Inhibition of JNK2 Disrupts Anaphase and Produces Aneuploidy in Mammalian Cells

Abstract

The JNK family members JNK1 and JNK2 regulate tumor growth and are essential for transformation by oncogenes such as constitutively activated Ras. The mechanisms downstream of JNK that regulate cell cycle progression and transformation are unclear. Here we show that inhibition of JNK2, but not JNK1, with either a dominant-negative mutant, a pharmacological inhibitor, or RNA interference caused an accumulation of mammalian cells with 4N DNA content. When observed by immunofluorescence, these cells progressed to metaphase without apparent defects in spindle formation or chromosome alignment to the metaphase plate, suggesting that the 4N accumulation is a result of postmetaphase defects. Consistent with this prediction, when JNK activity was suppressed, we observed defects in central spindle formation and chromosome segregation during anaphase. In contrast, cyclin-dependent kinase 1 activity, cyclin B1 protein, and Polo-like kinase 1 protein turnover remained intact when JNK was inhibited. In addition, continued inhibition of JNK activity did not block reentry into subsequent cell cycles but instead resulted in polyploidy. This evidence suggests that JNK2 functions in maintaining the genomic stability of mammalian cells by signaling that is independent of cyclin-dependent kinase 1/cyclin B1 down-regulation. JNK11 and JNK2 are members of the mitogen-activated protein kinase family, which also includes the prototypical family members extracellular signal-regulated kinase and p38. Mitogen-activated protein kinases are components of signal transduction pathways that connect extracellular stimuli to intracellular responses such as modulation of cell viability, cell cycle regulation, and gene expression (1-3). JNK1 and JNK2 are ubiquitously expressed and are generally considered to share redundant functions in apoptosis and transformation and differential functions in T cell differentiation (3-6). However, recent studies have shown that the stress-induced proapoptotic function previously attributed to both isoforms is actually a function specific to JNK1 (7,8). The function of JNK2, therefore, has become less clear. This suggests that JNK functions in transformation may also be isoform-specific and should be addressed in a way that differentiates between the contributions of JNK1 and JNK2.Ras-activating mutations have been identified in close to 30% of human cancers (9). Transformation by Ras requires JNK activity, which has made JNK an attractive target for cancer therapy (10 -12). Targeting JNK for cancer therapy is also supported by studies showing that JNK activity is elevated in human tumors and that loss of JNK function inhibits tumor growth in mice (13-16). The transforming mechanism downstream of JNK is not entirely clear, but it may work through the phosphorylation of c-Jun, which in turn regulates transcription of cell cycle regulators. In addition to this pathway, JNK may have a more direct function in cell cycle regulation. We have shown that JNK localizes to centrosomes and is active in this compartment from...

Kinesin-1 Regulates Microtubule Dynamics via a c-Jun N-terminal Kinase-dependent Mechanism

“…Besides the clear effects we demonstrated for the kinesin-1/JNK pathway in interphase cells, its role in mitosis would also be worth studying. Indeed, although kinesin-1 knockdown was not found to produce an obvious defective mitotic phenotype (39,40), JNK basal activity is required to allow mitotic progression and correct anaphase (41,42).…”

Regulation of MAP Kinases by the VHR Dual-Specific Phosphatase – Implications for Cell Growth and Differentiation

“…75 In several cellular models reduction of JNK1 and JNK2 levels or activity results in S phase accumulation. [76][77][78][79] The role of JNK in the G 2 phase of the cell-cycle is documented by RNA interference and the use of the specific JNK inhibitor SP600125. In both cases, cell treatment induces an accumulation of cells with 4N DNA content.…”

“…Depending on the particular cell type, cell-cycle arrest is accompanied by increasing DNA accumulation or by cell death. 16,76,77 This mechanism may be responsible for the anti-cancer activity of several natural compounds with anti-JNK activity. 80,81 Interestingly, mouse embryonic fibroblasts derived from JNK1 -/-and JNK2 -/-mice revealed that these two kinases have somewhat different roles in cell cycle regulation: JNK2 -/-cells exhibit a shorter G 1 cell cycle phase, i.e., they enter S phase earlier than wild-type counterparts, while JNK1 -/-cells show the inverse phenotype, a longer G 1 phase than wild-type cells.…”

JNK2: A Negative Regulator of Cellular Proliferation

“…In addition to increased proliferation due to lack of JNK2, a recent report also suggested that specific inhibition of JNK2 resulted in aneuploidy in human cells. 25 This was proposed to be due to defects in chromosome segregation and central spindle formation during anaphase, which eventually resulted in polyploidy. Together, the emerging trend points to increased proliferation in multiple cell types in the absence of JNK2 (Table 1).…”