NeuroMolecular Medicine

, Volume 17, Issue 1, pp 12–23 | Cite as

Sumoylation of p35 Modulates p35/Cyclin-Dependent Kinase (Cdk) 5 Complex Activity

  • Anja Büchner
  • Petranka Krumova
  • Sundar Ganesan
  • Mathias Bähr
  • Katrin Eckermann
  • Jochen H. Weishaupt
Original Paper


Cyclin-dependent kinase (Cdk) 5 is critical for central nervous system development and neuron-specific functions including neurite outgrowth as well as synaptic function and plasticity. Cdk5 activity requires association with one of the two regulatory subunits, called p35 and p39. p35 redistribution as well as misregulation of Cdk5 activity is followed by cell death in several models of neurodegeneration. Posttranslational protein modification by small ubiquitin-related modifier (SUMO) proteins (sumoylation) has emerged as key regulator of protein targeting and protein/protein interaction. Under cell-free in vitro conditions, we found p35 covalently modified by SUMO1. Using both biochemical and FRET-/FLIM-based approaches, we demonstrated that SUMO2 is robustly conjugated to p35 in cells and identified the two major SUMO acceptor lysines in p35, K246 and K290. Furthermore, different degrees of oxidative stress resulted in differential p35 sumoylation, linking oxidative stress that is encountered in neurodegenerative diseases to the altered activity of Cdk5. Functionally, sumoylation of p35 increased the activity of the p35/Cdk5 complex. We thus identified a novel neuronal SUMO target and show that sumoylation is a likely candidate mechanism for the rapid modulation of p35/Cdk5 activity in physiological situations as well as in disease.


Sumoylation p35/Cdk5 activity FRET/FLIM Oxidative stress 



We thank Christine Poser and Claudia Fokken for excellent technical support. We thank Ron Hay (University of Dundee, Dundee, UK) for providing us with the His6-SUMO1 and His6-SUMO2 plasmids, Frauke Melchior (ZMBH, Heidelberg, Germany) for the components used for the in vitro sumoylation assay and Gertrude Bunt (University Medical Center Göttingen, Göttingen, Germany) for mVenus and mTFP plasmids. This work was supported by the Cluster of Excellence and DFG Research Center Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB).

