FIG. 8.

The binding activity of CUG-BP1 is regulated by EGFR-induced phosphorylation. (A and B) Binding of CUG-BP1 to C/EBPβ mRNA is increased by stimulation of cells with EGF and inhibited by the EGF antagonists AG1478 and OSI-774 and the MEK1/2 inhibitor U0126. MCF10A cells were serum starved for 24 h and treated with either AG1478 (A), OSI-774 (B), or U0126 (C, left part) for 30 min prior to addition of EGF. Cells were harvested at 16 h, and whole-cell protein extracts were incubated with [γ-³²P]ATP-labeled C/EBPβ RNA probes (Fig. 1C) and cross-linked with UV light. RNA-bound proteins were analyzed via electrophoresis on 8 to 16% denaturing polyacrylamide gradient gels, transferred onto nitrocellulose membranes, and visualized by autoradiography. (C, right part) CUG-BP1 interacts specifically with GCN repeats located in the 5′ region of C/EBPβ mRNA. Addition of an excess of an unlabeled RNA oligonucleotide (sORF), but not that of an RNA oligonucleotide (AU-rich), abolished the binding of CUG-BP1 to the labeled (sORF-LAP) probe, as detected by PAGE and autoradiography. (D) Phosphatase treatment of CUG-BP1 abolishes binding to C/EBPβ mRNA, as determined by UV cross-link-immunoprecipitation assay. As described above, MCF10A cells were stimulated with EGF and whole-cell extracts were treated with phosphatase. CUG-BP1 was then immunoprecipitated from both EGF and non-EGF-stimulated cells (lanes 1 and 2) and from CIP-treated, EGF-activated extracts (lanes 3 and 4). The immunoprecipitate was incubated with a [γ-³²P]ATP-labeled C/EBPβ sORF probe and analyzed via SDS-PAGE and autoradiography.