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Biochem Biophys Res Commun
2019 Sep 10;5171:140-145. doi: 10.1016/j.bbrc.2019.07.033.
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Autophosphorylation of MAP kinase disables the MAPK pathway in apoptotic Xenopus eggs.
Tokmakov AA
,
Akino K
,
Iguchi S
,
Iwasaki T
,
Stefanov VE
,
Sato KI
.
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Mitogen-activated protein kinases (MAPKs) are involved in the regulation of various cellular processes, including cell survival and apoptosis. Here, we report that Xenopus p42MAPK becomes phosphorylated in apoptotic eggs, however this modification does not activate the enzyme. Using phosphorylation residue-specific antibodies, we demonstrate that this modification occurs on the Tyr residue in the MAPK activation segment, pinpointing the autophosphorylation mechanism. Notably, MAPK phosphorylation in apoptotic Xenopus eggs coincides with prominent intracellular acidification accompanying apoptosis in these cells. Furthermore, autophosphorylation of recombinant Xenopus MAPK is stimulated and phosphorylation of a protein substrate is inhibited under low pH conditions. Thus, acidic intracellular conditions inactivate MAPK and effectively disable the MAPK-mediated survival pathway in the apoptotic eggs. Given that cell acidification is a rather common feature of apoptosis, we hypothesize that stimulation of MAPK autophosphorylation and shutdown of the MAPK pathway may represent universal traits of apoptotic cell death.
Fig. 1. Cell death events in ionophore-treated Xenopus eggs. In vitro matured Xenopus eggs were treated with 1 μM calcium ionophore A23187 for 5 min and monitored over the indicated times. Caspase activation (A), cytochrome c release (B), intracellular pH (C), and egg diameter (D) are shown. Western blots of phospho-MAPK and MAPK and their quantification are presented in panels E and F. Fractions I, II, III, IV in panel E denote the pooled samples prepared from meiotically arrested, post-meiotic, apoptotic, and post-apototic eggs, respectively. Bars in panels A, C, D, F indicate SDs of three to five measurements obtained in different experiments.
Fig. 2. MAPK phosphorylation and activity in ionophore-treated Xenopus eggs. Fractions I, II, III, IV represent the samples prepared from meiotically arrested, post-meiotic, apoptotic, and post-apototic eggs, respectively. Western blots of fractions I-IV with the indicated antibodies and their quantification are shown in panels A and B. MAPK activity in the fractions, as assessed by MBP phosphorylation, and its quantification are presented in panels C and D. Bars in panel B indicate SDs of three to five measurements using eggs obtained from three different female frogs. Bars in panel D represent SDs of four MAPK activity measurements.
Fig. 3. Activity of recombinant MAPK at different pH and Xenopus MAPK structure. Specific phosphate incorporation into the recombinant Xenopus GST-MAPK (Auto, red bars) and the exogenous substrate (MBP, blue bars) at pH 7.5 and 6.5, as determined in the in vitro kinase assay, are presented in panel A. Modeled 3D structure of Xenopus MAPK is shown in B. The activation segment is shown in cyan, the catalytic residue (D152) is colored in magenta, and phospho-regulatory residues T188 and Y190 are shown in red. Bars in panel A represent SDs of four measurements. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 4. Activation segment of Xenopus MAPK. (A) Amino acid sequence of the activation segment. Phosphoregulatory residues are shown in red and histidine residues protonated at ∼ pH 6.0 are presented in green. (B) Electrostatic potentials in the activation segment. Positive potentials are shown in blue and negative potentials in red. (C) Catalytic cleft of Xenopus MAPK. The activation segment is presented in yellow, phosphoregulatory tyrosine T190 is shown in red, and negatively charged residues of the activation segment and loop L16 are in cyan. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
