 3, B and C). From these data we conclude that the membrane-associated p62cplx contains an additional 100-kD subunit that is detected only when the SGF is not high pH washed before detergent solubilization. Both the 62- and 100-kD subunits are likely to be phosphorylated by the same kinase because their phosphorylation displays the same calcium activation curve.

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