Autophagy is a cellular degradation process that is up-regulated upon starvation. Nutrition-dependent regulation of mTOR (mammalian target of rapamycin) is a major determinant of autophagy. RTK (receptor tyrosine kinase) signalling and AMPK (AMP-activated protein kinase) converge upon mTOR to suppress or activate autophagy. Nutrition-dependent regulation of autophagy is mediated via mTOR phosphorylation of the serine/threonine kinase ULK1 (unc51-like kinase 1). In the present study, we also describe ULK1 as an mTOR-independent convergence point for AMPK and RTK signalling. We initially identified ULK1 as a 14-3-3-binding protein and this interaction was enhanced by treatment with AMPK agonists. AMPK interacted with ULK1 and phosphorylated ULK1 at Ser(555) in vitro. Mutation of this residue to alanine abrogated 14-3-3 binding to ULK1, and in vivo phosphorylation of ULK1 was blocked by a dominant-negative AMPK mutant. We next identified a high-stringency Akt site in ULK1 at Ser(774) and showed that phosphorylation at this site was increased by insulin. Finally, we found that the kinase-activation loop of ULK1 contains a consensus phosphorylation site at Thr(180) that is required for ULK1 autophosphorylation activity. Collectively, our results suggest that ULK1 may act as a major node for regulation by multiple kinases including AMPK and Akt that play both stimulatory and inhibitory roles in regulating autophagy.
Mitophagy is a selective pathway, which targets and delivers mitochondria to the lysosomes for degradation. Depolarization of mitochondria by the protonophore CCCP is a strategy increasingly used to experimentally trigger not only mitophagy, but also bulk autophagy. Using live-cell fluorescence microscopy we found that treatment of HeLa cells with CCCP caused redistribution of mitochondrially targeted dyes, including DiOC6, TMRM, MTR, and MTG, from mitochondria to the cytosol, and subsequently to lysosomal compartments. Localization of mitochondrial dyes to lysosomal compartments was caused by retargeting of the dye, rather than delivery of mitochondrial components to the lysosome. We showed that CCCP interfered with lysosomal function and autophagosomal degradation in both yeast and mammalian cells, inhibited starvation-induced mitophagy in mammalian cells, and blocked the induction of mitophagy in yeast cells. PARK2/Parkin-expressing mammalian cells treated with CCCP have been reported to undergo high levels of mitophagy and clearance of all mitochondria during extensive treatment with CCCP. Using correlative light and electron microscopy in PARK2-expressing HeLa cells, we showed that mitochondrial remnants remained present in the cell after 24 h of CCCP treatment, although they were no longer easily identifiable as such due to morphological alterations. Our results showed that CCCP inhibits autophagy at both the initiation and lysosomal degradation stages. In addition, our data demonstrated that caution should be taken when using organelle-specific dyes in conjunction with strategies affecting membrane potential.
ABSTRACT:In addition to its function as carnitine transporter, novel organic cation transporter type 2 (OCTN2; human gene symbol SLC22A5) is widely recognized as a transporter of drugs. This notion is based on several reports of direct measurement of drug accumulation. However, a rigorous, comparative, and comprehensive analysis of transport efficiency of OCTN2 has not been available so far. In the present study, OCTN2 orthologs from human, rat, and chicken were expressed in 293 cells using an inducible expression system. Uptake of trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (ASP ؉ ), cephaloridine, ergothioneine, gabapentin, mildronate, pyrilamine, quinidine, spironolactone, tetraethylammonium, verapamil, and vigabatrin was determined by liquid chromatography/mass spectrometry. For reference, uptake of carnitine was measured in parallel. Our results indicate that OCTN2-mediated uptake of drugs was not significantly different from zero or, with tetraethylammonium and ergothioneine, was minute relative to carnitine. The carnitine congener mildronate, by contrast, was transported very efficiently. Thus, OCTN2 is not a general drug transporter but a highly specific carrier for carnitine and closely related molecules. Transport parameters (cellular accumulation, transporter affinity, sodium dependence) were similar for mildronate and carnitine. Efficiency of transport of mildronate was even higher than that of carnitine. Hence, our results establish that OCTN2 is a key target of the cardioprotective agent mildronate because it controls, as integral protein of the plasma membrane, cellular entry of mildronate and enables efficient access to intracellular targets. The highest levels of human OCTN2 mRNA were detected by real-time reverse transcription-polymerase chain reaction in kidney, ileum, breast, small intestine, skeletal muscle, and ovary but also in some heart and central nervous system tissues.
The function of human proteins is commonly analyzed by heterologous expression in cultured cell lines. Regulated expression, i.e. a system to switch on expression on demand, has clear advantages over constitutive expression. With constitutive expression, cells may die during antibiotic selection because of toxic effects of the expressed protein [1]. Also, for a close match of backgrounds, it is better to compare two states of a single cell line rather than two separately transfected and selected cell lines.Several widely used systems for regulated expression in mammalian cell lines are based on the tetracycline We have developed a novel plasmid vector, pEBTetD, for full establishment of doxycycline-inducible protein expression by just a single transfection. pEBTetD contains an Epstein-Barr virus origin of replication for stable and efficient episomal propagation in human cell lines, a cassette for continuous expression of the simple tetracycline repressor, and a cytomegalovirus-type 2 tetracycline operator (tetO2)-tetO2 promoter. As there is no integration of vector into the genome, clonal isolation of transfected cells is not necessary. Cells are thus ready for use 1 week after transfection; this contrasts with 3-12 weeks for other systems. Adequate regulation of protein expression was accomplished by abrogation of mRNA polyadenylation. In northern analysis of seven cDNAs coding for transport proteins, pools of transfected human embryonic kidney 293 cells showed on ⁄ off mRNA ratios in the order of 100 : 1. Cell pools were also analyzed for regulation of protein function. With two transport proteins of the plasma membrane, the on ⁄ off activity ratios were 24 : 1 and 34 : 1, respectively. With enhanced green fluorescent protein, a 23 : 1 ratio was observed based on fluorescence intensity data from flow cytometry. The unique advantage of our system rests on the unmodified tetracycline repressor, which is less likely, by relocation upon binding of doxycycline, to cause cellular disturbances than chimera of tetracycline repressor and eukaryotic transactivation domains. Thus, in a comprehensive comparison of on-and off-states, a steady cellular background is provided. Finally, in contrast to a system based on Flp recombinase, the set-up of our system is inherently reliable.Abbreviations CMV, cytomegalovirus; EBV, Epstein-Barr virus; ETTh, ergothioneine transporter from human; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; eGFP, enhanced green fluorescent protein; MPP + , 1-methyl-4-phenylpyridinium; rtTA, reverse tetracycline-controlled transcriptional activator; tetO2, type 2 tetracycline operator; TetR, tetracycline repressor; tTA, tetracycline-controlled transcriptional activator; tTS, tetracycline-controlled transcriptional silencer.
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