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J Biol Chem, Vol. 275, Issue 2, 992-998, January 14, 2000
,,¶
From 3-Methyladenine which stops macroautophagy at the
sequestration step in mammalian cells also inhibits the
phosphoinositide 3-kinase (PI3K) activity raising the possibility that
PI3K signaling controls the macroautophagic pathway (Blommaart, E. F. C., Krause, U., Schellens, J. P. M.,
Vreeling-Sindelárová, H., and Meijer, A. J. (1997)
Eur. J. Biochem. 243, 240-246). The aim of this study was to identify PI3Ks involved in the control of macroautophagic sequestration in human colon cancer HT-29 cells. An increase of class I
PI3K products (phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate) caused by either feeding cells
with synthetic lipids (dipalmitoyl phosphatidylinositol 3,4-bisphosphate and dipalmitoyl phosphatidylinositol
3,4,5-triphosphate) or by stimulating the enzymatic activity by
interleukin-13 reduced macroautophagy. In contrast, an increase in the
class III PI3K product (phosphatidylinositol 3-phosphate), either by
feeding cells with a synthetic lipid or by overexpressing the p150
adaptor, stimulates macroautophagy. Transfection of a specific class
III PI3K antisense oligonucleotide greatly inhibited the rate of
macroautophagy. In accordance with a role of class III PI3K, wortmannin
(an inhibitor of PI3Ks) inhibits macroautophagic sequestration and
protein degradation in the low nanomolar range (IC50 5-15
nM). Further in vitro enzymatic assay showed
that 3-methyladenine inhibits the class III PI3K activity. Dipalmitoyl
phosphatidylinositol 3-phosphate supplementation or p150 overexpression
rescued the macroautophagic pathway in HT-29 cells overexpressing a
GTPase-deficient mutant of the G INSERM U504, Glycobiologie et Signalisation
Cellulaire, 16 avenue Paul-Vaillant-Couturier, 94807 Villejuif, Cedex,
France and the § Department of Biochemistry, Academic
Medical Centre, University of Amsterdam,
Amsterdam, The Netherlands
i3 protein suggesting
that both class III PI3K and trimeric Gi3 protein signaling
are required in the control macroautophagy in HT-29 cells. In
conclusion, our results demonstrate that distinct classes of PI3K
control the macroautophagic pathway in opposite directions. The roles
of PI3Ks in macroautophagy are discussed in the context of membrane recycling.
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