Cytosolic Retention of Phosphorylated Extracellular Signal-Regulated Kinase and a Rho-Associated Kinase-Mediated Signal Impair Expression of p21^Cip1/Waf1 in Phorbol 12-Myristate-13- Acetate-Induced Apoptotic Cells

Abstract

In response to treatment with phorbol-12-myristate-13-acetate (PMA), the half-population of erythromyeloblast D2 cells, a cytokine-independent variant of TF-1 cells, displayed adhesion and differentiated into a monocyte/macrophage-like morphology, while the other half-population remained in suspension and underwent apoptosis. Expression of the cell cycle inhibitor p21^Cip1/Waf1 was induced after PMA treatment in the adherent cells but not in the proapoptotic cells. We investigated the mechanism responsible for the impairment of p21^Cip1/Waf1 induction in PMA-induced proapoptotic cells. We demonstrated that in PMA-induced adherent cells, upregulation of p21^Cip1/Waf1 requires the activation and nuclear translocation of phosphorylated extracellular signal-regulated kinase (phospho-ERK). Although ERK was phosphorylated to comparable levels in PMA-induced proapoptotic and adherent cells, nuclear distribution of phospho-ERK was seen only in the adherent, not in the proapoptotic cells. We also found that only PMA-induced proapoptotic cells contained the phosphorylated form of myosin light chain, which is dependent on Rho-associated kinase (ROCK) activation, and that expression of a dominant-active form of ROCK suppressed activation of the p21^Cip1/Waf1 promoter during PMA induction. Finally, we demonstrated that inhibition of ROCK restores nuclear distribution of phospho-ERK and activation of p21^Cip1/Waf1 expression. Based on these findings, we propose that a ROCK-mediated signal is involved in interfering with the process of ERK-mediated p21^Cip1/Waf1 induction in PMA-induced proapoptotic TF-1 and D2 cells.

We are grateful to H.-F. Yang-Yen (Academia Sinica, Taipei, Taiwan) for providing D2 and TF-1 cells, T.-S. Jou (National Taiwan University, Taipei, Taiwan) for expression plasmids RhoAV14, and ROCK(CAT), which were originally obtained from K. Kaibuchi (Nara Institute of Science and Technology, Ikoma, Japan), B. Vogelstein (Johns Hopkins University School of Medicine, Baltimore, Md.) for pWWP-Luc, J. Pouyssegur (Université de Nice, Nice, France) for expression vectors MKP3(wt) and MKP3(C/S), and J. M. Staddon (University College London, London, United Kingdom) for antibody against ppMLC. We also thank C.-L. Chien (National Taiwan University, Taipei, Taiwan) for guidance on confocal microscopy, which was supported by grant 89-B-FA01-1-4, J.-Y. Chen and W.-N. Wen for critical reading of the manuscript, and A. Hall for the suggestion on MLC phosphorylation.

This research is supported by grant NSC90-2320-B-002-171 from the National Science Council, Taiwan, Republic of China.