Alfred J. Meijer

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

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 Gαi3 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.

The Tumor Suppressor PTEN Positively Regulates Macroautophagy by Inhibiting the Phosphatidylinositol 3-Kinase/Protein Kinase B Pathway

The tumor suppressor PTEN is a dual protein and phosphoinositide phosphatase that negatively controls the phosphatidylinositol (PI) 3-kinase/protein kinase B (Akt/PKB) signaling pathway. Interleukin-13 via the activation of the class I PI 3-kinase has been shown to inhibit the macroautophagic pathway in the human colon cancer HT-29 cells. Here we demonstrate that the wild-type PTEN is expressed in this cell line. Its overexpression directed by an inducible promoter counteracts the interleukin-13 down-regulation of macroautophagy. This effect was dependent upon the phosphoinositide phosphatase activity of PTEN as determined by using the mutant G129E, which has only protein phosphatase activity. The role of Akt/PKB in the signaling control of interleukin-13-dependent macroautophagy was investigated by expressing a constitutively active form of the kinase (MyrPKB). Under these conditions a dramatic inhibition of macroautophagy was observed. By contrast a high rate of autophagy was observed in cells expressing a dominant negative form of PKB. These data demonstrate that the signaling control of macroautophagy overlaps with the well known PI 3-kinase/PKB survival pathway and that the loss of PTEN function in cancer cells inhibits a major catabolic pathway.

AMP-activated Protein Kinase and the Regulation of Autophagic Proteolysis

Interruption of mTOR-dependent signaling by rapamycin is known to stimulate autophagy, both in mammalian cells and in yeast. Because activation of AMPK also inhibits mTOR-dependent signaling one would expect stimulation of autophagy by AMPK activation. According to the literature, this is true for yeast but, unexpectedly, not for mammalian cells on the basis of the use of AICAR, a pharmacological activator of AMPK. In the present study, carried out with hepatocytes, HT-29 cells, and HeLa cells, we have reexamined the possible role of AMPK in the control of mammalian autophagy. Inhibition of AMPK activity by compound C or by transfection with a dominant negative form of AMPK almost completely inhibited autophagy. These results suggest that the inhibition of autophagy by AICAR is not related to its ability to activate AMPK. We conclude that in mammalian cells, as in yeast, AMPK is required for autophagy.

Autophagic proteolysis: control and specificity

The rate of proteolysis is an important determinant of the intracellular protein content. Part of the degradation of intracellular proteins occurs in the lysosomes and is mediated by macroautophagy. In liver, macroautophagy is very active and almost completely accounts for starvation-induced proteolysis. Factors inhibiting this process include amino acids, cell swelling and insulin. In the mechanisms controlling macroautophagy, protein phosphorylation plays an important role. Activation of a signal transduction pathway, ultimately leading to phosphorylation of ribosomal protein S6, accompanies inhibition of macroautophagy. Components of this pathway may include a heterotrimeric Gi3-protein, phosphatidylinositol 3- kinase and p70S6 kinase. Recent evidence indicates that lysosomal protein degradation can be selective and occurs via ubiquitin- dependent and -independent pathways.

Activity of peroxisomal enzymes and intracellular distribution of catalase in Zellweger syndrome

The activity of peroxisomal enzymes was studied in human liver and cultured human skin fibroblasts in relation to the finding (Goldfischer, S. et al. (1973) Science 182, 62-64) that morphologically distinct peroxisomes are not detectable in patients with the cerebro-hepato-renal (Zellweger) syndrome. In homogenates of liver from the patients, dihydroxyacetone phosphate acyltransferase, a membrane-bound peroxisomal enzyme, is deficient (Schutgens, R.B.H., et al. (1984) Biochem. Biophys. Res. Commun. 120, 179-184). In contrast, there is no deficiency of the soluble peroxisomal matrix enzymes catalase, L-alpha-hydroxyacid oxidase and E-aminoacid oxidase. Catalase is also not deficient in homogenates of cultured skin fibroblasts from the patients. The results of digitonin titration experiments showed that in control fibroblasts at least 70% of the catalase activity is present in subcellular particles distinct from mitochondria or lysosomes. In contrast, all of the catalase activity in fibroblasts from Zellweger patients is found in the same compartment as the cytosolic marker enzyme lactate dehydrogenase.

