Proteomic Identification of Bcl2-associated Athanogene 2 as a Novel MAPK-activated Protein Kinase 2 Substrate

Koji Ueda,Hidetaka Kosako,Yasuhisa Fukui,Seisuke Hattori

doi:10.1074/jbc.m406049200

Abstract

The p38 MAPK cascade is activated by various stresses or cytokines. Downstream of p38 MAPKs, there are diversification and extensive branching of signaling pathways. Fluorescent two-dimensional difference gel electrophoresis of phosphoprotein-enriched samples from HeLa cells in which p38 MAPK activity was either suppressed or activated enabled us to detect approximately 90 candidate spots for factors involved in p38-dependent pathways. Among these candidates, here we identified four proteins including Bcl-2-associated athanogene 2 (BAG2) by peptide mass fingerprintings. BAG family proteins are highly conserved throughout eukaryotes and regulate Hsc/Hsp70-mediated molecular chaperone activities and apoptosis. The results of two-dimensional immunoblots suggested that the phosphorylation of BAG2 was specifically controlled in a p38 MAPK-dependent manner. Furthermore, BAG2 was directly phosphorylated at serine 20 in vitro by MAPK-activated protein kinase 2 (MAPKAP kinase 2), which is known as a primary substrate of p38 MAPK and mediates several p38 MAPK-dependent processes. We confirmed that MAPKAP kinase 2 is also required for phosphorylation of BAG2 in vivo. Thus, p38 MAPK-MAPKAP kinase 2-BAG2 phosphorylation cascade may be a novel signaling pathway for response to extracellular stresses.

Highlights

Mitogen-activated protein kinases (MAPKs)1 regulate a wide variety of cellular processes by phosphorylating their specific substrates
Identification of Signaling Molecules in the p38 MAPK Cascade—To globally identify factors involved in the p38 MAPK cascade, we developed a system consisting of phosphoprotein purification, fluorescent 2D-DIGE, and mass spectrometric identification of proteins
We confirmed that phosphorylated forms of p38 MAPK, MAPKAPK2, and heat shock protein 27 (Hsp27) bound to the column and were eluted with relatively good recoveries from the column

Summary

EXPERIMENTAL PROCEDURES

Cell Lines and Materials—HeLa and 293 cells were purchased from the ATCC (Manassas, VA). For pEF-BOS/HA-BAG2, the DNA fragment coding N-terminal HAtagged human BAG2 was amplified with PCR using primers (5Ј-CATGGTACCCAGACCGTGCATCATGTACCCATACGACGTCCCAGACTACGCTGCTCAGGCGAAGATCAACGCT-3Ј and 5Ј-CATGGATCCCTAATTGAATCTGCTTTCAGCATTT-3Ј) and cloned into pEF-BOS vector [19]. For pGEX-4T-1/Hsp, the DNA fragment coding human Hsp was amplified with PCR using primers (5Ј-CATGGATCCACCGAGCGCCGCGTCCCCTTCT-3Ј and 5Ј-CATCTCGAGTTACTTGGCGGCAGTCTCATC-3Ј) and cloned into pGEX-4T-1 vector (Amersham Biosciences). Three purified samples (50 ␮g each) were labeled with Cy2, Cy3, and Cy5 minimal dyes (Amersham Biosciences), respectively, following the manufacturer’s instructions. Determination of Proteins of Interest Using 2D-DIGE and Mass Spectrometry—Phosphoprotein-enriched sample prepared as above from anisomycin-treated cells (650 ␮g, nonlabeled) was subjected to second dimension gel, transferred to ProBlott membranes (Applied Biosystems), and stained with Coomassie Brilliant Blue. Construction and Transfection of siMAPKAPK2—A short interfering RNA (siRNA) targeting endogenous MAPKAPK2 was generated using BLOCK-iT Dicer RNAi kits (Invitrogen) following recommended protocols, except that the target sequence was amplified by PCR using primers (5Ј-CAAGAAGAACGCCATCATC-3Ј and 5Ј-GAGGGTTGGATGCATCTT-3Ј). Immunoblotting was performed using the following antibodies: anti-phospho-MAPKAPK2 (Thr334), anti-phospho-HSP27 (Ser82), anti-phospho-c-Jun (Ser63), anti-phosphop44/p42 MAPK (Thr202/Tyr204), and anti-MAPKAPK2 (Cell Signaling Technology); anti-actin (Chemicon International); and anti-HA High Affinity (3F10) (Roche Applied Science)

RESULTS

Sequence coverageb

DISCUSSION