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aPKC phosphorylates NuMA-related LIN-5 to position the mitotic spindle during asymmetric division

Abstract

The position of the mitotic spindle controls the plane of cell cleavage and determines whether polarized cells divide symmetrically or asymmetrically1,2,3. In animals, an evolutionarily conserved pathway of LIN-5 (homologues: Mud and NuMA), GPR-1/2 (homologues: Pins, LGN, AGS-3) and Gα mediates spindle positioning, and acts downstream of the conserved PAR-3–PAR-6–aPKC polarity complex1,2,3,4,5,6. However, molecular interactions between polarity proteins and LIN-5–GPR–Gα remain to be identified. Here we describe a quantitative mass spectrometry approach for in vivo identification of protein kinase substrates. Applying this strategy to Caenorhabditis elegans embryos, we found that depletion of the polarity kinase PKC-3 results in markedly decreased levels of phosphorylation of a cluster of four LIN-5 serine residues. These residues are directly phosphorylated by PKC-3 in vitro. Phospho-LIN-5 co-localizes with PKC-3 at the anterior cell cortex and temporally coincides with a switch from anterior- to posterior-directed spindle movements in the one-cell embryo. LIN-5 mutations that prevent phosphorylation increase the extent of anterior-directed spindle movements, whereas phosphomimetic mutations decrease spindle migration. Our results indicate that anterior-located PKC-3 inhibits cortical microtubule pulling forces through direct phosphorylation of LIN-5. This molecular interaction between polarity and spindle-positioning proteins may be used broadly in cell cleavage plane determination.

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Figure 1: PKC-3 aPKC directs LIN-5 phosphorylation in vivo.
Figure 2: PKC-3 phosphorylates LIN-5 at the anterior cortex of the one-cell embryo.
Figure 3: PKC-3 phosphorylates LIN-5 in mitosis and requires CDK-1.
Figure 4: PKC-3-mediated phosphorylation of LIN-5 directs mitotic spindle positioning in the one-cell embryo.
Figure 5: Model illustrating how phosphorylation of LIN-5 by PKC-3 may control the position of the mitotic spindle in the one-cell embryo.

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Acknowledgements

We thank J. Krijgsveld for help in the initial phases of the project, and A. Thomas and M. Tanenbaum for critically reading the manuscript, helpful advice and support throughout the project. We thank members of the van den Heuvel and Boxem laboratories for fruitful discussions. We are grateful to C. Frokjaer-Jensen and E. Jorgensen for reagents and advice on MosSCI. M.G. was supported by a predoctoral fellowship from the Boehringer Ingelheim Fonds and foundation ‘De Drie Lichten’ in the Netherlands. This work was also supported by the Netherlands Proteomics Centre, which is part of the Netherlands Genomics Initiative.

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Contributions

M.G. and S.v.d.H. designed the project and wrote the manuscript. J.M. and A.J.R.H. helped design the quantitative mass spectrometry experiments, J.M. carried out the mass spectrometry experiments and analysed the data. M.G. carried out all other experiments. V.P. and M.B. provided help in molecular cloning and yeast two-hybrid experiments. S.W.G. guided the spindle severing experiments. All authors discussed the results and commented on the manuscript.

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Correspondence to Sander van den Heuvel.

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Galli, M., Muñoz, J., Portegijs, V. et al. aPKC phosphorylates NuMA-related LIN-5 to position the mitotic spindle during asymmetric division. Nat Cell Biol 13, 1132–1138 (2011). https://doi.org/10.1038/ncb2315

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