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 p21Cip1/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 p21Cip1/Waf1 induction in PMA-induced proapoptotic cells. We demonstrated that in PMA-induced adherent cells, upregulation of p21Cip1/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 p21Cip1/Waf1 promoter during PMA induction. Finally, we demonstrated that inhibition of ROCK restores nuclear distribution of phospho-ERK and activation of p21Cip1/Waf1 expression. Based on these findings, we propose that a ROCK-mediated signal is involved in interfering with the process of ERK-mediated p21Cip1/Waf1 induction in PMA-induced proapoptotic TF-1 and D2 cells

Subcellular Localization Determines the Protective Effects of Activated ERK2 against Distinct Apoptogenic Stimuli in Myeloid Leukemia Cells

“…As such the protective effect against growth factor depletion is carried out by cytoplasmic ERK, whereas the anti-apoptotic response against STI571 takes place in the nucleus. In line with our data, in D2 myeloid cells phosphorylated ERK accumulates in the cytoplasm of phorbol ester-induced pro-apoptotic cells (63), and recent results indicate that PEA-15, a protein that retains activated ERK in the cytoplasm, can prevent tumor necrosis factor ␣-induced apoptosis in astrocytes (64). On the other hand, we show that constitutive ERK activation, irrespective of its localization, is insufficient to prevent apoptosis induced by stimuli such as UV light, probably as a consequence of the apoptogenic response against this type of radiation being primarily unleashed by processes that occur downstream from ERKs.…”

Activation of Rho-associated coiled-coil protein kinase 1 (ROCK-1) by caspase-3 cleavage plays an essential role in cardiac myocyte apoptosis

“…Rho kinase's proapoptotic role may be mediated via regulation of actin cytoskeletal rearrangement, which in turn induced the activation of the caspase cascade via the assembly of the deathinducing signaling (28,29,33,34). In addition, we demonstrated that constitutively active ROCK-1 generated by cleavage was sufficient to activate caspase-3 and lead to myocyte apoptosis, as outlined in a schematic model (Fig.…”

Rho kinase in the regulation of cell death and survival

“…ROCK-mediated MLC phosphorylation acts as an upstream event required for membrane contraction and the subsequent caspase 8, 10 and 3 activation during phorbol-12-myristate-13-acetate (PMA) stimulation in erythroblastic TF-1 cells [67]. In addition to MLC phosphorylation, ROCK activation mediates other molecular events in PMA-treated apoptotic cells such as prevention of nuclear distribution of phospho-ERK, inhibition of induction of the cell cycle inhibitor p21 (Cip1/Waf1) [68], and cytosolic translocation of heterogeneous nuclear ribonucleoprotein C1 and C2, which are two nuclear restricted pre-mRNA binding proteins [72]. In primary thymocytes, apoptosis induced by activation of the receptor for thromboxane A(2), which is coupled to Gα(12/13) subunits, is inhibited by deletion of Lsc RhoGEF (an activator of GTPases of the Rho family), associated with inactivation of Rho/ROCK and increased Akt phosphorylation [47].…”