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Bcl-2 phosphorylation required for anti-apoptosis function
1Sealy Center for Oncology and Hematology and Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555-1048, USA.
Abstract:
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.
Insights
Phosphorylation of the proto-oncogene Bcl-2 is essential for its role in suppressing apoptosis. Mutating key phosphorylation sites impairs Bcl-2 function, demonstrating phosphorylation
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- The proto-oncogene Bcl-2 inhibits apoptosis in growth factor-dependent cells.
- A post-receptor signaling mechanism for Bcl-2 function remained unknown.
- Interleukin-3 (IL-3), erythropoietin, and bryostatin-1 (Bryo) suppress apoptosis and stimulate Bcl-2 phosphorylation.
Purpose of the Study:
- To investigate whether Bcl-2 phosphorylation is required for its anti-apoptotic function.
- To determine the role of specific protein kinase C phosphorylation sites in Bcl-2 activity.
Main Methods:
- Conservative serine to alanine mutations were introduced at seven putative protein kinase C phosphorylation sites in Bcl-2.
- The phosphorylation status and anti-apoptotic function of wild-type and mutant Bcl-2 were assessed.
- Cell survival was evaluated under conditions of IL-3 deprivation and etoposide treatment.
- Heterodimerization of Bcl-2 mutants with Bax was analyzed.
Main Results:
- The S70A Bcl-2 mutant, lacking phosphorylation at serine 70, failed to suppress apoptosis upon IL-3 withdrawal or etoposide exposure.
- The S70E mutant, mimicking a phosphate charge, enhanced suppression of etoposide-induced apoptosis.
- The S70A mutant retained the ability to heterodimerize with Bax, indicating this interaction is insufficient for full function.
Conclusions:
- Bcl-2 phosphorylation, specifically at serine 70, is crucial for its full death suppressor activity.
- Bcl-2:Bax heterodimerization alone is not sufficient to mediate Bcl-2's anti-apoptotic signaling.
- Phosphorylation is a key post-translational modification regulating Bcl-2's role in cell survival.