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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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Wortmannin binding  identified a 100-kD protein in the  p62cplx as the PI3-kinase catalytic subunit. Total cytosol (100  μg, cytosol) or immunoprecipitates from SGF using preimmune sera (preimmune), or antibodies against TGN38 (αTGN38)  were incubated with 1 μM wortmannin at 30°C for 10 min and  prepared for SDS-PAGE. The  gels were transferred to nitrocellulose and blotted with affinity-purified antibodies against wortmannin, detected with 125I-protein A and autoradiography. SDS-PAGE molecular weight  markers are shown at the left and the mobility of the 100-kD protein is noted at the right of the panel.
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Figure 4: Wortmannin binding identified a 100-kD protein in the p62cplx as the PI3-kinase catalytic subunit. Total cytosol (100 μg, cytosol) or immunoprecipitates from SGF using preimmune sera (preimmune), or antibodies against TGN38 (αTGN38) were incubated with 1 μM wortmannin at 30°C for 10 min and prepared for SDS-PAGE. The gels were transferred to nitrocellulose and blotted with affinity-purified antibodies against wortmannin, detected with 125I-protein A and autoradiography. SDS-PAGE molecular weight markers are shown at the left and the mobility of the 100-kD protein is noted at the right of the panel.

Mentions: Wortmannin inhibits PI3-kinases by covalently coupling to the active site of the catalytic subunit and this interaction is stable after resolution by SDS-PAGE (Wymann et al., 1996). The bound wortmannin can be identified by using either [3H]wortmannin and autoradiography or an antibody against wortmannin and immunoblotting. Brunn et al. (1996) (using rat brain extracts and in vitro incubations) showed wortmannin covalently coupled to a very broad band of proteins that span 100–110 kD, as well as several proteins at ∼200 kD. To identify the catalytic subunit associated with the membrane-bound, p62cplx-associated PI3-kinase, fractions were incubated with wortmannin, resolved by SDS-PAGE, and then immunoblotted with antibodies against wortmannin. In rat liver, cytosol bands of 100, ∼130, and ∼200 kD bind wortmannin. Immunoprecipitates of the membrane-bound, p62cplx-associated PI3-kinase (the same as those used for the PI3-kinase assays) contains only the 100-kD wortmannin binding band and this band is not precipitated by the preimmune sera (Fig. 4). The findings that the immunoprecipitates have PI3-kinase activity and a 100-kD wortmannin binding protein, support the identification of the 100-kD band as the PI3-kinase catalytic subunit.


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

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

Wortmannin binding  identified a 100-kD protein in the  p62cplx as the PI3-kinase catalytic subunit. Total cytosol (100  μg, cytosol) or immunoprecipitates from SGF using preimmune sera (preimmune), or antibodies against TGN38 (αTGN38)  were incubated with 1 μM wortmannin at 30°C for 10 min and  prepared for SDS-PAGE. The  gels were transferred to nitrocellulose and blotted with affinity-purified antibodies against wortmannin, detected with 125I-protein A and autoradiography. SDS-PAGE molecular weight  markers are shown at the left and the mobility of the 100-kD protein is noted at the right of the panel.
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Related In: Results  -  Collection

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

Figure 4: Wortmannin binding identified a 100-kD protein in the p62cplx as the PI3-kinase catalytic subunit. Total cytosol (100 μg, cytosol) or immunoprecipitates from SGF using preimmune sera (preimmune), or antibodies against TGN38 (αTGN38) were incubated with 1 μM wortmannin at 30°C for 10 min and prepared for SDS-PAGE. The gels were transferred to nitrocellulose and blotted with affinity-purified antibodies against wortmannin, detected with 125I-protein A and autoradiography. SDS-PAGE molecular weight markers are shown at the left and the mobility of the 100-kD protein is noted at the right of the panel.
Mentions: Wortmannin inhibits PI3-kinases by covalently coupling to the active site of the catalytic subunit and this interaction is stable after resolution by SDS-PAGE (Wymann et al., 1996). The bound wortmannin can be identified by using either [3H]wortmannin and autoradiography or an antibody against wortmannin and immunoblotting. Brunn et al. (1996) (using rat brain extracts and in vitro incubations) showed wortmannin covalently coupled to a very broad band of proteins that span 100–110 kD, as well as several proteins at ∼200 kD. To identify the catalytic subunit associated with the membrane-bound, p62cplx-associated PI3-kinase, fractions were incubated with wortmannin, resolved by SDS-PAGE, and then immunoblotted with antibodies against wortmannin. In rat liver, cytosol bands of 100, ∼130, and ∼200 kD bind wortmannin. Immunoprecipitates of the membrane-bound, p62cplx-associated PI3-kinase (the same as those used for the PI3-kinase assays) contains only the 100-kD wortmannin binding band and this band is not precipitated by the preimmune sera (Fig. 4). The findings that the immunoprecipitates have PI3-kinase activity and a 100-kD wortmannin binding protein, support the identification of the 100-kD band as the PI3-kinase catalytic subunit.

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