Autophagy: Regulation and role in disease

Autophagy, a lysosomal process involved in the maintenance of cellular homeostasis, is responsible for the turnover of long-lived proteins and organelles that are either damaged or functionally redundant. The process is tightly controlled by the insulin-amino acid-mammalian target of the rapamycin-dependent signal-transduction pathway. Research in the last decade has indicated not only that autophagy provides cells with oxidizable substrate when nutrients become scarce but also that it can provide protection against aging and a number of pathologies such as cancer, neurodegeneration, cardiac disease, diabetes, and infections.

AMP-Activated Protein Kinase and Autophagy

Autophagy is inhibited by TOR-dependent signaling. Interruption of signalling by rapamycin is known to stimulate autophagy, both in mammalian cells and in yeast. However, inactivation of TOR by AMPK has yielded controversial results in the literature with regard to its effect on autophagy: activation of autophagy in yeast but inhibition in hepatocytes. In a recent study, carried out with hepatocytes, HT-29 cells, and HeLa cells, the possible role of AMPK in the control of mammalian autophagy was reexamined. The data suggest that in mammalian cells, as in yeast, AMPK is required for autophagy. Addendum to: AMP-Activated Protein Kinase and the Regulation of Autophagic Proteolysis D. Meley, C. Bauvy, J.H.P.M. Houben-Weerts, P.F. Dubbelhuis, M.T.J. Helmond, P. Codogno and A.J. Meijer J Biol Chem 2006; 281:34870-79

Amino-acid-dependent signal transduction.

Recent research carried out in several laboratories has indicated that, in addition to their role as intermediates in many metabolic pathways, amino acids can interact with insulin-dependent signal transduction. In this short review, the current state of this rapidly expanding field is discussed.

Effect of sulphydryl-blocking reagents on mitochondrial anion-exchange reactions involving phosphate

Chappell and coworkers [l-4] have proposed that the transport of anions across the mitochondrial membrane is brought about by specific carrier systems that mediate exchange-diffusion processes. Two of these carrier systems, or translocators, mediate an exchange between phosphate and hydroxyl and an exchange between phosphate and dicarboxylate, respectively. Fonyo [5] and Tyler [6] have shown that certain sulphydryl-binding reagents specifically interfere with the transport of phosphate across the mitochondrial membrane. The problem arises of whether these inhibitors not only specifically block the exchange diffusion between phosphate and hydroxyl, but also that between dicarboxylate and phosphate. We have recently presented evidence for inhibition of both transport systems by the sulphydryl-binding reagent mersalyl [7] . According to Johnson and Chappell [8] , however, these sulphydryl-blocking reagents inhibit only the phosphate-hydroxyl exchange. We have now found that there is a difference in sensitivity of the phosphate-hydroxyl and phosphatedicarboxylate exchange reactions, depending on the sulphydryl-blocking reagent used. Mersalyl [6] and p-hydroxymercuribenzoate [5] inhibit both of these anion-exchange reactions (see ref. [7] ). On the other hand, N-ethylmaleimide inhibits the phosphatehydroxyl exchange only. These results provide additional evidence for the postulate [l-4] that the phosphate-hydroxyl and the phosphate-dicarboxylate exchange reactions are mediated by distinct translo-

Amino Acids as Regulators and Components of Nonproteinogenic Pathways

Amino acids are not only important precursors for the synthesis of proteins and other N-containing compounds, but also participate in the regulation of major metabolic pathways. Glutamate and aspartate, for example, are components of the malate/aspartate shuttle and their concentrations control the rate of mitochondrial oxidation of glycolytic NADH. Glutamate also controls the rate of urea synthesis, not only as the precursor of ammonia and aspartate, but as substrate for synthesis of N-acetylglutamate, the essential activator of carbamoyl-phosphate synthase. This mechanism allows large variations in urea synthesis at relatively constant ammonia concentrations. Increases in intracellular amino acid concentration increase cell volume. Cell swelling per se has anabolic effects on protein, carbohydrate and lipid metabolism: enhanced synthesis of macromolecules compensates for increases in intracellular osmolarity. Mechanisms responsible for cell swelling-induced changes in pathway fluxes include changes in intracellular ion concentrations and in signal transduction. Specific amino acids (e.g., leucine) stimulate protein synthesis and inhibit (autophagic) protein degradation independent of changes in cell volume because they stimulate mTOR (mammalian target of rapamycin), a protein kinase, which is one of the components of a signal transduction pathway used by insulin. When the cellular energy state is low, stimulation of mTOR by amino acids is prevented by activation of AMP-dependent protein kinase. Amino acid-dependent signaling also promotes insulin production by beta-cells. This further adds to the anabolic properties of amino acids. It is concluded that amino acids are important regulators of major metabolic pathways.