Distinct Classes of Phosphatidylinositol 3′-Kinases Are Involved in Signaling Pathways That Control Macroautophagy in HT-29 Cells

doi:10.1074/jbc.275.2.992

Distinct Classes of Phosphatidylinositol 3′-Kinases Are Involved in Signaling Pathways That Control Macroautophagy in HT-29 Cells*

From ^‡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

Abstract

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 Gα_i3 protein suggesting that both class III PI3K and trimeric G_i3 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.

Footnotes

↵* This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM) and Association pour la Recherche sur le Cancer Grant 9716. This work is part of a bilateral project of INSERM/NWO.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ To whom correspondence should be addressed: INSERM U504, Glycobiologie et Signalisation Cellulaire, 16 Avenue Paul-Vaillant-Couturier, 94807 Villejuif Cedex, France. Tel.: 33-1-45-59-50-41; Fax: 33-1-46-77-02-33; E-mail: codogno@vjf.inserm.fr.
Abbreviations:

PI3K

phosphatidylinositol 3-kinase

diC16PtdIns(3)P

dipalmitoyl phosphatidylinositol 3-phosphate

diC16PtdIns(4)P

dipalmitoyl phosphatidylinositol 4-phosphate

diC16PtdIns(3

4)P₂, dipalmitoyl phosphatidylinositol 3,4-bisphosphate

diC16PtdIns(4

5)P₂, dipalmitoyl phosphatidylinositol 4,5-bisphosphate

diC16PtdIns(3

4,5)P₃, dipalmitoyl phosphatidylinositol 3,4,5-triphosphate

IL-13

interleukin-13

LDH

lactate dehydrogenase

3-MA

3-methyladenine

PKB

protein kinase B, PtdIns(3)P, phosphatidylinositol 3-phosphate

PtdIns(4)P

phosphatidylinositol 4-phosphate

PtdIns(3

4)P₂, phosphatidylinositol 3,4-bisphosphate

PtdIns(4

5)P₂, phosphatidylinositol 4,5-bisphosphate

PtdIns(3

4,5)P₃, phosphatidylinositol 3,4,5-triphosphate

WT

wortmannin

RT-PCR

reverse transcriptase-polymerase chain reaction

SH2

Src homology domain 2
- Received August 27, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.