Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

SADB phosphorylation of γ-tubulin regulates centrosome duplication

Nature Cell Biology volume 11, pages 1081–1092 (2009)Cite this article

Abstract

Symmetrical cell division requires duplication of DNA and protein content to generate two daughter cells. Centrosomes also duplicate during cell division, but the mechanism controlling this process is incompletely understood. We describe an alternative splice form of SadB encoding a short SADB Ser/Thr kinase whose activity fluctuates during the cell cycle, localizes to centrosomes, and controls centrosome duplication. Reduction of endogenous SADB levels diminished centrosome numbers, whereas enhanced SADB expression induced centrosome amplification. SADB exerted this action through phosphorylation of γ-tubulin on Ser 131, as expression of a phosphomimetic Ser 131-to-Asp γ-tubulin mutant alone increased centrosome numbers, whereas non-phosphorylatable Ala 131-γ-tubulin impaired centrosome duplication. We propose that SADB kinase activity controls centrosome homeostasis by regulating phosphorylation of γ-tubulin.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: SadB exhibits alternative splicing.
Figure 2: SADB kinase is a centrosomal protein that regulates centrosome numbers.
Figure 3: SADB knockdown inhibits centrosome duplication.
Figure 4: SADB-deficient cells express an SadBS mRNA splice variant.
Figure 5: SADB kinases phosphorylate γ-tubulin on Ser 131.
Figure 6: SADB kinases modulate cellular levels of pSer 131γ-tubulin.
Figure 7: Increased centrosome numbers in Asp 131-γ-tubulin-expressing cells.
Figure 8: SADB kinases affect CP110 localization and microtubule nucleation.

Similar content being viewed by others

References

  1. Hinchcliffe, E. H. & Sluder, G. “It takes two to tango”: understanding how centrosome duplication is regulated throughout the cell cycle. Genes Dev. 15, 1167–1181 (2001).

    Article  CAS  Google Scholar 

  2. Nigg, E. A. Centrosome aberrations: cause or consequence of cancer progression? Nature Rev. Cancer 2, 815–825 (2002).

    Article  CAS  Google Scholar 

  3. Hemerly, A. S., Prasanth, S. G., Siddiqui, K. & Stillman, B. Orc1 controls centriole and centrosome copy number in human cells. Science 323, 789–793 (2009).

    Article  CAS  Google Scholar 

  4. Meraldi, P., Lukas, J., Fry, A. M., Bartek, J. & Nigg, E. A. Centrosome duplication in mammalian somatic cells requires E2F and Cdk2-cyclin A. Nature Cell Biol. 1, 88–93 (1999).

    Article  CAS  Google Scholar 

  5. Hergovich, A., Lamla, S., Nigg, E. A. & Hemmings, B. A. Centrosome-associated NDRK regulates centrosome duplication. Mol. Cell 25, 625–634 (2007).

    Article  CAS  Google Scholar 

  6. Kleylein-Sohn, J., Westendorf, J., Le Clech, M., Habedanck, R., Stierhof, Y. D. & Nigg, E. A. Plk4-induced centriole biogenesis in human cells. Dev. Cell 13, 190–202 (2007).

    Article  CAS  Google Scholar 

  7. Kim, H. K. et al. De novo formation of basal bodies in Naegleria gruberi: regulation by phosphorylation. J. Cell Biol. 169, 719–724 (2005).

    Article  CAS  Google Scholar 

  8. Muller, H., Fogeron, M. L., Lehmann, V., Lehrach, H. & Lange, B. M. A centrosome-independent role for γ-TuRC proteins in the spindle assembly checkpoint. Science 314, 654–657 (2006).

    Article  Google Scholar 

  9. Ruiz, F., Beisson, J., Rossier, J. & Dupuis-Williams, P. Basal body duplication in Paramecium requires γ-Tubulin. Curr. Biol. 9, 43–46 (1999).

    Article  CAS  Google Scholar 

  10. Schiebel, E. γ-Tubulin complexes: binding to the centrosome, regulation and microtubule nucleation. Curr. Opin. Cell Biol. 12, 113–118 (2000).

    Article  CAS  Google Scholar 

  11. Lew, D. J. Cell-cycle checkpoints that ensure coordination between nuclear and cytoplasmic events in S. cerevisiae. Curr. Opin. Genet. Dev. 10, 47–53 (2000).

    Article  CAS  Google Scholar 

  12. Lu, R., Niida, H. & Nakanishi, M. hSAD1 kinase is involved in UV-induced DNA damage checkpoint function. J. Biol. Chem. 279, 31164–31170 (2004).

    Article  CAS  Google Scholar 

  13. Kishi, M., Pan, Y. A., Crump, J. G. & Sanes, J. R. Mammalian SAD kinases are required for neuronal polarization. Science 307, 929–932 (2005).

