FIGURE 2.

Abl kinase inhibition impairs the degradation of long-lived proteins. A, sub-confluent A549-GFP-LC3 cells were treated with 10 μm STI571 or 2 μg/ml rapamycin for 24 h or 48 h and analyzed for activation markers of Abl kinase signaling (phospho-CrkL) and mTOR signaling (phospho-p70S6K). The results are representative of three independent experiments. B, the same lysates were also analyzed for GFP-LC3-I and GFP-LC3-II levels by Western blotting with an anti-GFP antibody. The -fold changes in LC3-II levels are indicated below the blot. C and D, analysis of basal and starvation-induced long-lived protein degradation in drug-treated A549-GFP-LC3 cells. A549-GFP-LC3 cells were metabolically labeled with [l-³H]leucine. The cells were then chased in complete medium for 6, 24, or 48 h (C) or in Earle's balanced salt solution media for 6 h (D). The media was supplemented with DMSO (control), 2 μg/ml rapamycin, 10 μm STI571, or 50 μg/ml vinblastine as indicated. At each time point the radioactivity released into the medium was measured by scintillation counting. [l-³H]Leucine turnover rates were determined by dividing the cpm in the medium by the total cpm (medium cpm + lysate cpm). E, A549 cells expressing control or Abl and Arg miRNAs were analyzed for starvation-induced long-lived protein degradation as in D except no drugs were added to the medium. F, Western blots of the corresponding cell lysates in E blotted with phospho-CrkL, CrkL and Abl-specific antibodies as indicated. G, analysis of starvation-induced long-lived protein degradation in Abl/Arg double knockout and Abl/Arg heterozygous MEFs. H, Western blots of the corresponding cell lysates in G blotted with phospho-CrkL, CrkL and Abl-specific antibodies as indicated. n = 3 ± S.D. ^**, p < 0.05 compared with the control (C and D), scramble (E), or Abl/Arg heterozygous (G) groups. The results for each autophagy assay are representative of three independent experiments.