Histone deacetylase 6 (HDAC6) deacetylates extracellular signal-regulated kinase 1 (ERK1) and thereby stimulates ERK1 activity

Jheng-Yu Wu,Shengyan Xiang,Mu Zhang,Bin Fang,He Huang,Oh Kwang Kwon,Yingming Zhao,Zhe Yang,Wenlong Bai,Gerold Bepler,Xiaohong Mary Zhang

doi:10.1074/jbc.m117.795955

Abstract

Histone deacetylase 6 (HDAC6), a class IIb HDAC, plays an important role in many biological and pathological processes. Previously, we found that ERK1, a downstream kinase in the mitogen-activated protein kinase signaling pathway, phosphorylates HDAC6, thereby increasing HDAC6-mediated deacetylation of α-tubulin. However, whether HDAC6 reciprocally modulates ERK1 activity is unknown. Here, we report that both ERK1 and -2 are acetylated and that HDAC6 promotes ERK1 activity via deacetylation. Briefly, we found that both ERK1 and -2 physically interact with HDAC6. Endogenous ERK1/2 acetylation levels increased upon treatment with a pan-HDAC inhibitor, an HDAC6-specific inhibitor, or depletion of HDAC6, suggesting that HDAC6 deacetylates ERK1/2. We also noted that the acetyltransferases CREB-binding protein and p300 both can acetylate ERK1/2. Acetylated ERK1 exhibits reduced enzymatic activity toward the transcription factor ELK1, a well-known ERK1 substrate. Furthermore, mass spectrometry analysis indicated Lys-72 as an acetylation site in the ERK1 N terminus, adjacent to Lys-71, which binds to ATP, suggesting that acetylation status of Lys-72 may affect ERK1 ATP binding. Interestingly, an acetylation-mimicking ERK1 mutant (K72Q) exhibited less phosphorylation than the WT enzyme and a deacetylation-mimicking mutant (K72R). Of note, the K72Q mutant displayed decreased enzymatic activity in an in vitro kinase assay and in a cellular luciferase assay compared with the WT and K72R mutant. Taken together, our findings suggest that HDAC6 stimulates ERK1 activity. Along with our previous report that ERK1 promotes HDAC6 activity, we propose that HDAC6 and ERK1 may form a positive feed-forward loop, which might play a role in cancer.

Highlights

Histone deacetylase 6 (HDAC6), a class IIb Histone deacetylases (HDACs), plays an important role in many biological and pathological processes
We have revealed that ERK1 and ERK2 are acetylated proteins and are novel substrates of the deacetylase HDAC6 and the acetyltransferases CBP and p300
We have shown that the ERK1 Lys-72 acetylation-mimicking mutant (K72Q) displayed reduced kinase activity as compared with the wildtype and deacetylationmimicking mutant (K72R)

Summary

To whom correspondence should be addressed

HDAC6 deacetylates ERK1 lates cell-signaling regulators K-Ras and ␤-catenin, leading to altered oncogenic activity and nuclear localization, respectively [8, 9]. All MAPKs, except ERK3/4 and NLK, contain a conserved Thr-Xaa-Tyr motif in their kinase domain Phosphorylation of both Thr and Tyr residues in this motif is a critical step for MAPK activation [10]. Several reports have shown that when cell lines, including A549, MB361, BT474, MV4-11, PC-3, SKBR-3, HN-9, and SQ20B, were treated with HDAC6 inhibitors, the level of phosphorylated ERK1/2 decreased [23,24,25,26], suggesting that acetylation of ERK1/2 compromises their activities and that HDAC6 inhibition may down-regulate ERK1/2 activities. We have determined that ERK1/2 are acetylated proteins and that ERK1/2 are novel substrates of HDAC6 Both ERK1/2 show the ability to physically interact with HDAC6 in vitro. Lys-72, was identified in ERK1 via mass spectrometry analysis, and the acetylationmimicking mutant of ERK1 exhibits reduced kinase activity, suggesting that the acetylation status of ERK1 plays an important role in regulating ERK1 enzymatic activity

Results

Discussion

Experimental procedures