Swainsonine is a useful tool to monitor the intracellular traffic of N‐linked glycoproteins as a function of the state of enterocytic differentiation of HT‐29 cells

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Activation of ERK1/2 stimulates macroautophagy in the human colon cancer cell line HT-29 by favoring the phosphorylation of the G␣-interacting protein (GAIP) in an amino acid-dependent manner (Ogier-Denis, E., Pattingre, S., El Benna, J., and Codogno, P. (2000) J. Biol. Chem. 275, 39090 -39095). Here we show that ERK1/2 activation by aurintricarboxylic acid (ATA) treatment induces the phosphorylation of GAIP in an amino acid-dependent manner. Accordingly, ATA challenge increased the rate of macroautophagy, whereas epidermal growth factor did not significantly affect macroautophagy and GAIP phosphorylation status. In fact, ATA activated the ERK1/2 signaling pathway, whereas epidermal growth factor stimulated both the ERK1/2 pathway and the class I phosphoinositide 3-kinase pathway, known to decrease the rate of macroautophagy. Amino acids interfered with the ATA-induced macroautophagy by inhibiting the activation of the kinase Raf-1. The role of the Ras/Raf-1/ERK1/2 signaling pathway in the GAIPand amino acid-dependent control of macroautophagy was confirmed in HT-29 cells expressing the Ras(G12V, T35S) mutant. Similar to the protein phosphatase 2A inhibitor okadaic acid, amino acids sustained the phosphorylation of Ser 259 , which is involved in the negative regulation of Raf-1. In conclusion, these results add a novel target to the amino acid signaling-dependent control of macroautophagy in intestinal cells.Macroautophagy is a lysosomal catabolic route characterized by the sequestration of cytoplasmic material by a membrane of uncertain origin to form an autophagosome that can receive input of material from the endocytic pathway before its fusion with the lysosomal compartment (1-4). There is now mounting evidence for the importance of macroautophagy in physiological processes such as the formation of pulmonary surfactant (5) and maturation of erythrocytes (6) and for its implication in tumor progression (7), cardiomyopathy (8), and programmed cell death (9). In recent years, our understanding of the molecular controls of macroautophagy has greatly increased because of the identification of a set of genes conserved from yeast to human that are involved in the signaling and formation of autophagic vacuoles (10 -12). However, apg/aut genes are not the only actors playing a role in macroautophagy that demonstrate the complexity of this cell process. Phosphoinositide 3-kinase enzymes are involved both in the signaling and formation of autophagic vacuoles (13-15). Similar to other cytoplasmic membrane dynamics, macroautophagy is controlled by cytoplasmic GTPases (16,17). When the cytoplasmic trimeric G i3 protein is bound to GDP, the formation of autophagic vacuoles is stimulated in human colon cancer HT-29 cells, whereas the binding of GTP to the G␣ i3 protein inhibits the formation of autophagic vacuoles (18,19). More recently, we have shown that GAIP, 1 a protein belonging to the RGS (regulator of Gprotein signaling) family (20), stimulates the macroautophagic pathway by accelerating the GTPase activity of the G␣...

Section: Discussionsupporting

confidence: 92%

Section: Discussionsupporting

confidence: 61%

Amino Acids Interfere with the ERK1/2-dependent Control of Macroautophagy by Controlling the Activation of Raf-1 in Human Colon Cancer HT-29 Cells

Pattingre¹,

Bauvy

Codogno

2003

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“…14 C]valine-labeled cells (40,46). Autophagic protein degradation could be inhibited to uninduced levels by the addition of either wortmannin or leupeptin to the EBSS starvation medium, while, in contrast, the proteasomal inhibitor lactacystin did not show any inhibition (data not shown).…”

Section: Screen Of Human Kinome For Autophagy Modulators-tomentioning

confidence: 88%

siRNA Screening of the Kinome Identifies ULK1 as a Multidomain Modulator of Autophagy

