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Localization of Protein Kinase NDR2 to Peroxisomes and Its Role in Ciliogenesis
Shoko Abe1, Tomoaki Nagai1, Moe Masukawa1
1From the Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578.
Abstract:
Nuclear Dbf2-related (NDR) kinases, comprising NDR1 and NDR2, are serine/threonine kinases that play crucial roles in the control of cell proliferation, apoptosis, and morphogenesis. We recently showed that NDR2, but not NDR1, is involved in primary cilium formation; however, the mechanism underlying their functional difference in ciliogenesis is unknown. To address this issue, we examined their subcellular localization. Despite their close sequence similarity, NDR2 exhibited punctate localization in the cytoplasm, whereas NDR1 was diffusely distributed within the cell. Notably, NDR2 puncta mostly co-localized with the peroxisome marker proteins, catalase and CFP-SKL (cyan fluorescent protein carrying the C-terminal typical peroxisome-targeting signal type-1 (PTS1) sequence, Ser-Lys-Leu). NDR2 contains the PTS1-like sequence, Gly-Lys-Leu, at the C-terminal end, whereas the C-terminal end of NDR1 is Ala-Lys. An NDR2 mutant lacking the C-terminal Leu, NDR2(ΔL), exhibited almost diffuse distribution in cells. Additionally, NDR2, but neither NDR1 nor NDR2(ΔL), bound to the PTS1 receptor Pex5p. Together, these findings indicate that NDR2 localizes to the peroxisome by using the C-terminal GKL sequence. Intriguingly, topology analysis of NDR2 suggests that NDR2 is exposed to the cytosolic surface of the peroxisome. The expression of wild-type NDR2, but not NDR2(ΔL), recovered the suppressive effect of NDR2 knockdown on ciliogenesis. Furthermore, knockdown of peroxisome biogenesis factor genes (PEX1 or PEX3) partially suppressed ciliogenesis. These results suggest that the peroxisomal localization of NDR2 is implicated in its function to promote primary cilium formation.
Insights
Nuclear Dbf2-related (NDR) kinase 2 (NDR2) localizes to peroxisomes via its C-terminal sequence, promoting primary cilium formation. This peroxisomal targeting is crucial for NDR2
Area of Science:
- Cell Biology
- Molecular Biology
Background:
- Nuclear Dbf2-related (NDR) kinases (NDR1 and NDR2) are serine/threonine kinases regulating cell proliferation, apoptosis, and morphogenesis.
- NDR2, but not NDR1, is implicated in primary cilium formation, yet the underlying mechanism remains unclear.
Purpose of the Study:
- To investigate the mechanism behind NDR2's specific role in ciliogenesis by examining its subcellular localization.
- To determine how NDR2's localization differs from NDR1 and its functional implications.
Main Methods:
- Comparative analysis of NDR1 and NDR2 subcellular localization using cell imaging.
- Identification of peroxisome-targeting signals (PTS1) and their role in NDR2 localization.
- Investigation of NDR2 interaction with peroxisomal receptor Pex5p.
- Functional rescue experiments involving NDR2 mutants and peroxisome biogenesis factors (PEX1, PEX3).
Main Results:
- NDR2 exhibits punctate cytoplasmic localization, co-localizing with peroxisomes, unlike the diffuse NDR1 distribution.
- NDR2 possesses a C-terminal peroxisome-targeting signal-like sequence (GKL) responsible for its peroxisomal localization.
- NDR2 binds to the peroxisomal receptor Pex5p, and its peroxisomal localization is essential for restoring ciliogenesis in NDR2-knockdown cells.
- Disruption of peroxisome biogenesis partially inhibits ciliogenesis, supporting the role of peroxisomes in this process.
Conclusions:
- NDR2 specifically localizes to the peroxisome via its C-terminal GKL sequence and interaction with Pex5p.
- Peroxisomal localization of NDR2 is critical for its function in promoting primary cilium formation.
- This study reveals a novel role for peroxisomes in regulating ciliogenesis through NDR2 localization.
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