Figure 5. Degradation of HK2 via CMA... | Rockefeller University Press

$Figure 5. Degradation of HK2 via CMA leads to metabolic stress and cell death. (A) Mutant p53 levels in the cytosolic fraction of ES2 cells treated with AC220 and for 16 h in the absence or presence of MG132. (B) Scatter plot depicting proteins identified and quantified in a quantitative proteomics experiment. Proteins significantly enriched as containing one or more stringent or loose motif biochemically related to KFERQ are highlighted as potential CMA substrates. (C) HK2, SGT1, and FAM3C protein levels in ES2 cells treated with AC220 and C43 up to 24 h. (D) WB analysis of HK2, SGT1, FAM3C, p53, and GAPDH levels in ES2 cells treated with AC220 and C43 for 16 h in the absence or presence of proteasome (MG132) or lysosomal inhibitor (ClQ). This experiment (bottom row) used the same blot as in the bottom row of Fig. 3 C with additional antibodies. (E) Cell viability (%) and cell death (fold) of scramble (SCR) or HK2 siRNA–transfected ES2 cells at 72 h after transfection. (F) The combined ribbon representation and stick model showing the overall structure and the CMA motif of HK2 protein in complex with glucose. The interaction of HK2 with Hsc70 or Lamp2A, after AC220 and/or C43 treatment, was analyzed by coimmunoprecipitation. (G) WB of HK2, p53, and GAPDH levels in ES2 cells transfected with nontargeting (N.T) or Hsc70 siRNA, treated with AC220 and C43 for 12 h. (H) The localization of HK2 in cellular endosomal/mitochondria (EM), lysosomal (L), or cytosolic (C) fractions in ES2 cells treated with AC220 and C43 for 16 h, in the presence of the lysosomal inhibitor (ClQ). Lamp2A, Tom40, or β-actin were used as markers for the fractions. (I) The interaction of wt or Q712A;R713A mutant GFP-HK2 with Hsc70, after AC220 and/or C43 treatment, analyzed in 293T cells by coimmunoprecipitation. (J) WB analysis, cell viability (%), and cell death (fold) of wt and Q712A;R713A mutant GPF-HK2–expressing ES2 cells after AC220 and C43 treatment for 16 h. Anti–α-tubulin was used as a loading control. Cells were treated with 0.1% DMSO (control: vehicle) or 1 µM AC220 and 10 µM C43, unless otherwise stated. In all the experiments, treatment groups were compared with the control group, unless otherwise shown. Error bars indicate ±SD. ***, P < 0.001.$

Figure 5.

Degradation of HK2 via CMA leads to metabolic stress and cell death. (A) Mutant p53 levels in the cytosolic fraction of ES2 cells treated with AC220 and for 16 h in the absence or presence of MG132. (B) Scatter plot depicting proteins identified and quantified in a quantitative proteomics experiment. Proteins significantly enriched as containing one or more stringent or loose motif biochemically related to KFERQ are highlighted as potential CMA substrates. (C) HK2, SGT1, and FAM3C protein levels in ES2 cells treated with AC220 and C43 up to 24 h. (D) WB analysis of HK2, SGT1, FAM3C, p53, and GAPDH levels in ES2 cells treated with AC220 and C43 for 16 h in the absence or presence of proteasome (MG132) or lysosomal inhibitor (ClQ). This experiment (bottom row) used the same blot as in the bottom row of Fig. 3 C with additional antibodies. (E) Cell viability (%) and cell death (fold) of scramble (SCR) or HK2 siRNA–transfected ES2 cells at 72 h after transfection. (F) The combined ribbon representation and stick model showing the overall structure and the CMA motif of HK2 protein in complex with glucose. The interaction of HK2 with Hsc70 or Lamp2A, after AC220 and/or C43 treatment, was analyzed by coimmunoprecipitation. (G) WB of HK2, p53, and GAPDH levels in ES2 cells transfected with nontargeting (N.T) or Hsc70 siRNA, treated with AC220 and C43 for 12 h. (H) The localization of HK2 in cellular endosomal/mitochondria (EM), lysosomal (L), or cytosolic (C) fractions in ES2 cells treated with AC220 and C43 for 16 h, in the presence of the lysosomal inhibitor (ClQ). Lamp2A, Tom40, or β-actin were used as markers for the fractions. (I) The interaction of wt or Q712A;R713A mutant GFP-HK2 with Hsc70, after AC220 and/or C43 treatment, analyzed in 293T cells by coimmunoprecipitation. (J) WB analysis, cell viability (%), and cell death (fold) of wt and Q712A;R713A mutant GPF-HK2–expressing ES2 cells after AC220 and C43 treatment for 16 h. Anti–α-tubulin was used as a loading control. Cells were treated with 0.1% DMSO (control: vehicle) or 1 µM AC220 and 10 µM C43, unless otherwise stated. In all the experiments, treatment groups were compared with the control group, unless otherwise shown. Error bars indicate ±SD. ***, P < 0.001.

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