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Death-associated protein kinase 2 mediates nocodazole-induced apoptosis through interaction with tubulin
Kinuka Isshiki1, Taishi Hirase1, Shinya Matsuda1
1Department of Biological Science and Technology, Tokushima University Graduate School, 2-1 Minamijosanjima, Tokushima 770-8506, Japan.
Abstract:
Death-associated protein kinase 2 (DAPK2) is a positive regulator of apoptosis. Although we recently reported that 14-3-3 proteins inhibit DAPK2 activity and its subsequent apoptotic effects via binding to DAPK2, the molecular mechanisms underlying the DAPK2-mediated apoptotic pathway remain unclear. Therefore, we attempted to further identify DAPK2-interacting proteins using pull-down assays and mass spectrometry. The microtubule β-tubulin was identified as a novel DAPK2-binding protein in HeLa cells. Pull-down assays revealed that DAPK2 interacted with the α/β-tubulin heterodimer, and that the C-terminal region of DAPK2, which differs from that of other DAPK family members, was sufficient for the association with β-tubulin. Although the microtubule-depolymerizing agent nocodazole induced apoptosis in HeLa cells, the level of apoptosis was significantly decreased in the DAPK2 knockdown cells. Furthermore, we found that treatment with nocodazole resulted in an increased binding of DAPK2 to β-tubulin. These findings indicate that DAPK2 mediates nocodazole-induced apoptosis via binding to tubulin.
Insights
Death-associated protein kinase 2 (DAPK2) binds to β-tubulin, a key component of microtubules. This interaction is crucial for mediating apoptosis induced by microtubule-disrupting agents like nocodazole.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Death-associated protein kinase 2 (DAPK2) is a known regulator of apoptosis.
- The precise molecular mechanisms of DAPK2-mediated apoptosis are not fully understood.
- Previous studies indicated 14-3-3 proteins inhibit DAPK2 activity.
Purpose of the Study:
- To identify novel DAPK2-interacting proteins.
- To elucidate the molecular mechanisms of DAPK2-mediated apoptosis.
- To investigate the role of DAPK2 in response to microtubule disruption.
Main Methods:
- Pull-down assays were employed to identify DAPK2-binding proteins.
- Mass spectrometry was utilized for protein identification.
- DAPK2 knockdown and apoptosis assays were performed.
Main Results:
- Microtubule β-tubulin was identified as a novel DAPK2-binding protein.
- DAPK2 directly interacts with the α/β-tubulin heterodimer via its C-terminal region.
- Nocodazole-induced apoptosis was significantly reduced in DAPK2 knockdown cells.
- Nocodazole treatment enhanced the binding between DAPK2 and β-tubulin.
Conclusions:
- DAPK2 interacts with tubulin, a novel finding in apoptosis regulation.
- DAPK2 plays a significant role in mediating apoptosis induced by microtubule-targeting agents.
- The interaction between DAPK2 and tubulin is a key event in the apoptotic pathway triggered by nocodazole.
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