Bcl-2 phosphorylation required for anti-apoptosis function.
The protooncogene Bcl-2 functions as a suppressor of apoptosis in growth factor-dependent cells, but a post-receptor signaling mechanism is not known. We recently reported that interleukin 3 (IL-3) and erythropoietin, or the protein kinase C activator bryostatin-1 (Bryo), not only suppresses apoptosis but also stimulates the phosphorylation of Bcl-2 (May, W. S., Tyler, P. G., Ito, T., Armstrong, D. K., Qatsha, K. A., and Davidson, N. E. (1994) J. Biol. Chem. 269, 26865-26870). To test whether phosphorylation is required for Bcl-2 function, conservative serine --> alanine mutations were produced at the seven putative protein kinase C phosphorylation sites in Bcl-2. Results indicate that the S70A Bcl-2 mutant fails to be phosphorylated after IL-3 or Bryo stimulation and is unable to support prolonged cell survival either upon IL-3 deprivation or etoposide treatment when compared with wild-type Bcl-2. In contrast, a Ser --> Glu mutant, S70E, which may mimic a potential phosphate charge, more potently suppressed the etoposide-induced apoptosis than wild type in the absence of IL-3. Since the loss of function S70A mutant can heterodimerize with its partner protein and death effector Bax, these findings demonstrate that Bcl-2:Bax heterodimerization is not sufficient and Bcl-2 phosphorylation is required for full Bcl-2 death suppressor signaling activity.
Ito
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T
Sealy Center for Oncology and Hematology and Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555-1048, USA.
Deng
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X
Carr
B
B
May
W S
WS
D000409:Alanine; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D054713:Bryostatins; D002460:Cell Line; D019281:Dimerization; D004789:Enzyme Activation; D005047:Etoposide / Q000494:pharmacology; D018698:Glutamic Acid; D007377:Interleukin-3 / Q000494:pharmacology; D007700:Kinetics; D007783:Lactones / Q000494:pharmacology; D018942:Macrolides; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D011493:Protein Kinase C / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine; D014162:Transfection
D054713:Bryostatins; D007377:Interleukin-3; D007783:Lactones; D018942:Macrolides; D010710:Phosphates; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; C046785:bryostatin 1; D018698:Glutamic Acid; D012694:Serine; D005047:Etoposide; D011493:Protein Kinase C; D000409:Alanine