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

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.The insulin-like growth factor (IGF) 2 system, consisting of two ligands (IGF-I and -II), two receptors (the IGF-I receptor and IGF-II receptor), and six high affinity IGF-binding proteins (IGFBPs), converges on a conserved signaling pathway that plays fundamental roles in vertebrate development and physiology and is implicated in several human diseases (1). The bioavailability and bioactivity of IGFs are regulated by their interactions with various members of the IGFBP family. IGFBPs all have a highly cysteine-rich N-terminal (N)-domain, a cysteine-rich C-terminal (C)-domain, and a middle linker (L)-domain with no cysteine residues except in IGFBP-4. The N-and C-domains are highly conserved within the IGFBP family, whereas the L-domain varies both within the family and across species.Besides binding to IGF and modulating its actions, IGFBP-5 has been reported to regulate cell proliferation (2, 3), migration (4 -6), and apoptosis/survival (7-11) independent of IGF. Although the ligand-dependent actions of IGFBP-5 are attributed to its interactions with the IGF ligand and other proteins (1), the mechanistic basis of the ligand-independent actions of IGFBP-5 is not yet understood.Andress et al. (12) used IGFBP-5 affinity chromatography to purify a 420-kDa membrane protein from human osteoblast cells and proposed that it was an IGFBP-5 receptor. The same study reported that IGFBP-5 ...

40 YEARS OF IGF1: IGF-binding proteins

“…Within the nucleus, IGFBP-5 had transactivation activity via 492 its N-terminal domain (Zhao, et al 2006) and it bound transcription factors such as FHL2 (Amaar, 493 et al 2002) and the nucleolar protein nucleolin (Su, et al 2015). Nuclear IGFBP-5 bound to the 494 vitamin D receptor and attenuated vitamin D-induced expression of bone differentiation markers 495 (Schedlich, et al 2007).…”

IGF binding protein 3 exerts its ligand-independent action by antagonizing BMP in zebrafish embryos

“…Studies from a number of labs have shown that IGFBP3 has a functional NLS in its C-domain and is capable of nuclear translocation in cultured human tumor cells, and it can interact with retinoid-X receptor-a and other nuclear proteins in the nucleus (Liu et al, 2000;Oufattole et al, 2006). In addition, the N-domain of mammalian IGFBP3 has been shown to possess intrinsic transactivation activity (Zhao et al, 2006). Despite these findings, the in vivo significance of the nuclear localization and transactivation activity of IGFBP3 has not been explored.…”

“…Mammalian IGFBP3 has a functional NLS and can be found in the nucleus of cultured tumor cells Jogie-Brahim et al, 2009), but the functional significance of this NLS in mediating the ligand-independent actions of IGFBP3 is also unclear and whether the NLS of IGFBP3 has a role in vivo is unknown. Likewise, the mammalian IGFBP3 N-domain contains a functional transactivation domain (Zhao et al, 2006). It is not clear whether this activity is conserved or whether it has any biological significance in vivo.…”

“…1D). We next compared the sequence of the zebrafish Igfbp3 N-domain with that of human IGFBP5 N-domain, whose transactivation domain (TA) has been extensively characterized (Xu et al, 2004;Zhao et al, 2006). Among the eight residues in the human IGFBP5 N-domain that are known to be crucial for its transactivation ability (Zhao et al, 2006), six (E8, D11, D12, E31, P32 and E44) are conserved in zebrafish Igfbp3 (Fig.…”

“…Previous studies suggest that mammalian IGFBP3 is not only secreted but is also present in the nucleus , and its N-domain has transactivation activity (Xu et al, 2004;Zhao et al, 2006). To determine whether this activity is conserved, the zebrafish Igfbp3 N-domain (amino acids 1-81) was fused to the Gal4 DNA-binding domain (Gal4DBD), and the activity was measured.…”