Polypyrimidine Tract-Binding Protein Enhances the Internal Ribosomal Entry Site-Dependent Translation of p27^Kip1 mRNA and Modulates Transition from G₁ to S Phase

Abstract

The p27^Kip1 protein plays a critical role in the regulation of cell proliferation through the inhibition of cyclin-dependent kinase activity. Translation of p27^Kip1 is directed by an internal ribosomal entry site (IRES) in the 5′ nontranslated region of p27^Kip1 mRNA. Here, we report that polypyrimidine tract-binding protein (PTB) specifically enhances the IRES activity of p27^Kip1 mRNA through an interaction with the IRES element. We found that addition of PTB to an in vitro translation system and overexpression of PTB in 293T cells augmented the IRES activity of p27^Kip1 mRNA but that knockdown of PTB by introduction of PTB-specific small interfering RNAs (siRNAs) diminished the IRES activity of p27^Kip1 mRNA. Moreover, the G₁ phase in the cell cycle (which is maintained in part by p27^Kip1) was shortened in cells depleted of PTB by siRNA knockdown. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced differentiation in HL60 cells was used to examine PTB-induced modulation of p27^Kip1 protein synthesis during differentiation. The IRES activity of p27^Kip1 mRNA in HL60 cells was increased by TPA treatment (with a concomitant increase in PTB protein levels), but the levels of p27^Kip1 mRNA remained unchanged. Together, these data suggest that PTB modulates cell cycle and differentiation, at least in part, by enhancing the IRES activity of p27^Kip1 mRNA.

ACKNOWLEDGMENTS

We thank E. Wimmer (State University of New York at Stony Brook) and M. Bachmann (Johannes-Gutenberg Universität, Mainz, Germany) for providing the anti-PTB and anti-La monoclonal antibodies, respectively. We are grateful to Tae-Don Kim for technical advices and helpful discussions.

The present study was supported in part by grants from the NRL (M10204000018-03J0000-01410), the MMRG (M10106000056-03B4500-01010) of MOST, the KHIDI (02-PJ2-PG1-CH16-0002), the KRF (KRF-2003-005-C00011), and POSCO.