p204 Protein Overcomes the Inhibition of the Differentiation of P19 Murine Embryonal Carcinoma Cells to Beating Cardiac Myocytes by Id Proteins

Abstract We reported in the accompanying article (Ding, B., Liu, C., Huang, Y., Hickey, R. P., Yu, J., Kong, W., and Lengyel, P. (2006) J. Biol. Chem. 281, 14882-14892) that (i) the p204 protein is required for the differentiation of murine P19 embryonal carcinoma stem cells to beating cardiac myocytes, and (ii) the expression of p204 in the differentiating P19 cells is synergistically transactivated by the cardiac transcription factors Gata4, Nkx2.5, and Tbx5. Here we report that endogenous or ectopic inhibitor of differentiation (Id) proteins inhibited the differentiation of P19 cells to myocytes. This was in consequence of the binding of Id1, Id2, or Id3 protein to the Gata4 and Nkx2.5 proteins and the resulting inhibitions (i) of the binding of these transcription factors to each other and to DNA and (ii) of their synergistic transactivation of the expression of various genes, including atrial natriuretic factor and Ifi204 (encoding p204). p204 overcame this inhibition by Id proteins in consequence of (i) binding and sequestering Id proteins, (ii) accelerating their ubiquitination and degradation by proteasomes, and (iii) decreasing the level of Id proteins in the nucleus by increasing their translocation from the nucleus to the cytoplasm. Points (ii) and (iii) depended on the presence of the nuclear export signal in p204. In the course of the differentiation, Gata4, Nkx2.5, and p204 were components of a positive feedback loop. This loop arose in consequence of it that p204 overcame the inhibition of the synergistic activity of Gata4 and Nkx2.5 by the Id proteins.