American Society of Hematology

Figure 4

$Figure 4. Nuclear PKR activates PP2A to inhibit ATM phosphorylation. (A) PKR co-IPs with PP2A from the nuclear fraction of REH and K562 cell lysates but this association is significantly reduced 30 minutes after exposure to 5 Gy IR (IR+). Western blotting for HSP90 (cytoplasm) and LSD1 (nucleus) was performed as a subcellular fractionation control. Input is 10% of total REH nuclear lysate used in the co-IP. (B) PP2A activity in the nuclear fraction of leukemia cell lines is reduced in cells with decreased PKR expression (sh-PKR) compared with cells transfected with a control shRNA (sh-ctrl). (C) co-IP of PP2A and ATM from the nuclear lysate of REH sh-ctrl and REH sh-PKR cells 30 minutes after treatment with 5 Gy IR and/or pretreatment with 2.5 μM of the PP2A activator FTY720 for 8 hours revealed that PP2A and ATM association is decreased after irradiation, decreased by reduced PKR expression, but increased by FTY720 treatment. In addition, p-ATM is correspondingly increased when ATM-PP2A nuclear association is decreased. Input is 10% of total REH sh-ctrl nuclear lysate used in the co-IP. (D) co-IP of PKR with ATM from the nuclear fraction of REH cells is increased by treatment with 2.5 μM of the PP2A activator FTY720 for 8 hours, whereas PKR-ATM association is decreased by treatment with 1 μM of the PP2A inhibitor OA for 8 hours. Vertical dashed line indicates a repositioned gel lane. (E) Expression of PP2A-B55α and PP2A-B56γ subunits in the cytoplasm and nucleus of REH cells were detected by western blotting. Knockdown of PKR decreased nuclear B55α and increased cytoplasmic B55α both before and after 5 Gy IR. (F) Western blotting demonstrates that B55α expression in REH cells was decreased by B55α-specific shRNA (sh-B55α) compared with control shRNA (sh-ctrl) cells. (G) γ-H2AX formation 30 minutes after 5 Gy IR was measured by western blotting. Pretreatment of cells with 0.5 μM PKRI for 8 hours prior to IR promotes γ-H2AX formation in REH sh-ctrl cells but not REH sh-B55α cells with decreased B55α expression. (H) Flow cytometry after IR reveals that PKRI promotes increased p-ATM in control REH cells (sh-ctrl) but not B55α knockdown cells (sh-B55α). (I) Proposed model by which nuclear PKR mediates PP2A activity and DDR signaling following IR. In undamaged cells, nuclear PKR indirectly antagonizes ATM activation by promoting nuclear localization of the PP2A B55α regulatory subunit that increases nuclear PP2A phosphatase activity to inhibit ATM autophosphorylation. Following IR, PKR and PP2A no longer interact with the ATM complex, and the PP2A-B55α subunit is sequestered in the cytoplasm allowing ATM to be activated and initiate DDR signaling events. *P < .05; **P < .01.$

Nuclear PKR activates PP2A to inhibit ATM phosphorylation. (A) PKR co-IPs with PP2A from the nuclear fraction of REH and K562 cell lysates but this association is significantly reduced 30 minutes after exposure to 5 Gy IR (IR+). Western blotting for HSP90 (cytoplasm) and LSD1 (nucleus) was performed as a subcellular fractionation control. Input is 10% of total REH nuclear lysate used in the co-IP. (B) PP2A activity in the nuclear fraction of leukemia cell lines is reduced in cells with decreased PKR expression (sh-PKR) compared with cells transfected with a control shRNA (sh-ctrl). (C) co-IP of PP2A and ATM from the nuclear lysate of REH sh-ctrl and REH sh-PKR cells 30 minutes after treatment with 5 Gy IR and/or pretreatment with 2.5 μM of the PP2A activator FTY720 for 8 hours revealed that PP2A and ATM association is decreased after irradiation, decreased by reduced PKR expression, but increased by FTY720 treatment. In addition, p-ATM is correspondingly increased when ATM-PP2A nuclear association is decreased. Input is 10% of total REH sh-ctrl nuclear lysate used in the co-IP. (D) co-IP of PKR with ATM from the nuclear fraction of REH cells is increased by treatment with 2.5 μM of the PP2A activator FTY720 for 8 hours, whereas PKR-ATM association is decreased by treatment with 1 μM of the PP2A inhibitor OA for 8 hours. Vertical dashed line indicates a repositioned gel lane. (E) Expression of PP2A-B55α and PP2A-B56γ subunits in the cytoplasm and nucleus of REH cells were detected by western blotting. Knockdown of PKR decreased nuclear B55α and increased cytoplasmic B55α both before and after 5 Gy IR. (F) Western blotting demonstrates that B55α expression in REH cells was decreased by B55α-specific shRNA (sh-B55α) compared with control shRNA (sh-ctrl) cells. (G) γ-H2AX formation 30 minutes after 5 Gy IR was measured by western blotting. Pretreatment of cells with 0.5 μM PKRI for 8 hours prior to IR promotes γ-H2AX formation in REH sh-ctrl cells but not REH sh-B55α cells with decreased B55α expression. (H) Flow cytometry after IR reveals that PKRI promotes increased p-ATM in control REH cells (sh-ctrl) but not B55α knockdown cells (sh-B55α). (I) Proposed model by which nuclear PKR mediates PP2A activity and DDR signaling following IR. In undamaged cells, nuclear PKR indirectly antagonizes ATM activation by promoting nuclear localization of the PP2A B55α regulatory subunit that increases nuclear PP2A phosphatase activity to inhibit ATM autophosphorylation. Following IR, PKR and PP2A no longer interact with the ATM complex, and the PP2A-B55α subunit is sequestered in the cytoplasm allowing ATM to be activated and initiate DDR signaling events. *P < .05; **P < .01.

This Feature Is Available To Subscribers Only