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

Interleukin 3 (IL-3) stimulates the net growth of murine factor-dependent NSF/N1.H7 and FDC-P1/ER myeloid cells by stimulating proliferation and suppressing 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 kinase (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 Bcl2's ability to suppress apoptosis after IL-3 withdrawal or toxic chemotherapy, We; now show that phosphorylation of Bc12 occurs rapidly after the addition 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 phosphatase (PP) A as the Bc12 regulatory phosphatase is supported by several observations: 1) dephosphorylation of Bc12 is blocked by OA, a potent PP1 and PP2A inhibitor; 2) intracellular PP2A, but not PP1, co-localizes with Bc12; 3) the purified PP2Ac catalytic subunit directly dephosphorylates Bc12 in vitro in an OA-sensitive manner; 4) the purified PP2Ac catalytic subunit preferentially dephosphorylates Bc12 in vitro compared with PP1 and PP2B; 5) reciprocal immunoprecipitation studies indicate a direct interaction between PP2A and hemagglutinin (HA)-Bcl2; and 6) treatment of factor-deprived cells with bryostatin 1 dramatically increases the association between PP2A and Bc12, Increased association between Bc12 and PP2A occurs 15 min after agonist stimulation when Bcl2 phosphorylation has peaked and immediately before dephosphorylation. 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 sequential action of an agonist-activated Bc12 kinase and PP2A.