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

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-Sindelá rová , H., and Meijer, A. J. Macroautophagy is a major intralysosomal catabolic process conserved during the evolution of eukaryotic cells (1-4). In mammalian cells, the macroautophagic pathway starts with the sequestration of cytoplasmic material (including mitochondria, peroxisomes, smooth and rough membranes, and cytosolic constituents: proteins, glycogen, neutral lipids, RNA, and ribosomes) to form an early autophagosome. The membrane of early autophagosomes is derived from ribosome-free regions of the rough endoplasmic reticulum (5) and/or a related organelle called the phagophore (see Ref. 6 and references therein). After acidification the early autophagosome is transformed in a late autophagosome. Fusion between late autophagosomes and lysosomes triggers the degradation of sequestered material into autolysosomes. Material from the endocytic pathway can be imported in autophagic vacuoles at different steps of their maturation (7,8).Recently, studies have shed light on the intracellular mechanisms that control membrane flow along the autophagic/lysosomal pathway in mammalian cells (reviewed in Refs. 9 and 10). It has been suggested that the phosphoinositide 3-kinase (PI3K) 1 signaling cascade controls macroautophagy (11) and that 3-MA, an inhibitor of macroautophagic sequestration (12), behaves as a PI3K inhibitor.PI3K belongs to a family of enzymes that phosphorylates the 3Ј-hydroxyl group on the inositol ring of phosphoinositides (reviewed in Refs. 13-16). These phospholipids are involved in a large array of signal transduction pathways controlling mitogenic responses, differentiation, apoptosis, cytoskeletal organization, and membrane flow along the secretory and endocytic pathways (reviewed in Ref. 17). PI3Ks are classified into three classes. Class I enzymes are composed of catalytic p110 subunits and p85 adaptors. The SH2 motif contained in p85 adaptors bind to phosphorylated Tyr residues thereby linking the catalytic subunit to the Tyr kinase signaling pathway (class I A ). A member of the class I PI3K family (class I B , p110␥/p101) is activated by subunits of trimeric G proteins (18,19). Class I PI3Ks phosphorylate PtdIns, PtdIns(4)P, PtdIns(4,5)P 2 but in vivo PtdIns(4,5)P 2 is likely to be the favorite substrate. Class II enzymes are large enzymes (Ͼ200 kDa) characterized by a C-terminal containing a C2 domain (20-23). These enzymes phosphorylate in vitro PtdIns, PtdIns(4)P, but not PtdIns(4,5)P 2 . Class III enzymes 1 The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; diC16PtdIns(3)P, dipalmitoyl phosphatidylinositol 3-phosphate; diC16PtdIns(4)P, dipalmitoyl phosphatidylinositol 4-phosphate; diC16Ptd-Ins(3,4)P 2 , dipalmitoyl phosphatidylinositol 3,4-bisphosphate; diC16Pt-dIns(4,5)P 2 ...

Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells

“…Class I PI3K is involved in inhibitory effect on autophagy, while the class III PI3K is involved in the sequestration of cytoplasmic material in autophagy. 31 Therefore, we suggest that the effect of 3-MA on TMZ-induced autophagy may be due to inhibition of the class III PI3K. Bafilomycin A1 is a specific inhibitor of the lysosomal protein pump (vacuolar H þ -ATPase).…”

The effect of phosphoinositide 3-kinase inhibitors on programmed nuclear degradation in Tetrahymena and fate of surviving nuclei

“…Wortmannin and LY294002 bind to the catalytic subunit of PI 3-kinase, while 3-MA interacts with the regulatory subunit. 15 The consistent results obtained with three different PI 3-kinase inhibitors argue that PI 3-kinase Cell Death and Differentiation (2004) 11,[1146][1147][1148][1149] activates programmed degradation of the parental macronucleus and unexchanged pronuclei.…”

Autophagy and signaling: their role in cell survival and cell death

“…These experiments have shown that the product of class-III PI3K, phosphatidylinositol 3-phosphate (PI(3)P), is required for autophagy, whereas the products of class-I PI3K, phosphatidylinositol 3,4, bisphosphate (PI(3,4)P 2 ), and phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P 3 ), have inhibitory effects. 27 Indeed, overexpression of phosphatase and tensin homolog deleted from chromosome 10 (PTEN), which removes the phosphate from the 3-position of PI(3,4)P 2 and PI(3,4,5)P 3 , increases autophagy. 28 The PI3K inhibitors do not distinguish between class-I and -III PI3K, and also inhibit the formation of PI(3)P, and thus inhibit autophagy.…”