Phosphorylation of BCL-2 after exposure of human leukemic cells to retinoic acid.
Serine phosphorylation of bcl-2 has been reported after treatment of cells with protein kinase C, okadaic acid, taxol, and other chemotherapeutic agents that attack microtubules. We report here that bcl-2 is phosphorylated on serine in acute myeloblastic leukemia (AML) blasts exposed to all trans retinoic acid (ATRA). Two-dimension gels (isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) disclosed a novel acidic isoform of bcl-2 in ATRA-treated blast cells from a continuous line and from two AML patients; when the cell lysates were digested with lambda-phosphatase, bcl-2 reverted to the control position, indicating that it was phosphorylated. Metabolic labeling experiments using 32Pi showed that, while control bcl-2 was labeled, incorporation was greatly increased when cells were treated with ATRA. A comparison of bcl-2 from blasts treated with ATRA or taxol showed that bcl-2 was phosphorylated on serine in cells treated with either agent; however, both qualitative and quantitative differences were seen. Qualitatively, the phosphorylated isoform from taxol-treated cells was slightly larger than the native isoform and could be distinguished on 10% to 20% SDS-polyacrylamide gradient gels, while the phosphorylated bcl-2 after ATRA ran as a single band on gradient gels at the same position as control bcl-2. Quantitatively, all bcl-2 from ATRA-treated cells was in the phosphorylated isoform, while after taxol, both phosphorylated and native bcl-2 was present; incorporation of 32Pi into bcl-2 was stimulated to greater extent in ATRA-treated compared with taxol-treated cells. We used immunoprecipitation experiments to ask if bcl-2 phosphorylated after ATRA or taxol had altered capacity to dimerize with bax. No change in dimerization was demonstrated. We conclude that: bcl-2 is phosphorylated on serine after treatment of AML blasts with ATRA; bcl-2 phosphorylation after ATRA is different from that seen after taxol; bcl-2 phosphorylated after either agent retains capacity to dimerize with bax. The ATRA or taxol-induced phosphorylation of bcl-2 can also be seen in blast cells obtained from AML patients.
The Ontario Cancer Institute and Princess Margaret Hospital, Toronto, Canada.
Z B
ZB
Hu
M D
MD
Minden
E A
EA
McCulloch
D019281:Dimerization; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007163:Immunosorbent Techniques; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism*; D017239:Paclitaxel / Q000494:pharmacology; D019606:Phosphoamino Acids / Q000032:analysis; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D011994:Recombinant Proteins; D014212:Tretinoin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D019606:Phosphoamino Acids; D010761:Phosphorus Radioisotopes; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D010768:Phosphoserine; D014212:Tretinoin; D010744:Phosphoric Monoester Hydrolases; D017239:Paclitaxel