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. Amor, S., Peferoen, L. A., Vogel, D. Y., Breur, M., van der Valk, P., Baker, D., et al. (2014). Inflammation in neurodegenerative diseases–an update. Immunology, 142(2), 151–166.CrossRefPubMedGoogle Scholar
  2. Asada, A., Yamamoto, N., Gohda, M., Saito, T., Hayashi, N., & Hisanaga, S. (2008). Myristoylation of p39 and p35 is a determinant of cytoplasmic or nuclear localization of active cyclin-dependent kinase 5 complexes. Journal of Neurochemistry, 106(3), 1325–1336.CrossRefPubMedGoogle Scholar
  3. Bernier-Villamor, V., Sampson, D. A., Matunis, M. J., & Lima, C. D. (2002). Structural basis for E2-mediated SUMO conjugation revealed by a complex between ubiquitin-conjugating enzyme Ubc9 and RanGAP1. Cell, 108(3), 345–356.CrossRefPubMedGoogle Scholar
  4. Bossis, G., & Melchior, F. (2006). Regulation of SUMOylation by reversible oxidation of SUMO conjugating enzymes. Molecular Cell, 21(3), 349–357.CrossRefPubMedGoogle Scholar
  5. Chae, T., Kwon, Y. T., Bronson, R., Dikkes, P., Li, E., & Tsai, L. H. (1997). Mice lacking p35, a neuronal specific activator of Cdk5, display cortical lamination defects, seizures, and adult lethality. Neuron, 18(1), 29–42.CrossRefPubMedGoogle Scholar
  6. Choe, E. A., Liao, L., Zhou, J. Y., Cheng, D., Duong, D. M., Jin, P., et al. (2007). Neuronal morphogenesis is regulated by the interplay between cyclin-dependent kinase 5 and the ubiquitin ligase mind bomb 1. Journal of Neuroscience, 27(35), 9503–9512.CrossRefPubMedGoogle Scholar
  7. Day, R. N., Booker, C. F., & Periasamy, A. (2008). Characterization of an improved donor fluorescent protein for Forster resonance energy transfer microscopy. Journal of Biomedial Optics, 13(3), 031203.CrossRefGoogle Scholar
  8. Desterro, J. M., Rodriguez, M. S., Kemp, G. D., & Hay, R. T. (1999). Identification of the enzyme required for activation of the small ubiquitin-like protein SUMO-1. Journal of Biological Chemistry, 274(15), 10618–10624.CrossRefPubMedGoogle Scholar
  9. Dhavan, R., & Tsai, L. H. (2001). A decade of CDK5. Nature Reviews Molecular Cell Biology, 2(10), 749–759.CrossRefPubMedGoogle Scholar
  10. Eckermann, K. (2013). SUMO and parkinson’s disease. Neuromolecular Med, 15(4), 737–759.CrossRefPubMedGoogle Scholar
  11. Feligioni, M., & Nistico, R. (2013). SUMO: a (oxidative) stressed protein. Neuromolecular Medicine, 15(4), 707–719.CrossRefPubMedGoogle Scholar
  12. Fu, X., Choi, Y. K., Qu, D., Yu, Y., Cheung, N. S., & Qi, R. Z. (2006). Identification of nuclear import mechanisms for the neuronal Cdk5 activator. Journal of Biological Chemistry, 281(51), 39014–39021.CrossRefPubMedGoogle Scholar
  13. Gill, G. (2004). SUMO and ubiquitin in the nucleus: different functions, similar mechanisms? Genes & Development, 18(17), 2046–2059.CrossRefGoogle Scholar
  14. Golebiowski, F., Matic, I., Tatham, M. H., Cole, C., Yin, Y., Nakamura, A., et al. (2009). System-wide changes to SUMO modifications in response to heat shock. Science Signaling, 2(72), ra24.Google Scholar
  15. Gong, L., Millas, S., Maul, G. G., & Yeh, E. T. (2000). Differential regulation of sentrinized proteins by a novel sentrin-specific protease. Journal of Biological Chemistry, 275(5), 3355–3359.CrossRefPubMedGoogle Scholar
  16. Halliwell, B., Clement, M. V., & Long, L. H. (2000). Hydrogen peroxide in the human body. FEBS Letters, 486(1), 10–13.CrossRefPubMedGoogle Scholar
  17. Hecker, C. M., Rabiller, M., Haglund, K., Bayer, P., & Dikic, I. (2006). Specification of SUMO1- and SUMO2-interacting motifs. Journal of Biological Chemistry, 281(23), 16117–16127.CrossRefPubMedGoogle Scholar
  18. Hsiao, K., Bozdagi, O., & Benson, D. L. (2014). Axonal cap-dependent translation regulates presynaptic p35. Developmental Neurobiology, 74(3), 351–364.CrossRefPubMedGoogle Scholar
  19. Jaffray, E. G., & Hay, R. T. (2006). Detection of modification by ubiquitin-like proteins. Methods, 38(1), 35–38.CrossRefPubMedGoogle Scholar
  20. Kim, E. T., Kim, K. K., Matunis, M. J., & Ahn, J. H. (2009). Enhanced SUMOylation of proteins containing a SUMO-interacting motif by SUMO-Ubc9 fusion. Biochemical and Biophysical Research Communications, 388(1), 41–45.CrossRefPubMedGoogle Scholar
