Mono- and multisite phosphorylation enhances Bcl2's antiapoptotic function and inhibition of cell cycle entry functions.
Bcl2 functions to suppress apoptosis and retard cell cycle entry. Single-site phosphorylation at serine 70 (S70) is required for Bcl2's antiapoptotic function, and multisite phosphorylation at threonine 69 (T69), S70, and S87 has been reported to inactivate Bcl2. To address this apparent conflict and identify the regulatory role for Bcl2 phosphorylation in cell death and cell cycle control, a series of serine/threonine (S/T) --> glutamate/alanine (E/A) mutants including T69E/A, S70E/A, S87E/A, T69E/S70A/S87A (EAA), T69A/S70E/S87A (AEA), T69A/S70A/S87E (AAE), T69E/S70E/S87E (EEE), and T69A/S70A/S87A (AAA) was created to mimic or abrogate, respectively, either single-site or multisite phosphorylation. The survival and cell cycle status of cells expressing the phosphomimetic or nonphosphorylatable Bcl2 mutants were compared. Surprisingly, all of the E but not the A Bcl2 mutants potently enhance cell survival after stress and retard G(1)/S cell cycle transition. The EEE Bcl2 mutant is the most potent, indicating a possible cumulative advantage for multisite phosphorylation of Bcl2 in survival and retardation of G(1)/S transition functions. Because the E-containing Bcl2 mutants, but not the A-containing mutants, can more potently block cytochrome c release from mitochondria during apoptotic stress, even at times when steady-state expression levels are similar for all mutants, we conclude that phosphorylation at one or multiple sites within the flexible loop domain of Bcl2 not only stimulates antiapoptotic activity but also can regulate cell cycle entry.
Deng
Xingming
X
Shands Cancer Center and Department of Medicine, University of Florida, Gainesville, FL 32610-0232, USA.
Gao
Fengqin
F
Flagg
Tammy
T
May
W Stratford
WS
D019943:Amino Acid Substitution; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001665:Binding Sites; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D045304:Cytochromes c / Q000378:metabolism; D004355:Drug Stability; D016193:G1 Phase / Q000502:physiology; D066298:In Vitro Techniques; D007377:Interleukin-3 / Q000494:pharmacology; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017239:Paclitaxel / Q000494:pharmacology; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016196:S Phase / Q000502:physiology
D007377:Interleukin-3; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D045304:Cytochromes c; D017239:Paclitaxel