Validation of Phospho-ATG14 (Ser29) and Total kits on the human HCT116 cell line using ULK1/2 and mTORC1 inhibitors

uman HCT116 cells  were plated in a 96-well culture-treated plate (200,000 cells/well) in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were treated with a dose-response of AZD2014 (Vistusertib) for 4h at 37°C, 5% CO2. After culture medium removal, cells were then lysed with 25 µl of supplemented lysis buffer #4 (1X) for 30 min at RT under gentle shaking. After cell lysis, 14 µL of lysate were transferred into a 384-well low volume white microplate. 2 µL of Activation Buffer, then 4 µL of the HTRF Phospho-ATG14 (Ser 29) or Total-ATG14 detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.

As expected, AZD2014, a potent mTOR inhibitor, alleviated ULK1 inhibition, increasing autophagy initiation machinery, and leading to an increase in ATG14 phosphorylation on Serine 29 without any effect on the expression level of the ATG14 total protein.

 

Specificity of HTRF Phospho-ATG14 assay using knockout ATG14 HAP1 cells

Human basal phospho (Ser29) ATG14 expression level was assessed with the Phospho (Ser29) HTRF kit in HAP1 cells with both Wild Type (WT) and ATG14 knock-out (KO). Normalization was done using the GAPDH Housekeeping HTRF kit. The cell lines were cultured in flasks at 37°C, 5% CO2 and after culture medium removal, cells were then lysed with 1 mL of supplemented lysis buffer #4 (1X) per 10 million cells, for 30 min at RT under gentle shaking. After cell lysis, 14 µL of lysate were transferred into a 384-well low volume white microplate, and 2 µL of Activation Buffer, then 4 µL of the HTRF Phospho-ATG14 (Ser 29) detection reagents were added. For GAPDH, 8 µL of lysate was prediluted with 60 µL diluent before mixing 16 µL of the diluted material with 4 µL of the GAPDH detection reagents. The HTRF signal was recorded after an overnight incubation at room temperature. In HAP1 KO ATG14 cells, the HTRF signal was equivalent to the non-specific signal (not shown) indicating a complete ATG14 gene silencing and thus demonstrating the specificity of the HTRF Phospho(Ser29) ATG14 kit for the ATG14 protein. GAPDH normalization was applied to take into account slight growth differences between parental and KO cells (the phospho-ATG14 over GAPDH ratio is presented here). HAP1 ATG14 KO (1bp del) cell line from Horizon Discovery # HZGHC024663c012 HAP1 WT from Horizon Discovery # C631

 

ATG14 signaling pathway in macro-autophagy

Cellular Autophagy is a specialized degradation and recycling process that is instrumental for cell homeostasis, being activated in response to several different stresses. There are 3 types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy. Macroautophagy pathways involve several key steps: initiation, nucleation, elongation of phagophore complexes, then sequestration of cytoplasmic cargos with LC3-PE recruitment followed by fusion with lysosome yielding to cargo degradations.
 

Biogenesis of the autophagosome is controlled by sequential and concerted actions of the so-called autophagy related proteins, ATGs, which are activated and recruited to the ER and autophagosome membranes. The recruitment of the ULK1 complex is the first event in the initiation step. ULK1 is a Ser/Thr kinase which forms a complex with the Atg13, Atg101 and FIP200 proteins. This complex is the most upstream component of the core autophagy machinery and is therefore the key initiator of autophagy in mammalian cells. ULK1 is regulated by the key nutrient/energy-sensitive kinases mTOR and AMPK, which are both able to phosphorylate ULK1 on Serine 317 and Serine 556, and directly regulate its kinase activity.
 

The Activated ULK1 complex then binds ATG14 via ATG13, and phosphorylates ATG14 on Serine 29. The kinase activity of phosphorylated and activated ATG14 stimulates other proteins of the PIK3C3 complex (nucleation) that is responsible for the critical step of phosphorylation of phosphatidylinositols (PI) into phosphatidylinositol-3-phosphate (PI3P). This in turn is responsible for the formation of the initial phagosomal membrane structure and later allows fixation of LC3-II using other ATG proteins (ATG16/12/5 complex), thus generating a support for elongation and closing steps.
 

This means tha phosphorylation of ATG14 protein on serine 29 is a key early marker of the nucleation step in the engagement of the macroautophagic process.