Reversible phosphorylation of Bcl2 following interleukin 3 or bryostatin 1is mediated by direct interaction with protein phosphatase 2A

Interleukin 3 (IL-3) stimulates the net growth of murine factor-dependent N SF/N1.H7 and FDC-P1/ER myeloid cells by stimulating proliferation and suppr essing apoptosis, Recently, we discovered that Bcl2 is phosphorylated at an evolutionarily conserved serine residue (Ser(70)) after treatment with the survival agonists IL-3 or bryostatin 1, a potent activator of protein kina se (Ito, T., Deng, X., Carr, B,, and May, W, S, (1997) J. Biol, Chem. 272, 11671-11673), In addition, an intact Ser(70) was found to be required for B cl2's ability to suppress apoptosis after IL-3 withdrawal or toxic chemothe rapy, We; now show that phosphorylation of Bc12 occurs rapidly after the ad dition of agonist to IL-3-deprived cells and can be reversed by the action of an okadaic acid (OA)-sensitive phosphatase. A role for protein phosphata se (PP) A as the Bc12 regulatory phosphatase is supported by several observ ations: 1) dephosphorylation of Bc12 is blocked by OA, a potent PP1 and PP2 A inhibitor; 2) intracellular PP2A, but not PP1, co-localizes with Bc12; 3) the purified PP2Ac catalytic subunit directly dephosphorylates Bc12 in vit ro in an OA-sensitive manner; 4) the purified PP2Ac catalytic subunit prefe rentially dephosphorylates Bc12 in vitro compared with PP1 and PP2B; 5) rec iprocal immunoprecipitation studies indicate a direct interaction between P P2A and hemagglutinin (HA)-Bcl2; and 6) treatment of factor-deprived cells with bryostatin 1 dramatically increases the association between PP2A and B c12, Increased association between Bc12 and PP2A occurs 15 min after agonis t stimulation when Bcl2 phosphorylation has peaked and immediately before d ephosphorylation. An agonist-induced increased association of PP2A and Bc12 fails to occur in cells expressing the inactive, phosphorylation-negative S70A Bcl2 mutant, which indicates that an intact Ser(70) site is necessary and sufficient for the interaction to occur. Functional phosphorylation of Bc12 at Ser(70) is proposed to be a dynamic process regulated by the sequen tial action of an agonist-activated Bc12 kinase and PP2A.