P204 Protein Overcomes the Inhibition of the Differentiation of P19 Murine Embryonal Carcinoma Cells to Beating Cardiac Myocytes by Id Proteins

Bo Ding,Chuan-Ju Liu,Yan Huang,Jin Yu,Weihua Kong,Peter Lengyel

doi:10.1074/jbc.m511748200

Bo Ding, Chuan-Ju Liu + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m511748200

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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.

Highlights

P204 and p202a are the best characterized members of the interferon-inducible murine p200 family proteins [1,2,3,4]. p202a modulates transcription, cell proliferation, and apoptosis primarily by binding and modulating the activity of various sequence-specific transcription factors [5,6,7,8,9]. p204, a sister protein of p202a, is growth-inhibitory [1, 10, 11]
Experiments involving the use of 204 antisense (204AS)2 RNA for decreasing the level of endogenous p204 revealed that p204 is required for the differentiation of C2C12 myoblasts to myotubes [16]. p204 enables the differentiation, at least in part, by overcoming the inhibition of the activities of MyoD, E12/E47, and other myogenic basic regionhelix-loop-helix transcription factors by the inhibitor of differentiation (Id) proteins Id1, Id2, and Id3 [17]
The fact that the level of p204 is even higher in mouse heart than in skeletal muscle [16] prompted us to explore whether the Id proteins affect the differentiation of P19 cells to cardiac-type myocytes

Summary

The abbreviations used are

␣Gata4), antiserum to the protein in question (e.g. to Gata4); ANF, atrial natriuretic factor; bHLH, basic region-helix-loop-helix; CHX, cycloheximide; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; GST, glutathione S-transferase; Id, inhibitor of differentiation; MHC, myosin heavy chain; NES, nuclear export signal; p204⌬NES, p204 lacking the NES; p204MNES, p204 with a mutated NES; GFP, green fluorescent protein; MBP, maltose-binding protein; E1, ubiquitin-activating enzyme; HA, hemagglutinin; FCS, fetal calf serum. Mechanisms of p204 Action in Cardiac Myocyte Differentiation the various mechanisms by which p204 overcomes these inhibitions are the topics of this study

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION