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The sIgG+ lymphoblastoid B cell line CESS spontaneously produces a high amount of nerve growth factor (NGF) and expresses both high affinity (p140Trk-A) and low affinity (p75NTR) NGF receptors. Autocrine production of NGF maintains the survival of CESS cells through the continuous deactivation of p38 MAPK, an enzyme able to induce Bcl-2 phosphorylation and subsequent cytochrome c release and caspase activation. In this paper, we show that NGF induces transcriptional activation and synthesis of MAPK phosphatase 1 (MKP1), a dual specificity phosphatase that dephosphorylates p38 MAPK, thus preventing Bcl-2 phosphorylation. Furthermore, NGF increases MKP-1 protein stability by preventing its degradation through the proteasome pathway. Following NGF stimulation, MKP- protein mainly localizes on mitochondria, suggesting an interaction with p38 MAPK in this compartment. Incubation of CESS cells with MKP-1-specific antisense oligonucleotides induces cell death, which was not prevented by exogenous NGF. By contrast, overexpression of native MKP-1, but not of its catalytically impaired form, inhibits apoptosis induced by NGF neutralization in CESS cells. Thus, the molecular mechanisms underlying the survival function of NGF in CESS B cell line predominantly consist in maintaining elevated levels of MKP-1 protein, which controls p38 MAPK activation. [ABSTRACT FROM AUTHOR]

The antiapoptotic role of Bcl-2 can be regulated by its phosphorylation in serine and threonine residues located in a non-structured 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, Ser87 and Thr56 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 p38a knock-out mice (p38α-/- MEF), whereas they occur within 12 h of serum withdrawal in p38α+/+ 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. [ABSTRACT FROM AUTHOR]