Several Acidic Amino Acids in the N-domain of Insulin-like Growth Factor-binding Protein-5 Are Important for Its Transactivation Activity

Yang Zhao,Leon A Bach,Cunming Duan,Ping Yin

doi:10.1074/jbc.m506941200

Yang Zhao, Leon A Bach + Show 2 more

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https://doi.org/10.1074/jbc.m506941200

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Abstract

Insulin-like growth factor-binding protein (IGFBP)-5 is a secreted protein that binds to IGFs and modulates IGF actions. IGFBP-5 is also found in the nuclei of cultured cells and has transactivation activity. Here we report the nuclear localization of endogenous IGFBP-5 in mouse embryonic skeletal cells. Chromatin immunoprecipitation experiments indicated that IGFBP-5 interacts with the nuclear histone-DNA complex. Using a series of deletion mutants, the transactivation domain of IGFBP-5 was mapped to its N-terminal region. Intriguingly, the transactivation activity of IGFBP-5 is masked by negative regulatory elements located in the L- and C-domains. Among the other IGFBPs, the N-domains of IGFBP-2 and -3 also had strong transactivation activity, whereas those of IGFBP-1 and -6 had no activity. The IGFBP-4 N-domain had modest activity. Sequence analysis revealed several amino acids in the IGFBP-5 N-domain that are not present in IGFBP-1. The activities of mutants in which these residues were changed to the corresponding IGFBP-1 sequence were determined. Mutations that changed acidic residues to neutral residues (e.g. E8A, D11S, E12A, E30S/P31A, E43L, and E52A) or a polar to a basic residue (e.g. Q56R) significantly reduced transactivation activity. The E8A/D11S/E12A triple mutant and E52A/Q56R double mutants showed further reduced activity. The combinatory mutants had essentially no transactivation activity. Taken together, our results indicate that there are several conserved residues in the IGFBP-5 N-terminal region that are critical for transactivation and that IGFBP-2 and -3 also have strong transactivation activity in their N-domains.

Highlights

Teine-rich C-terminal (C)-domain, and a middle linker (L)-domain with no cysteine residues except in Insulin-like growth factor-binding protein (IGFBP)-4
IGFBP-5 Is Physically Associated with the Histone-DNA Complex in the Nucleus—To determine whether endogenous IGFBP-5 is present in the nucleus in an in vivo setting, immunocytochemical analysis was performed in mouse embryo costal cartilage sections using a specific IGFBP-5 polyclonal antibody
The anti-FLAG antibody pulled down histone 3 (Fig. 1C, lower panel) in the transfected cells but not in the control cells. These results suggest that IGFBP-5 is localized in the nucleus and that the nuclear IGFBP-5 is physically associated with the histone-DNA complex

Summary

Introduction

Teine-rich C-terminal (C)-domain, and a middle linker (L)-domain with no cysteine residues except in IGFBP-4. We have shown that 1) endogenous IGFBP-5 is localized in the nucleus of cultured porcine vascular smooth muscle cells; 2) several basic residues in the IGFBP-5 C-domain are necessary and sufficient for nuclear localization of the intact protein; and 3) the IGFBP-5 N-domain activates transcription independent of IGF [18]. These findings suggest that IGFBP-5 is present in the nucleus and may affect gene expression independent of the IGF ligand. In addition to IGFBP-5, the N-domains of IGFBP-3 and IGFBP-2 possess strong transactivation activity

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