  21. Krumova, P., Meulmeester, E., Garrido, M., Tirard, M., Hsiao, H. H., Bossis, G., et al. (2011). Sumoylation inhibits alpha-synuclein aggregation and toxicity. Journal of Cell Biology, 194(1), 49–60.CrossRefPubMedCentralPubMedGoogle Scholar
  22. Krumova, P., & Weishaupt, J. H. (2013). Sumoylation in neurodegenerative diseases. Cellular and Molecular Life Sciences, 70(12), 2123–2138.CrossRefPubMedGoogle Scholar
  23. Li, Z., David, G., Hung, K. W., DePinho, R. A., Fu, A. K., & Ip, N. Y. (2004). Cdk5/p35 phosphorylates mSds3 and regulates mSds3-mediated repression of transcription. Journal of Biological Chemistry, 279(52), 54438–54444.CrossRefPubMedGoogle Scholar
  24. Macauley, M. S., Errington, W. J., Scharpf, M., Mackereth, C. D., Blaszczak, A. G., Graves, B. J., et al. (2006). Beads-on-a-string, characterization of ETS-1 sumoylated within its flexible N-terminal sequence. Journal of Biological Chemistry, 281(7), 4164–4172.CrossRefPubMedGoogle Scholar
  25. Mahajan, R., Delphin, C., Guan, T., Gerace, L., & Melchior, F. (1997). A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell, 88(1), 97–107.CrossRefPubMedGoogle Scholar
  26. Matunis, M. J., Coutavas, E., & Blobel, G. (1996). A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. Journal of Cell Biology, 135(6 Pt 1), 1457–1470.CrossRefPubMedGoogle Scholar
  27. Mukhopadhyay, D., & Dasso, M. (2007). Modification in reverse: the SUMO proteases. Trends in Biochemical Sciences, 32(6), 286–295.CrossRefPubMedGoogle Scholar
  28. Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., & Miyawaki, A. (2002). A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nature Biotechnology, 20(1), 87–90.CrossRefPubMedGoogle Scholar
  29. Nguyen, M. D., Lariviere, R. C., & Julien, J. P. (2001). Deregulation of Cdk5 in a mouse model of ALS: toxicity alleviated by perikaryal neurofilament inclusions. Neuron, 30(1), 135–147.CrossRefPubMedGoogle Scholar
  30. Nikolic, M., Dudek, H., Kwon, Y. T., Ramos, Y. F., & Tsai, L. H. (1996). The cdk5/p35 kinase is essential for neurite outgrowth during neuronal differentiation. Genes & Development, 10(7), 816–825.CrossRefGoogle Scholar
  31. O’Hare, M. J., Kushwaha, N., Zhang, Y., Aleyasin, H., Callaghan, S. M., Slack, R. S., et al. (2005). Differential roles of nuclear and cytoplasmic cyclin-dependent kinase 5 in apoptotic and excitotoxic neuronal death. Journal of Neuroscience, 25(39), 8954–8966.CrossRefPubMedGoogle Scholar
  32. Osuga, H., Osuga, S., Wang, F., Fetni, R., Hogan, M. J., Slack, R. S., et al. (2000). Cyclin-dependent kinases as a therapeutic target for stroke. Proceedings of the National Academy of Sciences USA, 97(18), 10254–10259.CrossRefGoogle Scholar
  33. Patrick, G. N., Zhou, P., Kwon, Y. T., Howley, P. M., & Tsai, L. H. (1998). p35, the neuronal-specific activator of cyclin-dependent kinase 5 (Cdk5) is degraded by the ubiquitin-proteasome pathway. Journal of Biological Chemistry, 273(37), 24057–24064.CrossRefPubMedGoogle Scholar
  34. Patrick, G. N., Zukerberg, L., Nikolic, M., de la Monte, S., Dikkes, P., & Tsai, L. H. (1999). Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration. Nature, 402(6762), 615–622.CrossRefPubMedGoogle Scholar
  35. Patzke, H., & Tsai, L. H. (2002). Calpain-mediated cleavage of the cyclin-dependent kinase-5 activator p39 to p29. Journal of Biological Chemistry, 277(10), 8054–8060.CrossRefPubMedGoogle Scholar
  36. Poon, R. Y., Lew, J., & Hunter, T. (1997). Identification of functional domains in the neuronal Cdk5 activator protein. Journal of Biological Chemistry, 272(9), 5703–5708.CrossRefPubMedGoogle Scholar
  37. Sahlgren, C. M., Pallari, H. M., He, T., Chou, Y. H., Goldman, R. D., & Eriksson, J. E. (2006). A nestin scaffold links Cdk5/p35 signaling to oxidant-induced cell death. EMBO Journal, 25(20), 4808–4819.CrossRefPubMedCentralPubMedGoogle Scholar
  38. Saitoh, H., & Hinchey, J. (2000). Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3. Journal of Biological Chemistry, 275(9), 6252–6258.CrossRefPubMedGoogle Scholar
  39. Sapetschnig, A., Rischitor, G., Braun, H., Doll, A., Schergaut, M., Melchior, F., et al. (2002). Transcription factor Sp3 is silenced through SUMO modification by PIAS1. EMBO Journal, 21(19), 5206–5215.CrossRefPubMedCentralPubMedGoogle Scholar
