Expression, purification and biochemical characterization of the N-terminal regions of human TIG3 and HRASLS3 proteins

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Abstract

Tarzarotene-induced gene 3 (TIG3) and HRAS-like suppressor (HRASLS3) are members of the HREV107 family of class II tumor suppressors, which are down-regulated in various cancer cells. TIG3 and HRASLS3 also exhibit phospholipase activities. Both proteins share a common domain architecture with hydrophilic N-terminal and hydrophobic C-terminal regions. The hydrophobic C-terminal region is important for tumor suppression. However, the function of the hydrophilic N-terminal region remains elusive. To facilitate biochemical characterizations of TIG3 and HRASLS3, we expressed and purified the N-terminal regions of TIG3 and HRASLS3, designated TIG3 (1–134) and HRASLS3 (1–133), in a bacterial system. We found that the N-terminal regions of TIG3 and HRASLS3 have calcium-independent phospholipase A2 activities. Limited proteolysis revealed that TIG3 (1–132) is a structural domain in the N-terminal region of TIG3. Our data suggest that the hydrophobic C-terminal regions might be crucial for cellular localization, while the hydrophilic N-terminal regions are sufficient for the enzymatic activity of both TIG3 and HRASLS3.

Introduction

Tarzarotene-induced gene 3 (TIG3)1, also known as retinoid-inducible gene 1 (RIG1) and retinoic-acid receptor responder 3 (RARRES3), is a member of HREV107 family proteins, class II tumor suppressors, in human. TIG3 induces cellular apoptosis, differentiation and growth suppression [1], [2], [3], [4], [5], [6]. Low-level or decreased expression of TIG3 has been noted in multiple carcinomas and psoriasis [7], [8], [9], [10]. Examples of carcinomas associated with the low-level expression of TIG3 include ovarian carcinoma, hepatocellular carcinoma, cholanggiocarcinoma, skin cancer, and poorly differentiated colorectal adenocarcinoma. TIG3 inhibits the Ras signaling pathway by suppressing Ras activation in HtTA cervical cancer cells [2]. Interferon-γ-induced TIG3 down-regulates HER2 by repression of promoter activity, resulting in down-regulation of the PI3K/Akt/mTOR/VEGF pathway in ovarian cancer cells [11].

Human HRAS-like suppressor 3 (HRASLS3), another HREV107 family member, also suppresses growth in HRAS-transformed cells and exhibits reduced expression levels in various tumor cells such as ovarian carcinoma, testicular germ cell tumors and nasopharyngeal carcinoma [12], [13], [14], [15]. HRASLS3 binds to the regulatory subunit A of protein phosphatase 2A (PR65α) and sequesters the catalytic subunit (PR36) from it, inhibiting its catalytic activity [16].

TIG3 and HRASLS3 share a common architecture featuring four domains: proline-rich motif, H-box, NC domain in the N-terminal region (1–133) and membrane-anchoring domain at the C-terminal region [17], [18], [19]. The exact function of the N-terminal region remains unclear while the C-terminal region is reported to be essential to localization at the Golgi, regulating the tumor suppression functioning of the HREV107 family proteins [2], [6].

Recent studies have suggested that TIG3 and HRASLS3 manifest phospholipase A (PLA) activity, implicating that the phospholipid metabolism might be closely related to their tumor suppressor function [20], [21], [22]. TIG3 and HRASLS3 show a relatively high (∼51%) sequence identity, which reflects the structural and functional similarity between them. Despite of their functional significance in carcinogenesis, no detailed three-dimensional structures for the HREV107 family proteins have been elucidated yet. To facilitate the biochemical characterizations of both TIG3 and HRASLS3, we expressed several constructs of human TIG3 and HRASLS3 in E. coli and purified the N-terminal regions of the TIG3 and HRASLS3. Limited proteolysis revealed a structural domain in the N-terminal region of both TI3 and HRASLS3. We were able to determine that the PLA activity resides in the N-terminal regions of both proteins.

Section snippets

Cloning

Constructs covering the N-terminal regions of TIG3 and HRASLS3 were prepared by subcloning the corresponding inserts into pET28b(+) (Novagen) at the NdeI/XhoI restriction enzyme sites. The resulting constructs have an extra, 21-residue (MGSSHHHHHH SSGLVPRGSH) protein sequence including a six-residue poly histidine tag on the amino-terminus of the recombinant proteins. Other constructs of HRASLS3 were prepared using the pHis vector (modified from Novagen’s pET32a) with NdeI/XhoI restriction

Expression and purification of recombinant human TIG3 and HRASLS3

We tested many TIG3 and HRASLS3 constructs (Table 1, Fig. 1A) for soluble expression in the bacterial system and chose the largest soluble construct for purification. Full-length TIG3 was expressed as insoluble, which might be due to the presence of the hydrophobic membrane-anchoring domain. Constructs lacking the N-terminal 10 residues (11–110, 11–134 for TIG3 and 11–133 for HRASLS3) were also insoluble, suggesting that the N-terminal 10 residues in both TIG3 and HRASLS3 might be important for

Discussion

TIG3 and HRASLS3 of the HREV107 family are down-regulated in various cancer cells and their cellular functions may be important for cancer development. Recently, it has been reported that TIG3 and HRASLS3 possess PLA activity [20], [21], [22], raising the possibility that the lipid metabolism by these proteins may be related to their tumor suppression function. Additional support for the possible link between the lipid metabolism and the tumor suppression comes from the finding that the

Acknowledgments

This work was supported by National Cancer Center in Korea research grant (Grant No. 0810183). H.K.C. and Y.H.J. were supported by the High Field NMR research program of Korea Basic Science Institute.

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