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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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p62 immunoprecipitates have PI3-kinase activity. (A)  PI3-kinase enzymatic assays were carried out on immunoprecipitates as described in Materials and Methods. The autoradiogram  of the TLC plate is shown: lane 1, nonstimulated PDGF receptor;  lane 2, stimulated PDGF receptor; lane 3, preimmune sera to p62;  lane 4, TGN38 polyclonal; lane 5, p62; lane 6, p62cplx; lane 7,  TGN38 monoclonal; and lane 8, pIgA-R. Immunoprecipitates of  stimulated PDGF receptor (i.e., ligand bound) are known to generate PI(3)P and together with the nonstimulated receptor, provide controls for the mobility of PI(3)P on the TLC plate. The  amount of PI(3)P formed by each immunoprecipitate was quantitated by PhosphorImager analysis and the data plotted in B (light  bars). The small GTPase-dependent stimulation of the PI3- kinase activity of the same set of immunoprecipitates was assayed  in the presence of GTPγS (1 μm) (dark bars) and plotted with  the nonstimulated values. The immunoprecipitated antigens are  labeled at the bottom of the bar graph.
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Figure 1: p62 immunoprecipitates have PI3-kinase activity. (A) PI3-kinase enzymatic assays were carried out on immunoprecipitates as described in Materials and Methods. The autoradiogram of the TLC plate is shown: lane 1, nonstimulated PDGF receptor; lane 2, stimulated PDGF receptor; lane 3, preimmune sera to p62; lane 4, TGN38 polyclonal; lane 5, p62; lane 6, p62cplx; lane 7, TGN38 monoclonal; and lane 8, pIgA-R. Immunoprecipitates of stimulated PDGF receptor (i.e., ligand bound) are known to generate PI(3)P and together with the nonstimulated receptor, provide controls for the mobility of PI(3)P on the TLC plate. The amount of PI(3)P formed by each immunoprecipitate was quantitated by PhosphorImager analysis and the data plotted in B (light bars). The small GTPase-dependent stimulation of the PI3- kinase activity of the same set of immunoprecipitates was assayed in the presence of GTPγS (1 μm) (dark bars) and plotted with the nonstimulated values. The immunoprecipitated antigens are labeled at the bottom of the bar graph.

Mentions: Enzymatic assays were carried out to directly determine whether PI3-kinase activity is associated with the p62cplx. Since the assay for PI3-kinase associated with PDGF receptors was established using immunoprecipitates of the activated receptor (Kazlauskas and Cooper, 1990), we used immunoprecipitates of activated and nonactivated PDGF receptor as controls. Activated PDGF receptors generate PI(3)P from PI, therefore, the mobility of the PI(3)P serves as a standard on the TLC plates. For the assay, immunoprecipitates were prepared from CHAPS-solubilized native SGF using antibodies raised against characterized components of the membrane-associated p62cplx: TGN38 (luminal domain, polyclonal); p62; p62cplx; and TGN38 (luminal domain, monoclonal). As shown in Fig. 1, A and B, PI3-kinase activity was readily detected in all immunoprecipitates containing the membrane-associated p62cplx. The level of activity measured was at least as robust as that obtained for the unstimulated PDGF receptor. This comparison is germaine because the p62cplx activity most likely represents the unstimulated endogenous activity. Controls for the specificity of the assays included immunoprecipitates derived from the same fraction using either preimmune sera or antibodies directed against the pIgA-R, an abundant transmembrane protein of SGF. The control immunoprecipitates had minimal kinase activity. The amount of p62cplx-associated PI3-kinase activity obtained with the immunoprecipitates correlated with the amount of p62 precipitated by the different antibodies (p62cplx > p62 alone > TGN38 polyclonal > TGN38 monoclonal [data not shown]).


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

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

p62 immunoprecipitates have PI3-kinase activity. (A)  PI3-kinase enzymatic assays were carried out on immunoprecipitates as described in Materials and Methods. The autoradiogram  of the TLC plate is shown: lane 1, nonstimulated PDGF receptor;  lane 2, stimulated PDGF receptor; lane 3, preimmune sera to p62;  lane 4, TGN38 polyclonal; lane 5, p62; lane 6, p62cplx; lane 7,  TGN38 monoclonal; and lane 8, pIgA-R. Immunoprecipitates of  stimulated PDGF receptor (i.e., ligand bound) are known to generate PI(3)P and together with the nonstimulated receptor, provide controls for the mobility of PI(3)P on the TLC plate. The  amount of PI(3)P formed by each immunoprecipitate was quantitated by PhosphorImager analysis and the data plotted in B (light  bars). The small GTPase-dependent stimulation of the PI3- kinase activity of the same set of immunoprecipitates was assayed  in the presence of GTPγS (1 μm) (dark bars) and plotted with  the nonstimulated values. The immunoprecipitated antigens are  labeled at the bottom of the bar graph.
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Related In: Results  -  Collection

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Figure 1: p62 immunoprecipitates have PI3-kinase activity. (A) PI3-kinase enzymatic assays were carried out on immunoprecipitates as described in Materials and Methods. The autoradiogram of the TLC plate is shown: lane 1, nonstimulated PDGF receptor; lane 2, stimulated PDGF receptor; lane 3, preimmune sera to p62; lane 4, TGN38 polyclonal; lane 5, p62; lane 6, p62cplx; lane 7, TGN38 monoclonal; and lane 8, pIgA-R. Immunoprecipitates of stimulated PDGF receptor (i.e., ligand bound) are known to generate PI(3)P and together with the nonstimulated receptor, provide controls for the mobility of PI(3)P on the TLC plate. The amount of PI(3)P formed by each immunoprecipitate was quantitated by PhosphorImager analysis and the data plotted in B (light bars). The small GTPase-dependent stimulation of the PI3- kinase activity of the same set of immunoprecipitates was assayed in the presence of GTPγS (1 μm) (dark bars) and plotted with the nonstimulated values. The immunoprecipitated antigens are labeled at the bottom of the bar graph.
Mentions: Enzymatic assays were carried out to directly determine whether PI3-kinase activity is associated with the p62cplx. Since the assay for PI3-kinase associated with PDGF receptors was established using immunoprecipitates of the activated receptor (Kazlauskas and Cooper, 1990), we used immunoprecipitates of activated and nonactivated PDGF receptor as controls. Activated PDGF receptors generate PI(3)P from PI, therefore, the mobility of the PI(3)P serves as a standard on the TLC plates. For the assay, immunoprecipitates were prepared from CHAPS-solubilized native SGF using antibodies raised against characterized components of the membrane-associated p62cplx: TGN38 (luminal domain, polyclonal); p62; p62cplx; and TGN38 (luminal domain, monoclonal). As shown in Fig. 1, A and B, PI3-kinase activity was readily detected in all immunoprecipitates containing the membrane-associated p62cplx. The level of activity measured was at least as robust as that obtained for the unstimulated PDGF receptor. This comparison is germaine because the p62cplx activity most likely represents the unstimulated endogenous activity. Controls for the specificity of the assays included immunoprecipitates derived from the same fraction using either preimmune sera or antibodies directed against the pIgA-R, an abundant transmembrane protein of SGF. The control immunoprecipitates had minimal kinase activity. The amount of p62cplx-associated PI3-kinase activity obtained with the immunoprecipitates correlated with the amount of p62 precipitated by the different antibodies (p62cplx > p62 alone > TGN38 polyclonal > TGN38 monoclonal [data not shown]).

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