Several acidic amino acids in the N-domain of insulin-like growth factor-binding protein-5 are important for its transactivation activity.
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.
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109, USA.
Yang
Y
Zhao
Ping
P
Yin
Leon A
LA
Bach
Cunming
C
Duan
D000595:Amino Acid Sequence; D002460:Cell Line; D006657:Histones / Q000737:chemistry; D006801:Humans; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000737:chemistry; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000737:chemistry; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D006657:Histones; D018971:Insulin-Like Growth Factor Binding Protein 2; D018972:Insulin-Like Growth Factor Binding Protein 3; D018975:Insulin-Like Growth Factor Binding Protein 5