    Article  CAS  Google Scholar 

  14. Hanks, S. K., Quinn, A. M. & Hunter, T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241, 42–52 (1988).

    Article  CAS  Google Scholar 

  15. Habedanck, R., Stierhof, Y. D., Wilkinson, C. J. & Nigg, E. A. The Polo kinase Plk4 functions in centriole duplication. Nature Cell Biol. 7, 1140–1146 (2005).

    Article  CAS  Google Scholar 

  16. Alvarado-Kristensson, M. et al. p38-MAPK signals survival by phosphorylation of caspase-8 and -3 in human neutrophils. J. Exp. Med. 199, 449–458 (2004).

    Article  CAS  Google Scholar 

  17. Alvarez, B., Martinez-A., C., Burgering, B. M. & Carrera, A. C. Forkhead transcription factors contribute to execution of the mitotic programme in mammals. Nature 413, 744–747 (2001).

    Article  CAS  Google Scholar 

  18. Martinez-Gac, L., Marques, M., Garcia, Z., Campanero, M. R. & Carrera, A. C. Control of cyclin G2 mRNA expression by forkhead TF: novel mechanism for cell cycle control by PI3K and forkhead. Mol. Cell. Biol. 24, 2181–2189 (2004).

    Article  CAS  Google Scholar 

  19. Aldaz, H., Rice, L. M., Stearns, T. & Agard, D. A. Insights into microtubule nucleation from the crystal structure of human γ-tubulin. Nature 435, 523–527 (2005).

    Article  CAS  Google Scholar 

  20. Hesse, J., Thierauf, M. & Ponstingl, H. Tubulin sequence region β 155–174 is involved in binding exchangeable guanosine triphosphate. J. Biol. Chem. 262, 15472–15475 (1987).

    CAS  PubMed  Google Scholar 

  21. Guarguaglini, G. et al. The forkhead-associated domain protein Cep170 interacts with Polo-like kinase 1 and serves as a marker for mature centrioles. Mol. Biol. Cell 16, 1095–1107 (2004).

    Article  Google Scholar 

  22. Chen, Z. et al. CP110, a cell cycle-dependent CDK substrate, regulates centrosome duplication in human cells. Dev. Cell 3, 339–350 (2002).

    Article  CAS  Google Scholar 

  23. Delgehyr, N., Sillibourne, J. & Bornens, M. Microtubule nucleation and anchoring at the centrosome are independent processes linked by ninein function. J. Cell Sci. 118, 1565–1575 (2005).

    Article  CAS  Google Scholar 

  24. Wu, J., Cho, H. P., Rhee, D. B., Johnson, D. K., Dunlap, J., Liu, Y. & Wang, Y. Cdc14B depletion leads to centriole amplification, and its overexpression prevents unscheduled centriole duplication. J. Cell Biol. 181, 475–483 (2008).

    Article  CAS  Google Scholar 

  25. Boutros, R., Lobjois, V. & Ducommun, B. CDC25B involvement in the centrosome duplication cycle and in microtubule nucleation. Cancer Res. 67, 11557–11564 (2007).

    Article  CAS  Google Scholar 

  26. Vogel, J. et al. Phosphorylation of γ-tubulin regulates microtubule organization in budding yeast. Dev. Cell 1, 621–631 (2001).

    Article  CAS  Google Scholar 

  27. Zhu, H. et al. Analysis of yeast protein kinases using protein chips. Nature Genet. 26, 283–289 (2000).

    Article  CAS  Google Scholar 

  28. Barbosa, V., Yamamoto, R. R., Henderson, D. S. & Glover, D. M. Mutation of a Drosophila γ-tubulin ring complex subunit encoded by discs degenerate-4 differentially disrupts centrosomal protein localization. Genes Dev. 14, 3126–3139 (2000).

    Article  CAS  Google Scholar 

  29. Haren, L. et al. NEDD1-dependent recruitment of the γ-tubulin ring complex to the centrosome is necessary for centriole duplication and spindle assembly. J. Cell Biol. 172, 505–515 (2006).

    Article  CAS  Google Scholar 

  30. Félix, M. A., Antony, C., Wright, M. & Maro, B. Centrosome assembly in vitro: role of γ-tubulin recruitment in Xenopus sperm aster formation. J. Cell Biol. 124, 19–31 (1994).

    Article  Google Scholar 

  31. Chakravarty, A., Howard, L. & Compton, D. A. A mechanistic model for the organization of microtubule asters by motor and non-motor proteins in a mammalian mitotic extract. Mol. Biol. Cell 15, 2116–2132 (2004).

    Article  CAS  Google Scholar 

  32. Cuschieri, L., Miller, R. & Vogel, J. g-tubulin is required for proper recruitment and assembly of Kar9-Bim1 complexes in budding yeast. Mol Biol Cell. 17, 4420–4434 (2006).

    Article  CAS  Google Scholar 

  33. Bright, N. J., Carling, D. & Thornton, C. Investigating the regulation of brain-specific kinases 1 and 2 by phosphorylation. J. Biol. Chem. 283, 14946–14954 (2008).

