Inhibition of JNK2 Disrupts Anaphase and Produces Aneuploidy in Mammalian Cells

Rebecca A Maccorkle,Tse-Hua Tan

doi:10.1074/jbc.m405481200

Rebecca A Maccorkle, Tse-Hua Tan

Open Access

https://doi.org/10.1074/jbc.m405481200

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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.

Highlights

JNK1 1 and JNK2 are members of the mitogen-activated protein kinase family, which includes the prototypical family members extracellular signal-regulated kinase and p38
We showed that while JNK activity was dispensable for chromosome condensation, chromosome alignment to the metaphase plate, and spindle formation, loss of JNK function interfered with chromosome segregation and central spindle formation in mammalian cell lines
In three different cell lines (HeLa, Calu-1, and Chinese hamster ovary (CHO)) the loss of JNK activity led to an accumulation of polyploid cells, indicating that there is a breakdown in genomic segregation and either a direct or an indirect block in cytokinesis

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Derivation of Calu-1 7-5, 8-5, and 8-30 clones has been described previously [20]. Synchronized cells were gently washed with fresh media, returned to 37 °C, and fixed at 15-min intervals. The JNK inhibitor SP600125 (Alexis Biochemicals, San Diego, CA) or Me2SO carrier alone was added to cultures 30 min prior to release from nocodazole treatment and to the fresh media following mitotic release as indicated. Coverslips were fixed in ice-cold methanol for 3 min followed by three 5-min washes in PEM buffer (80 mM K-PIPES (pH 7.6), 5 mM EGTA, 2 mM MgCl2) at room temperature. Flow Cytometry—Cells were fixed in ice-cold 70% ethanol and incubated with 200 ␮g/ml RNase (Sigma) in phosphate-buffered saline for 30 min at 37 °C. Coverslips were transferred to fresh preheated media containing inhibitors or carrier alone for 5–15 min at 37 °C in 5% CO2 and gently fixed in ice-cold methanol for 3 min. Sample proteins were separated by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography

RESULTS

Percent S phase

DISCUSSION