CARS senses cysteine deprivation to activate AMPK for cell survival

A

His pull‐down assay was performed with His‐EV‐ or His‐fused CARS protein and HA‐tagged AMPK subunit proteins overexpressed in 293T cells.

B, C

GST pull‐down assay was performed with GST‐EV, GST‐AMPKγ1, GST‐AMPKγ2 (B) or GST‐EV, GST‐AMPKγ2, GST‐AMPKγ2‐N, GST‐AMPKγ2‐C (C) proteins and His‐tagged CARS protein purified from E. coli. The segments of AMPKγ2 are shown in the bar graph above (C). The red arrows indicate the target bands.

D, E

WB analysis of p‐AMPK, p‐ACC, p‐Raptor and total AMPK, ACC, Raptor, AMPKγ2 protein expression in 293T cells transfected with shRNAs targeting AMPKγ2 that were further treated with cystine‐deficient medium for 8 h (D) or transfected with Flag‐CARS for 48 h (E). Actin served as the loading control.

F

293T cells were transfected with HA‐AMPKγ2 alone or with Flag‐CARS for 48 h and then cultured with cystine‐deficient medium or complete medium for 8 h. Cell lysates were immunoprecipitated with anti‐Flag, and a WB analysis was performed.

G

293T cells were transfected with HA‐AMPKγ2 for 48 h and then cultured with cystine‐deficient medium or complete medium for 8 h. Cell lysates were immunoprecipitated with anti‐CARS, followed by WB analysis with anti‐HA and anti‐CARS.

H

GST pull‐down of His‐CARS by GST‐AMPKγ2 using protein purified from E. coli, followed by WB analysis. Cysteine (0, 2, 10, 50 and 250 μM) was added to the experimental buffer.

I

The schematic diagram shows that cystine deficiency‐induced AMPK activation is mediated by CaMKK2, which is recruited by CARS and the AMPKγ2 complex.

Figure 3. CARS activates AMPK by sensing cystine deficiency upon binding to AMPKγ2.