Protein kinase C bound to the Golgi apparatus supports the formation of constitutive transport vesicles

Constitutive secretion of heparan sulphate proteoglycans (HSPGs) was stimulated in human hepatoma HepG2 cells by phorbol 12-myristate 13-acetate (PMA) and inhibited by calphostin C, a specific inhibitor of protein kinase C (PKC). To delineate more closely the site of PKC action, the packaging in vit...

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Bibliographic Details
Published in:Biochemical journal 1996-12, Vol.320 ( Pt 2) (2), p.651-658
Main Authors: Westermann, P, Knoblich, M, Maier, O, Lindschau, C, Haller, H
Format: Article
Language:English
Subjects:
Carcinoma, Hepatocellular
Cell Fractionation
Cell Line
Cell-Free System
Cytoplasmic Granules - metabolism
Egtazic Acid - pharmacology
Enzyme Inhibitors - pharmacology
Golgi Apparatus - enzymology
Heparan Sulfate Proteoglycans
Heparitin Sulfate - biosynthesis
Humans
Indoles - pharmacology
Intracellular Membranes - enzymology
Isoenzymes - analysis
Isoenzymes - metabolism
Kinetics
Liver Neoplasms
Naphthalenes - pharmacology
Protein Binding
Protein Kinase C - analysis
Protein Kinase C - metabolism
Protein Kinase C-alpha
Proteoglycans - biosynthesis
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Summary:Constitutive secretion of heparan sulphate proteoglycans (HSPGs) was stimulated in human hepatoma HepG2 cells by phorbol 12-myristate 13-acetate (PMA) and inhibited by calphostin C, a specific inhibitor of protein kinase C (PKC). To delineate more closely the site of PKC action, the packaging in vitro of 35SO4-labelled HSPGs into transport vesicles was investigated. Formation of transport vesicles at the trans-Golgi network was stimulated by PMA and inhibited by calphostin C or Ro 31-8220 by using a post-nuclear supernatant. Treatment of either isolated Golgi-enriched membranes or cytosolic proteins with calphostin C provided evidence that membrane-bound PKC forms strongly supported vesicle formation, whereas cytosolic PKC forms showed a marginal effect. The PKC isoforms PKC-alpha and PKC-zeta were attached to highly purified Golgi membranes, as shown by Western blotting. Both isoforms were localized by confocal immunofluorescence microscopy in the Golgi area of HepG2 cells. Immunoelectron microscopy of ultrathin cryosections of HepG2 cells showed that PKC-zeta predominantly attaches to the trans-Golgi region, whereas PKC-alpha binds to the cis- and trans-Golgi area.
ISSN:0264-6021
1470-8728
DOI:10.1042/bj3200651