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Ribonucleic Acid Polymerase II Binding Subunit 3 (Rpb3), a Potential Nuclear Target of Insulin-Like Growth Factor Binding Protein-3

Department of Animal Sciences (M.O., S.W.-J.L., B.D.R.), Washington State University, Pullman, Washington 99164; Division of Pediatric Endocrinology (B.L., P.C.), Mattel Children’s Hospital, David Geffen School of Medicine at the University of California, Los Angeles, California 90095; and Protigen, Inc. (D.M.), Mountainview, California 94043

Address all correspondence and requests for reprints to: Buel D. Rodgers, Ph.D., Department of Animal Sciences, 124 ASLB, Washington State University, Pullman, Washington 99164-6351. E-mail: danrodgers{at}wsu.edu.

IGF-binding protein (IGFBP)-3 has intrinsic antiproliferative and proapoptotic functions that are independent of IGF binding and may involve nuclear localization. We determined that exogenous IGFBP-3 rapidly translocates to myoblast nuclei and that a 22-residue peptide containing the metal binding domain (MBD) and nuclear localization sequence (NLS) can similarly direct chimeric GFP into myoblast nuclei. Furthermore, a non-IGF-binding IGFBP-3 mutant inhibited myoblast proliferation without stimulating apoptosis. These results suggest that IGFBP-3 inhibits muscle cell growth in an IGF-independent manner that may be influenced by its rapid nuclear localization. We therefore identified IGFBP-3 interacting proteins by screening a rat L6 myoblast cDNA library using the yeast two-hybrid assay and two N-terminal deletion mutants as bait: BP3/231 (231 residues, L61 to K291) and BP3/111 (K181-K291). Proteins previously known to interact with IGFBP-3 as well as several novel proteins were identified, including RNA polymerase II binding subunit 3 (Rpb3). The domain necessary for Rpb3 binding was subsequently identified using different IGFBP-3 deletion mutants and was localized to the MBD/NLS epitope. Rpb3/IGFBP-3 binding was confirmed by coimmunoprecipitation assays with specific antisera, whereas a NLS mutant IGFBP-3 did not associate with Rpb3, suggesting that a functional NLS is required. Rpb3 facilitates recruitment of the polymerase complex to specific transcription factors and is necessary for the transactivation of many genes. Its association with IGFBP-3 provides a functional role for IGFBP-3 in the direct modulation of gene transcription.

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