Chan

Kir

Tooze

2007

432

407

Autophagy is a vital response to nutrient starvation. Here, we screened a kinase-specific siRNA library using an autophagy assay in human embryonic kidney 293 cells that measures lipidation of the marker protein GFP-LC3 following amino acid starvation. This screen identified ULK1 in addition to other novel candidates that could be confirmed with multiple siRNAs. Knockdown of ULK1, but not the related kinase ULK2, inhibited the autophagic response. Also, ULK1 knockdown inhibited rapamycin-induced autophagy consistent with a role downstream of mTOR. Overexpression of ULK1 inhibited autophagy and this inhibition was independent of its kinase activity. Deletion of the PDZ domain-binding Val-Tyr-Ala motif at the ULK1 C terminus generated a more potent dominant-negative protein. Further deletions revealed that the minimal ULK1 dominant-negative region could be mapped to residues 1-351. Fulllength ULK1 localized to cytoplasmic structures, some of which were GFP-LC3-positive, and this localization required the conserved C-terminal domain. In contrast, ULK1-(1-351) was diffuse in the cytoplasm. These experiments reveal at least two domains in ULK1 which likely function via unique sets of effectors to regulate autophagy.During macroautophagy, a membrane cisterna wraps around cytoplasmic material to form a nascent autophagosome, which subsequently fuses with degradative endosomes. The targets of autophagy can include long-lived proteins, damaged organelles, ubiquitinated cellular substrates, and aberrant protein aggregates (1-4). Macroautophagy, which we refer to here as autophagy, is rapidly induced in response to amino acid deprivation and acts to recycle free molecular building blocks to the starving cell. Reflecting this cellular metabolic role, autophagy also appears to be critical in the maintenance of proper nutritional balance in the context of neonatal mice, developing Dictyostelium, and Caenorhabditis elegans undergoing development arrest (1, 5-7). Autophagy is also a major component of type-II non-apoptotic programmed cell death, and this may form part of the response to certain types of cell stress (8 -12). The recent implication of autophagy in medical contexts such as cancer, neurodegeneration and immunity have increased interest in elucidating the basic molecular mechanisms of the process (4, 13-16).Two yeast screens pioneered the identification of molecular components required for autophagy and a unified nomenclature has been devised for these genes (17)(18)(19). Although the function for many of these genes is unknown, a large proportion can be grouped into distinct biochemical pathways. Studies in yeast and mammalian cells have established the stepwise progression of these pathways, which appear to be essential at an early stage of autophagy (20, 21). For example, the gene products of Atg3, Atg4, Atg5, Atg7, Atg8, Atg10, Atg12, and Atg16 function coordinately in two interrelated ubiquitin-like conjugation systems (22-24). Importantly, yeast Atg8p and its mammalian homologs become covalently linked to a ph...

“…20 and references therein for a recent discussion), these vacuoles acquire lysosomal enzymes by either direct fusion with lysosomes (21)(22)(23) or late endosomes/prelysosomes (24). We have previously shown that the impairment of N-linked glycoprotein processing (25)(26)(27) in undifferentiated HT-29 cells is the consequence of a constitutive autophagic pathway (28). Recently, we have shown that autophagic sequestration is controlled by a G i3 protein in this cell line (29).…”

mentioning

confidence: 99%

Guanine Nucleotide Exchange on Heterotrimeric Gi3 Protein Controls Autophagic Sequestration in HT-29 Cells

Ogier–Denis

Houri

Bauvy

et al. 1996

Self Cite

107

Recent results have shown that autophagic sequestration in the human colon cancer cell line HT-29 is controlled by the pertussis toxin-sensitive heterotrimeric G i3 protein. Here we show that transfection of an antisense oligodeoxynucleotide to the ␣ i3 -subunit markedly inhibits autophagic sequestration, whereas transfection of an antisense oligodeoxynucleotide to the ␣ i2 -subunit does not change the rate of autophagy in HT-29 cells. Autophagic sequestration was arrested in cells transfected with a mutant of the ␣ i3 -subunit (Q204L) that is restricted to the GTP-bound form. In Q204L-expressing cells, 3-methyladenine-sensitive degradation of long lived [14 C]valine-labeled proteins was severely impaired and could not be stimulated by nutrient deprivation. Autophagy was also reduced when dissociation of the ␤␥ dimer from the GTP-bound ␣ i3 -subunit was impaired in cells transfected with the G203A mutant. In contrast, a high rate of pertussis toxin-sensitive autophagy was observed in cells transfected with an ␣ i3 -subunit mutant (S47N) which has an increased guanine nucleotide exchange rate and increased preference for GDP over GTP. Cells that express pertussis toxin-insensitive mutants of either wild-type ␣ i3 -subunit (C351S) or S47N ␣ i3 -subunit (S47N/C351S) exhibit a high rate of autophagy.