Article

Bcl-2 Phosphorylation by p38 MAPK: Identification of target sites and biologic consequences

University of Florence, Florens, Tuscany, Italy
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2006; 281(30):21353-61. DOI: 10.1074/jbc.M511052200
Source: PubMed

ABSTRACT

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, 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 p38α 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.

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    • "Previous studies have demonstrated that MAPK pathway is active in Bcl-2 overexpressing cancer cells under stressful conditions[25]. Bcl-2 phosphorylation by activated p38-MAPK is a key event in the early induction of apoptosis under conditions of cellular stress[26]. PQ1 has been reported to induce apoptosis in human breast cancer cells through the upregulation of caspases and an alteration in Bax/Bcl-2 expression ratio[21,27]. "

    Preview · Article · Jan 2016 · International Journal of Molecular Sciences
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    • "This identifies p38 MAPK as a novel regulator of ER–mitochondria interaction. p38 MAPK is involved in ER stress–induced apoptosis through IRE1-TRAF2-ASK1-MKK6 signaling (Wang and Ron, 1996; Maytin et al., 2001; Oyadomari and Mori, 2004; De Chiara et al., 2006). On the other hand, p38 MAPK is activated downstream of the ER stress response element PERK (Liang et al., 2006; Zhang et al., 2010) and mediates the protective response against ER stress through a number of mechanisms, including activation of the ER stress sensor ATF6α, up-regulation of Bip/GRP78, activation of Akt, and downregulation of TRB3 expression, probably by phosphorylation and decreased transactivation activity of proapoptotic transcription factor CHOP/GADD153 (Ranganathan et al., 2006; Seimon et al., 2009; Zou et al., 2009; Egawa et al., 2010). "
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    ABSTRACT: Gp78 is an ERAD-associated E3 ubiquitin ligase that induces degradation of the mitofusin mitochondrial fusion proteins and mitochondrial fission. Gp78 is localized throughout the ER, however the anti-Gp78 3F3A monoclonal antibody (mAb) recognizes Gp78 selectively in mitochondria-associated ER domains. Epitope mapping localized the epitope of 3F3A and a commercial anti-Gp78 mAb to an eight amino acid (533-541 in mouse Gp78 isoform 2) motif, that forms part of a highly conserved 41 amino acid region containing 14-3-3 and WW binding domains and a p38 MAP kinase (p38 MAPK) consensus site on serine 538 (S538). 3F3A binds selectively to non-phosphorylated S538 Gp78. Using 3F3A as a reporter, Gp78 S538 phosphorylation was induced by serum starvation and shown to be mediated by p38 MAPK. Mass spectroscopy analysis of Gp78 phosphopeptides confirmed S538 as a major p38 MAPK phosphorylation site on Gp78. Gp78 S538 phosphorylation limited its ability to induce mitochondrial fusion and degrade MFN1 and MFN2 but did not impact in vitro Gp78 ubiquitin E3 ligase activity. Phosphomimetic Gp78 S538D mutation prevented Gp78 promotion of ER-mitochondria interaction and SB203580 inhibition of p38 MAPK increased ER-mitochondria association. p38 MAPK phosphorylation of Gp78 S538 therefore regulates Gp78-dependent ER-mitochondria association and mitochondria motility. © 2015 by The American Society for Cell Biology.
    Preview · Article · Sep 2015 · Molecular biology of the cell
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    • "Members of the Bcl-2 family proteins with either proapoptotic (e.g., Bax, and Bak) or antiapoptotic (e.g., Bcl-2, and Bcl-xL) functions regulate the mitochondrial membrane permeability (MMP) in apoptosis, and decreases in antiapoptotic and increases in proapoptotic Bcl-2 family proteins were observed during apoptosis of cancer cells under chemical stimulation. Previous papers indicated that the subtle balance of the Bcl-2/Bax complex led to an anti- or proapoptotic effect, and the overexpression of Bax may induce loss of the MMP that initiates apoptosis progression [17], [18]. It was indicated that disruption of the MMP via disturbing the Bcl-2/Bax balance leading to activation of caspases-9 and -3 plays an important role in apoptosis induced by chemotherapeutic agents. "
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    ABSTRACT: Evodiamine (EVO; 8,13,13b,14-tetrahydro-14-methylindolo[2'3'-3,4]pyrido[2,1-b]quinazolin-5-[7H]-one derived from the traditional herbal medicine Evodia rutaecarpa was reported to possess anticancer activity; however, the anticancer mechanism is still unclear. In this study, we investigated the anticancer effects of EVO on human colon COLO205 and HT-29 cells and their potential mechanisms. MTT and lactate dehydrogenase (LDH) release assays showed that the viability of COLOL205 and HT-29 cells was inhibited by EVO at various concentrations in accordance with increases in the percentage of apoptotic cells and cleavage of caspase-3 and poly(ADP ribose) polymerase (PARP) proteins. Disruption of the mitochondrial membrane potential by EVO was accompanied by increased Bax, caspase-9 protein cleavage, and cytochrome (Cyt) c protein translocation in COLO205 and HT-29 cells. Application of the antioxidant N-acetyl-L-cysteine (NAC) inhibited H2O2-induced reactive oxygen species (ROS) production and apoptosis, but did not affect EVO-induced apoptosis of COLO205 or HT-29 cells. Significant increases in the G2/M ratio and cyclinB1/cdc25c protein expression by EVO were respectively identified in colon carcinoma cells via a flow cytometric analysis and Western blotting. Induction of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) protein phosphorylation was detected in EVO-treated cells, and the JNK inhibitor, SP600125, but not the ERK inhibitor, U0126, inhibited EVO-induced phosphorylated JNK protein expression, apoptosis, and G2/M arrest of colon carcinoma cells. Data of the structure-activity analysis showed that EVO-related chemicals containing an alkyl group at position 14 were able to induce apoptosis, G2/M arrest associated with increased DNA ladder formation, cleavage of caspase-3 and PARP, and elevated cycB1 and cdc25c protein expressions in COLO205 and HT-29 cells. Evidence supporting JNK activation leading to EVO-induced apoptosis and G2/M arrest in colon carcinoma cells is provided, and alkylation at position 14 of EVO is a critical substitution for treatment of colonic cancer.
    Full-text · Article · Jun 2014 · PLoS ONE
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