DNA Damage Stress and Inhibition of Jak2-V617F Cause Its Degradation and Synergistically Induce Apoptosis through Activation of GSK3β
Figure 3
Possible involvement of ubiquitin-proteasome pathway and Cbls in downregulation of Jak2.
(A) 32D/EpoR cells deprived of Epo for 2 h were pretreated with 10 µM MG132 or left untreated as control, as indicated for 1 h in the absence of Epo. Cells were then treated for 6 h with or without 5 µM etoposide, as indicated. Cells were lysed and subjected to immunoblot analysis using indicated antibodies. (B) 293T cells were transfected on 6-well plate with 0.1 µg of pRK5-Ubiquitin-WT and 0.002 µg of pRK5-Jak2-Wt (Wt) or pRK5-Jak2-KE (KE) along with 0.1 µg of pXM-EpoR-Wt or empty plasmid, as indicated. Two days after transfection, cells were lysed, and Jak2 was immunoprecipitated. Immunoprecipitates were analyzed by immunoblotting using antibodies against polyubiquitin (poly-Ubi), Jak2, and Jak2 phosphorylated on Y1007 (Jak2-PY), as indicated. The vertical line indicates the smeary pattern characteristic of ubiquitination. (C) 293T cells were transfected on 6-well plate with 0.3 µg of pRevTRE-Jak2V617F and 0.2 µg of pTet-On along with 0.2 µg of pRK5-Ubiquitin-WT (Ubi), 0.5 µg of pRevTRE-c-Cbl (c-Cbl), or 0.5 µg of pRevTRE-Cbl-b (Cbl-b), as indicated. The amounts of plasmid DNA transfected were equalized by adding pRevTRE. Two days after transfection, cells were lysed, and lysates were analyzed by immunoblotting. The position of unmodified Jak2-V617F and those of ubiquitinated Jak2-V617F are indicated by asterisks and arrows, respectively. (D) Ton.32D/Flt3-Wt (Cont.) and Ton.32D/Flt3-Wt/c-Cbl-RQ/Cbl-b-CA (c-Cbl-RQ/Cbl-b-CA) cells, cultured in doxycycline-containing medium, were cultured for 3 h with or without 5 µM LY294002 (LY), as indicated, in the absence of IL-3. Cells were further treated for 6 h with 10 µM etoposide (VP16) or 1 µM doxorubicin (DXR), as indicated, and lysed. Cell lysates were analyzed by immunoblotting.