AT motif binding factor 1 (ATBF1) is highly phosphorylated in embryonic brain and protected from cleavage by calpain-1

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Abstract

ATBF1 is a transcription factor that regulates genes responsible for repairing tissues and the protection of cells from oxidative stress. Therefore reduction of ATBF1 promotes susceptibility to varieties of human diseases including neurodegenerative diseases and malignant tumors. The instability of the protein was found to be an important background of diseases. Because ATBF1 is composed of a large 404-kDa protein, it can be easily targeted by proteinases. The protein instability should be a serious problem for the function in the cells and practically for our biochemical study of ATBF1. We have found that calpain-1 is a protease responsible for the degeneration of ATBF1. We observed distinct difference between embryo and adult brain derived ATBF1 regarding the sensitivity to calpain-1. The comparative study showed that eight phosphorylated serine residues (Ser1600, Ser2634, Ser2795, Ser2804, Ser2900, Ser3431, Ser3613, Ser3697) in embryonic brain, but only one site (Ser2634) in adult brain. As long as these amino acids were phosphorylated, ATBF1 derived from embryonic mouse brain showed resistance to cleavage; however, treatment with calf intestine alkaline phosphatase sensitized ATBF1 to be digested by calpain-1. An inhibitor (FK506) against calcineurin, which is a serine/threonine specific phosphatase enhanced the resistance of ATBF1 against the digestion by calpain-1. Taken together, these results demonstrate that these phosphorylation sites on ATBF1 function as a defensive shield to calpain-1.

Highlights

► Adult mouse derived ATBF1 is highly sensitive to calpain-1. ► Embryonic mouse derived ATBF1 is resistant to calpain-1. ► ATBF1 is hyperphosphorylated in embryonic brain, but not in adult mouse brain. ► Dephosphorylation of ATBF1 with phosphatase increases the sensitivity to calpain-1. ► FK506 enhanced the resistance of ATBF1 to be digested by calpain-1.

Introduction

AT motif binding factor 1 (ATBF1, also called ZFHX3) is a 404-kDa transcription factor that contains four homeodomains and 23 zinc finger motifs [1]. A small alternatively-spliced product from the ATBF1 gene was first identified as one of the DNA binding factors interacting with an AT-rich element located upstream of alpha-fetoprotein (AFP) promoter to suppress gene transcription [2]. Because of its ability to suppress AFP gene transcription in hepatic cells, ATBF1 is a factor associated with differentiation of the hepatocyte. We observed expression of ATBF1 in the brains of developing embryos, but its expression is dramatically reduced in adult brain [3], [4]. Therefore we suggested that ATBF1 is specifically important for embryonic brain, but that it might not play an essential role in adult brains [4].

ATBF1 is expressed in a variety of adult organs, and abnormalities of ATBF1 have been linked to many human chronic diseases. ATBF1 may have another role in adult organs to maintain a healthy body. In fact, ATBF1 plays an essential role as an oncosuppressor in adult tissues. The ATBF1 gene, which has been assigned to chromosome 16q22.3-23.1, was identified as a plausible candidate tumor suppressor for prostatic cancer [5]. Accordingly, abnormalities of ATBF1 are associated with various cancers, including gastric cancer [6], [7], [8], hepatocellular carcinoma [9], breast cancer [10], [11], [12], and neuroblastoma [13]. Besides cancers, abnormalities of ATBF1 induce susceptibility to Kawasaki disease [14], atrial fibrillation and ischemic stroke [15], [16], schizophrenia [17], and ataxia telangiectasia [18]. Because ATBF1 regulates more than 200 genes responsible for repairing tissues and organizing protective responses to oxidative stress, it should be involved in a variety of human chronic diseases [18].

This report presents results on studies of the stability of ATBF1 regulated by phosphorylations.

Section snippets

Animals

Fourteen-day’s pregnant ICR mice were used to extract the adult mouse brain proteins and embryonic mouse brain proteins from the mother and its babies, respectively. All experiments were performed in accordance with the Guidelines for Animal Experiments of the Animal Experimentation Committee of the National Center for Geriatrics and Gerontology. Mouse brains were rapidly taken out from the skull on ice. Mouse brain tissues were washed with ice-cold PBS washing buffer [137 mM NaCl, 2.7 mM KCl, 4.3

Serine protease inhibitors block the degradation of ATBF1

To screen for the enzymes responsible for the degradation of ATBF1, a set of protease inhibitors, including bestatin, E-64, leupeptin, pepstatin, phosphoramidon, Pefabloc SC and EDTA were applied to lysates from adult mouse brain in vitro. Among the protease inhibitors, E-64, leupeptin, and EDTA specifically inhibited the degradation of ATBF1 (Fig. 1). E-64 irreversibly inhibits a wide range of cysteine peptidases including papain, cathepsin B, cathepsin L, staphopain and calpains [19].

Discussion

This result suggested that the cleavage of ATBF1 was strongly associated with the elevation of calcium ions. Although calpains were good candidates for the digestion of ATBF1, we could not determine whether ATBF1 was really a target of calpain-1 simply by searching its amino acid sequence. There is no specific amino acid sequence uniquely recognized by calpain-1. Amongst protein substrates, tertiary structure elements rather than amino acid sequence are likely to be responsible for directing

Acknowledgments

This study was supported by a Grant-in-Aid for Scientific Research [C-21590389] (T.N) [C-23590692] (H.M.), [C-23592343] (M.K.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; a Grant for Project Research from the High-Technology Center of Kanazawa Medical University [H2011-10] (T.N); a Grant-in-Aid from The New Energy and Industrial Technology Development Organization of Japan (Y.M.); and a Grant-in-Aid from the Japan Science and Technology Agency (Y.M.).

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    Sheng Zhang, Tae-Sun Kim, Yu Dong contributed equally.

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