    Article  CAS  Google Scholar 

  34. Kramer, A. et al. Centrosome-associated Chk1 prevents premature activation of cyclin-B-Cdk1 kinase. Nature Cell Biol. 6, 884–891 (2004).

    Article  Google Scholar 

  35. Peng, C. Y. et al. Mitotic and G2 checkpoint control: regulation of 14.3.3 protein binding by phosphorylation of Cdc25C on S216. Science 277, 1501–1505 (1997).

    Article  CAS  Google Scholar 

  36. Campbell, R. E. et al. A monomeric red fluorescent protein. Proc. Natl Acad. Sci. USA 99, 7877–7882 (2002).

    Article  CAS  Google Scholar 

  37. Piel, M., Nordberg, J., Euteneuer, U. & Bornens, M. Centrosome-dependent exit of cytokinesis in animal cells. Science 291, 1550–1553 (2001).

    Article  CAS  Google Scholar 

  38. Kishi, M., Pan, Y. A., Crump, J. G. & Sanes, J. R. Mammalian SAD kinases are required for neuronal polarization. Science 307, 929–932 (2005).

    Article  CAS  Google Scholar 

  39. Guarguaglini, G. et al. The forkhead-associated domain protein Cep170 interacts with Plk 1 and serves as a marker for mature centrioles. Mol. Biol. Cell 16, 1095–1107 (2004).

    Article  Google Scholar 

  40. Chen, Z. et al. CP110, a cell cycle-dependent CDK substrate, regulates centrosome duplication in human cells. Dev. Cell 3, 339–350 (2002).

    Article  CAS  Google Scholar 

  41. Martinez-Gac, L., Marques, M., Garcia, Z., Campanero, M. R. & Carrera, A. C. Control of cyclin G2 mRNA expression by forkhead TF: novel mechanism for cell cycle control by PI3K and forkhead. Mol. Cell. Biol. 24, 2181–2189 (2004).

    Article  CAS  Google Scholar 

  42. Marques, M. et al. PI3K p110α and β regulate cell cycle entry, exhibiting distinct activation kinetics in G1 phase. Mol. Cell. Biol. 28, 2803–2814 (2008).

    Article  CAS  Google Scholar 

  43. Alvarado-Kristensson, M. et al. p38-MAPK signals survival by phosphorylation of caspase-8 and -3 in human neutrophils. J. Exp. Med. 199, 449–458 (2004).

    Article  CAS  Google Scholar 

  44. Sui, G. et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl Acad. Sci. USA 99, 5515–5520 (2002).

    Article  CAS  Google Scholar 

  45. van de Wetering, M. et al. Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector. EMBO Rep. 4, 609–615 (2003).

    Article  CAS  Google Scholar 

  46. Sankaran, S., Starita, L. M., Groen, AC., Ko, M. J. & Parvin, J. D. Centrosomal MT nucleation activity is inhibited by BRCA1-dependent ubiquitination. Mol. Cell. Biol. 25, 8656–8668 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank York-Dieter Stierhof and Dagmar Ripper for their generous help with electron microscopy. We thank Y. Shi, M. van de Wetering, M. Marqués, M. Bornens and T. Strearns for reagents, J. Sanes and B. Lilley for SADB-deficient cells and comments, L. Kremer and L. Gómez for antibody production, C. Hernández for help in biochemistry assays, C. Mark and R. Hartong for editorial assistance. This work was supported by the Spanish Ministry of Science and Innovation (SAF200405955, 200763624 to ACC, CSD 2006-00023 to JMV), the Spanish Association against Cancer, the Spanish Ministry of Health (PI050964), the Royal Physiographic Society in Lund, the Ake Wibergs, Thelma Zoegas, OE och Edla Johanssons and U-MAS Cancer Research Found, and fellowships from Teggers Fond, EMBO and the Swedish Society for Medical Research.

Author information

Authors and Affiliations

  1. Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Darwin 3, E-28049, Madrid, Spain

    María Alvarado-Kristensson, Virginia Silió & Ana C. Carrera

  2. Center for Molecular Pathology, Dept. Laboratory Medicine, Lund University, Malmoe University Hospital, Sweden

    María Alvarado-Kristensson

  3. Department of Macromolecular Structures, Centro Nacional de Biotecnología/CSIC, Darwin 3, E-28049, Madrid, Spain

    María Josefa Rodríguez & José M. Valpuesta

Authors
  1. María Alvarado-Kristensson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. María Josefa Rodríguez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Virginia Silió
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. José M. Valpuesta
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ana C. Carrera
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.A.K. performed most experiments; M.J.R. and J.M.V. performed EM experiments; V.S. performed some experiments and A.C.C. directed the work, performed some assays and wrote the manuscript.

Corresponding authors

Correspondence to María Alvarado-Kristensson or Ana C. Carrera.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 1375 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Alvarado-Kristensson, M., Rodríguez, M., Silió, V. et al. SADB phosphorylation of γ-tubulin regulates centrosome duplication. Nat Cell Biol 11, 1081–1092 (2009). https://doi.org/10.1038/ncb1921

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb1921

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing