Bcl-2 Phosphorylation by p38 MAPK: identification of target sites and biologic consequences.
The antiapoptotic role of Bcl-2 can be regulated by its phosphorylation in serine and threonine residues located in a nonstructured loop that links BH3 and BH4 domains. p38 MAPK has been identified as one of the kinases able to mediate such phosphorylation, through direct interaction with Bcl-2 protein in the mitochondrial compartment. In this study, we identify, by using mass spectrometry techniques and specific anti-phosphopeptide antibodies, Ser(87) and Thr(56) as the Bcl-2 residues phosphorylated by p38 MAPK and show that phosphorylation of these residues is always associated with a decrease in the antiapoptotic potential of Bcl-2 protein. Furthermore, we obtained evidence that p38 MAPK-induced Bcl-2 phosphorylation plays a key role in the early events following serum deprivation in embryonic fibroblasts. Both cytochrome c release and caspase activation triggered by p38 MAPK activation and Bcl-2 phosphorylation are absent in embryonic fibroblasts from p38alpha knock-out mice (p38alpha(-/-) MEF), whereas they occur within 12 h of serum withdrawal in p38alpha(+/+) MEF; moreover, they can be prevented by p38 MAPK inhibitors and are not associated with the synthesis of the proapoptotic proteins Bax and Fas. Thus, Bcl-2 phosphorylation by activated p38 MAPK is a key event in the early induction of apoptosis under conditions of cellular stress.
De Chiara
Giovanna
G
Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Viale Regina Elena 299, I-00161 Rome, Italy. Electronic address: dechiara@uniroma2.it.
Marcocci
Maria Elena
ME
Department of Experimental Medicine and Biochemical Sciences, University of Rome "Tor Vergata", Via Montpellier 1, I-00133 Rome, Italy.
Torcia
Maria
M
Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, I-50139 Florence, Italy.
Lucibello
Maria
M
Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, I-50139 Florence, Italy; Institute of Neurobiology and Molecular Medicine, National Research Council, Via Fosso del Cavaliere 100, I-00133 Rome, Italy.
Rosini
Paolo
P
Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, I-50139 Florence, Italy.
Bonini
Paolo
P
Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, I-50139 Florence, Italy.
Higashimoto
Yukiro
Y
Laboratory of Cell Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892.
Damonte
Gianluca
G
DIMES Biochemistry Section, Mass Spectrometry Facility, Center of Excellence for Biomedical Research, University of Genoa, Viale Benedetto XV 7, I-16132 Genoa, Italy.
Armirotti
Andrea
A
DIMES Biochemistry Section, Mass Spectrometry Facility, Center of Excellence for Biomedical Research, University of Genoa, Viale Benedetto XV 7, I-16132 Genoa, Italy.
Amodei
Sarah
S
Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, I-50139 Florence, Italy.
Palamara
Anna Teresa
AT
Department of Public Health Science "G. Sanarelli," University of Rome "La Sapienza," Piazzale A. Moro 5, I-00185 Rome, Italy.
Russo
Tommaso
T
CEINGE Biotecnologie Avanzate, Department of Biochemistry and Medical Biotechnologies, University of Naples "Federico II," Via Comunale Margherita 482, I-80131 Naples, Italy.
Garaci
Enrico
E
Department of Experimental Medicine and Biochemical Sciences, University of Rome "Tor Vergata", Via Montpellier 1, I-00133 Rome, Italy.
Cozzolino
Federico
F
Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, I-50139 Florence, Italy. Electronic address: f.cozzolino@dfc.unifi.it.
D000818:Animals; D020169:Caspases / Q000378:metabolism; D045304:Cytochromes c / Q000737:chemistry; D004285:Dogs; D004789:Enzyme Activation; D006801:Humans; D020935:MAP Kinase Signaling System; D051379:Mice; D018345:Mice, Knockout; D010455:Peptides / Q000737:chemistry; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000378:metabolism; D019014:fas Receptor / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D010455:Peptides; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D019014:fas Receptor; D045304:Cytochromes c; D048051:p38 Mitogen-Activated Protein Kinases; D020169:Caspases