Phospho-p62/SQSTM1 (Ser403) Cellular Kit HTRF®

Human HeLa and SH-SY5Y cells, and mouse Neuro2a cells were plated at 100,000 and 120,000 and 50,000 cells/well in a 96 well plate, respectively .

After an incubation of 24h at 37°C, 5% CO2, the cell culture medium was discarded, and 50µL of supplemented lysis buffer #4 (1X) added.

After 30min lysis at RT under gentle shaking, lysates were transferred into a low volume detection microplate as follows:

16µL of lysate for the detection of phospho-p62/SQSTM1 (S403)

4 µl of lysate, then 12 µL of supplemented lysis buffer #4 (1X) for the detection of total p62/SQSTM1.

Finally, 4µL of the HTRF phospho (Ser403) or total p62/SQSTM1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

Whereas the expression of p62/SQSTM1 is evidenced in untreated cell lines assessed here, its phosphorylation level is elevated in Neuro2A cells only.

Beside demonstrating the compatibility of the assays with human and mouse samples, these results suggest differences in p62/SQSTM1 behavior depending on the cellular background.

Human HeLa and SH-SY5Y cells were plated at respectively 100,000 and 120,000 cells/well in a 96 well plate, and incubated 24h at 37°C, 5% CO2.

After overnight incubation with increasing concentrations of Bafilomycin A1, which is a well established autophagic flux blocker, the cell culture medium was removed, and cells were lysed with 50 µL of supplemented lysis buffer #4 (1X). After 30min lysis at RT under gentle shaking, lysates were transferred into a low volume detection microplate as follows:

16µL of lysate for the detection of phospho-p62/SQSTM1 (S403).

4 µl of lysate then 12 µL of supplemented lysis buffer #4 (1X) for the detection of total p62/SQSTM1.

Finally, 4µL of the HTRF phospho (Ser403) or total p62/SQSTM1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

As expected, increasing concentrations of Bafilomycin A1 are associated with a dose dependent increase of both expression and S403 phosphorylation of p62/SQSTM1.

Note that Bafilomycin EC50 is approximately 20nM, and comparable between Hela and SH-SY5Y cells.

Human HeLa cells were plated at 100,000 cells/well in a 96 well plate. After 24h incubation, cell culture medium was removed and replaced by EBSS (an amino acid and vitamin deprived medium) or fresh culture medium for 6h.

Next, cells were lysed with 50 µL of supplemented lysis buffer #4 (1X). After 30min lysis at RT under gentle shaking, lysates were transferred into a low volume detection microplate as follows:

16µL of lysate for the detection of phospho-p62/SQSTM1 (S403).

4 µl of lysate, then 12 µL of supplemented lysis buffer #4 (1X) for the detection of total p62/SQSTM1.

Finally, 4µL of the HTRF phospho (Ser403) or total p62/SQSTM1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

As expected, amino acid deprivation conditions are associated with a decrease in total p62/SQSTM1, which results from an autophagy-induced degradation mechanism.

Human HeLa cells and SH-SY5Y cells were plated at respectively 100,000 and 120,000 cells/well in a 96 well plate.

After cell treatment (Hela: 6H; SH-SY5Y: ON) with increasing concentrations of an autophagic inducer, PP242, the culture medium was removed.

Next, cells were lysed with 50 µL of supplemented lysis buffer #4 (1X). After 30 min lysis at RT under gentle shaking, lysates were transferred into a low volume detection microplate as follows:

16 µL of lysate for the detection of phospho-p62/SQSTM1 (S403).

4 µL of lysate, then 12 µL of supplemented lysis buffer #4 (1X) for the detection of total p62/SQSTM1.

Finally, 4 µL of the HTRF phospho (Ser403) or total p62/SQSTM1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

These results show that the p62/SQSTM1 level is reduced upon PP242 treatment, resulting from a degradation induced by the autophagic pathway.

Human HeLa cells were grown for 2 days, until 80% confluency was reached. Then the cells were stimulated with MG-132 (4 µM) overnight, before lysis with supplemented lysis buffer, and soluble supernatants were collected via centrifugation.

Serial dilutions of the cell lysate in supplemented lysis buffer #4 (1X) were performed, and 16µL of lysates were transferred into a HTRF low volume detection microplate prior to the addition of 4µL of HTRF phospho-p62/SQSTM1 (S403) detection reagents.

A side by side comparison of Western Blot and HTRF demonstrates that the HTRF assay is 4-fold more sensitive than the Western Blot, at least under these experimental conditions.

Autophagy is a physiological cellular process enabling the clearing out of misfolded or aggregated proteins, or damaged organelles like mitochondria, also called mitophagy. Activated upon oxidative stress, nutrient deprivation, infections (xenophagy), or during the development of cancers or neurodegenerative diseases, the autophagy pathway relies on key players such as p62/SQSTM1, ATG proteins, or LC3.

p62 / SQSTM1 is an adaptor protein which is initially phosphorylated by ULK1 on Serine 407, then on Serine 403 by casein kinase 2 or TBK1. The phosphorylation on Ser403 increases its affinity for ubiquitin chains, thus enabling p62/SQSTM1 to bind to ubiquitinated cargo proteins. Ubiquitinated proteins or ubiquitin coated mitochondria associated with p62SQSTM1 proteins are taken away in phagosomes, whose content is cleared out after lysosomal fusion.

In addition, another p62/SQSTM1 phosphorylation residue has been reported on Ser349 upon oxidative stress or amino acid deprivation. This phosphorylation event abrogates the interaction between Keap1 and the transcription factor Nrf2 which translocates into the nucleus, where it activates the transcription of antioxidant genes including p62/SQSTM1. In parallel, cytoplasmic Keap1 is captured by p62/SQSTM1 and is degraded by autophagic clearance.

Apart from the role of p62/SQSTM1 in addressing unwanted cargoes to autophagic clearance, other components are essential in the autophagy process. Among them are ULK1 and VSP34 complexes, as well as the conjugation machinery composed of the ATG protein family and LC3-II which are sequentially involved in the formation of phagophores. Phagophores evolve to autophagosomes which fuse with lysosomes, giving rise to autolysosomes where enzymes like protease or phosphatases are active.