PHOSPHORYLATION OF BCL-2 AFTER EXPOSURE OF HUMAN LEUKEMIC-CELLS TO RETINOIC ACID

Serine phosphorylation of bcl-2 has been reported after treatment of c ells with protein kinase C, okadaic acid, taxol, and other chemotherap eutic agents that attack microtubules. We report here that bcl-2 is ph osphorylated on serine in acute myeloblastic leukemia (AML) blasts exp osed to all trans retinoic acid (ATRA). Two-dimension gels (isoelectri c focusing followed by sodium dodecyl sulfate-polyacrylamide gel elect rophoresis [SDS-PAGE]) disclosed a novel acidic isoform of bcl-2 in AT RA-treated blast cells from a continuous line and from two AML patient s; when the cell lysates were digested with lambda-phosphatase, bcl-2 reverted to the control position, indicating that it was phosphorylate d. Metabolic labeling experiments using P-32(i) showed that, while con trol bcl-2 was labeled, incorporation was greatly increased when cells were treated with ATRA. A comparison of bcl-2 from blasts treated wit h ATRA or taxol showed that bcl-2 was phosphorylated on serine in cell s treated with either agent; however, both qualitative and quantitativ e differences were seen. Qualitatively, the phosphorylated isoform fro m 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 gra dient 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; incorp oration of P-32(i) info bcl-2 was stimulated to greater extent in ATRA -treated compared with taxol-treated cells. We used immunoprecipitatio n experiments to ask if bcl-2 phosphorylated after ATRA or taxol had a ltered capacity to dimerize with bar. No change in dimerization was de monstrated. 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 eith er agent retains capacity to dimerize with bar. The ATRA or taxol-indu ced phosphorylation of bcl-2 can also be seen in blast cells obtained from AML patients. (C) 1998 by The American Society of Hematology.