Regulation of Upstream Binding Factor 1 Activity by Insulin-like Growth Factor I Receptor Signaling

An Wu,Xiao Tu,Marco Prisco,Renato Baserga

doi:10.1074/jbc.m406138200

Abstract

The upstream binding factor 1 (UBF1) is one of the proteins in a complex that regulates the activity of RNA polymerase I, which controls the rate of ribosomal RNA (rRNA) synthesis. We have shown previously that insulin receptor substrate-1 (IRS-1) can translocate to the nuclei and nucleoli of cells and bind UBF1. We report here that activation of the type I insulin-like growth factor receptor (IGF-IR) by IGF-I increases transcription from the ribosomal DNA (rDNA) promoter in both myeloid cells and mouse fibroblasts. The increased activity of the rDNA promoter is accompanied by increased phosphorylation of UBF1, a requirement for UBF1 activation. Phosphorylation occurs on a number of UBF1 peptides, most prominently on the highly acidic, serine-rich C terminus. In myeloid cells (but not in mouse embryo fibroblasts) IRS-1 signaling stabilizes the levels of UBF1 protein. These findings demonstrate that IGF-IR signaling can increase the activity of UBF1 and transcription from the rDNA promoter, providing one explanation for the reported effects of the IGF/IRS-1 axis on cell and body size in animals and cells in culture.

Highlights

The upstream binding factor 1 (UBF1)1 is part of the complex of proteins that regulate the activity of RNA polymerase I at the ribosomal DNA promoter [1]
Because the IGF-IR itself is involved in the control of cell and body size, it was necessary to determine the respective contributions of the receptor and insulin receptor substrate-1 (IRS-1) in regulating UBF1 activity and the ribosomal DNA (rDNA) promoter
We had to use two different cell culture systems. 32D-derived cells have the advantage that the parental cells do not express insulin receptor substrate (IRS)-1 or IRS-2 [43, 44] and can be used to distinguish between the effects of the IGF-IR and those of IRS-1 on UBF1 activity. 32D cells are myeloid cells that require IL-3 for growth and undergo apoptosis upon removal of IL-3 [45,46,47]

Summary

EXPERIMENTAL PROCEDURES

IRS-1 Mutants and Miniribosome Genes—PHPTB IRS-1 comprises only the PH and phosphotyrosine binding (PTB) domains of IRS-1, approximately the first 300 amino acids [18]. The cells were collected in radioimmune precipitation assay buffer (50 mM TrisHCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and the protease mixture from Roche) to obtain whole cell lysates, which were immunoprecipitated with an antibody to UBF, followed by Western blots. The cells were labeled for 4 h with 32Pi at a final concentration of 1 mCi/ml (ICN Biochemicals) in phosphate-free RPMI 1640 medium. The cells were labeled for 4 h with 32Pi at a final concentration of 1 mCi/ml (ICN Biochemicals, Inc.) in phosphate-free medium (Invitrogen). Aliquots of lysates (50 ␮g/sample) were resolved by 4 –15% SDS-polyacrylamide gels and transferred to nitrocellulose membranes They were probed with the antibodies and developed with the ECL system (Amersham Biosciences). The expression of T antigen with the reporter gene was monitored with a monoclonal antibody to SV40 T antigen [37]

RESULTS

Molecular weight

DISCUSSION