MAPK-activated Protein Kinase-2 (MK2)-mediated Formation and Phosphorylation-regulated Dissociation of the Signal Complex Consisting of p38, MK2, Akt, and Hsp27

Xun Shen,Chunlei Zheng,Yajun Yang,Ziyang Lin,Zhizhuang Joe Zhao,Hanben Niu

doi:10.1074/jbc.m603622200

Abstract

The p38 MAPK and heat shock protein 27 (hsp27) form a signaling complex with serine/threonine kinase Akt and MAPK-activated protein kinase-2 (MK2), which plays an important role in controlling stress-induced apoptosis and reorganizing actin cytoskeleton. However, regulation of the complex is poorly understood. In this study, the interaction between p38 and hsp27 was visualized in single living L929 cells using fluorescence resonance energy transfer technology, while their association with Akt was examined by immunoprecipitation analysis. Under normal growth conditions, p38 kinase constitutively interacts with hsp27. When cells were exposed to H(2)O(2) or stimulated by arachidonic acid, this interaction was disrupted. However, inhibition of the activation of p38 and Akt by selective inhibitors or overexpression of the kinase-dead mutant of p38 diminished such effects. Furthermore, mutation of phosphorylation sites of hsp27 renders the interaction resistant to H(2)O(2) and arachidonic acid. It was interesting to find that the interaction disappeared in the cells from MK2-knock-out mice or the cells treated with lemptomycin B that blocks export of MK2 from nucleus to cytosol. However, MK2 is not required for the association of hsp27 with Akt. This study suggests that MK2 mediates the incorporation of p38 into the pre-existing complex of hsp27 with Akt. Phosphorylation of hsp27 finally breaks the signaling complex.

Highlights

cyan fluorescent protein (CFP)-tagged Hsp27 Still Protects the Transfected L929 Cells from Heat Shock-induced Death—To examine if the CFPtagged hsp27 still functions as a stress-response protein, the cells transiently transfected with either pECFP-hsp27 or pECFP-hsp27-3A and the non-transfected wild-type cells were subjected to heat shock at 44 °C for 30 min and imaged 30 h later
The results indicate that yellow fluorescent protein (YFP)-tagged p38 still functions as mitogen-activated protein kinase (MAPK), whereas the YFPp38AGF acts as a competitor to suppress activation of the endogenous p38 kinase in the cells
The signaling complex consisting of p38 kinase, MAPK-activated protein kinase-2 (MK2), Akt, and hsp27 represents a good model for studying protein-protein interaction in signal transduction and the regulation of the complex

Summary

Cell Culture and Transfection

The mouse embryonic fibroblast (MEF) MK2Ϫ/Ϫ cells from MK2 knock-out mice and its wild-type control cells (MEF MK2ϩ/ϩ) were kindly provided by Prof. M. Gaestel (Medical School Hanover, Germany) [30]. Gaestel (Medical School Hanover, Germany) [30] These cells and mouse fibroblast L929 cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum at 37 °C in a CO2 incubator. For FRET experiments L929 or MEF cells were co-transfected with a total of 1 ␮g of DNA plasmids (CFP to YFP constructs ratio was 1:2) using Lipofectamine 2000 (Invitrogen). Cells were grown in CO2 incubators at 37°C for 20 h before FRET observation. For analysis of hsp phosphorylation in the cells expressing hsp or hsp27-3A, the cells were stably transfected with pcDNA3.0-hsp and pcDNA3.0-hsp27-3A using Lipofectamine 2000 and selected with 400 ␮g/ml G418

EXPERIMENTAL PROCEDURES

Fluorescence Resonance Energy Transfer Microscopy

Fluorescence Lifetime Imaging Microscopy

Immunoprecipitation Analysis

RESULTS

DISCUSSION