  40. Shea, T. B., Zheng, Y. L., Ortiz, D., & Pant, H. C. (2004). Cyclin-dependent kinase 5 increases perikaryal neurofilament phosphorylation and inhibits neurofilament axonal transport in response to oxidative stress. Journal of Neuroscience Research, 76(6), 795–800.CrossRefPubMedGoogle Scholar
  41. Shin, E. J., Shin, H. M., Nam, E., Kim, W. S., Kim, J. H., Oh, B. H., et al. (2012). DeSUMOylating isopeptidase: a second class of SUMO protease. EMBO Reports, 13(4), 339–346.CrossRefPubMedCentralPubMedGoogle Scholar
  42. Shukla, V., Mishra, S. K., & Pant, H. C. (2011). Oxidative stress in neurodegeneration. Advance Pharmacology Science, 2011, 572634.Google Scholar
  43. Smith, P. D., Crocker, S. J., Jackson-Lewis, V., Jordan-Sciutto, K. L., Hayley, S., Mount, M. P., et al. (2003). Cyclin-dependent kinase 5 is a mediator of dopaminergic neuron loss in a mouse model of Parkinson’s disease. Proceedings of the National Academy of Sciences USA, 100(23), 13650–13655.CrossRefGoogle Scholar
  44. Su, S. C., & Tsai, L. H. (2011). Cyclin-dependent kinases in brain development and disease. Annual Review of Cell and Developmental Biology, 27, 465–491.CrossRefPubMedGoogle Scholar
  45. Sun, K. H., Chang, K. H., Clawson, S., Ghosh, S., Mirzaei, H., Regnier, F., et al. (2011). Glutathione-S-transferase P1 is a critical regulator of Cdk5 kinase activity. Journal of Neurochemistry, 118(5), 902–914.CrossRefPubMedGoogle Scholar
  46. Tan, T. C., Valova, V. A., Malladi, C. S., Graham, M. E., Berven, L. A., Jupp, O. J., et al. (2003). Cdk5 is essential for synaptic vesicle endocytosis. Nature Cell Biology, 5(8), 701–710.CrossRefPubMedGoogle Scholar
  47. Tang, D., Chun, A. C., Zhang, M., & Wang, J. H. (1997). Cyclin-dependent kinase 5 (Cdk5) activation domain of neuronal Cdk5 activator. Evidence of the existence of cyclin fold in neuronal Cdk5a activator. Journal of Biological Chemistry, 272(19), 12318–12327.CrossRefPubMedGoogle Scholar
  48. Tang, D., Yeung, J., Lee, K. Y., Matsushita, M., Matsui, H., Tomizawa, K., et al. (1995). An isoform of the neuronal cyclin-dependent kinase 5 (Cdk5) activator. Journal of Biological Chemistry, 270(45), 26897–26903.CrossRefPubMedGoogle Scholar
  49. Tarricone, C., Dhavan, R., Peng, J., Areces, L. B., Tsai, L. H., & Musacchio, A. (2001). Structure and regulation of the CDK5-p25(nck5a) complex. Molecular Cell, 8(3), 657–669.CrossRefPubMedGoogle Scholar
  50. Tatham, M. H., Jaffray, E., Vaughan, O. A., Desterro, J. M., Botting, C. H., Naismith, J. H., et al. (2001). Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9. Journal of Biological Chemistry, 276(38), 35368–35374.CrossRefPubMedGoogle Scholar
  51. Tsai, L. H., Delalle, I., Caviness, V. S, Jr, Chae, T., & Harlow, E. (1994). p35 is a neural-specific regulatory subunit of cyclin-dependent kinase 5. Nature, 371(6496), 419–423.CrossRefPubMedGoogle Scholar
  52. van den Heuvel, S., & Harlow, E. (1993). Distinct roles for cyclin-dependent kinases in cell cycle control. Science, 262(5142), 2050–2054.CrossRefPubMedGoogle Scholar
  53. Verstegen, A. M., Tagliatti, E., Lignani, G., Marte, A., Stolero, T., Atias, M., et al. (2014). Phosphorylation of synapsin I by cyclin-dependent kinase-5 sets the ratio between the resting and recycling pools of synaptic vesicles at hippocampal synapses. Journal of Neuroscience, 34(21), 7266–7280.CrossRefPubMedGoogle Scholar
  54. Weishaupt, J. H., Kussmaul, L., Grotsch, P., Heckel, A., Rohde, G., Romig, H., et al. (2003). Inhibition of CDK5 is protective in necrotic and apoptotic paradigms of neuronal cell death and prevents mitochondrial dysfunction. Molecular and Cellular Neuroscience, 24(2), 489–502.CrossRefPubMedGoogle Scholar
  55. Wittmann, C., Chockley, P., Singh, S. K., Pase, L., Lieschke, G. J., & Grabher, C. (2012). Hydrogen peroxide in inflammation: messenger, guide, and assassin. Advances in Hematology, 2012, 541471.CrossRefPubMedCentralPubMedGoogle Scholar
  56. Zukerberg, L. R., Patrick, G. N., Nikolic, M., Humbert, S., Wu, C. L., Lanier, L. M., et al. (2000). Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth. Neuron, 26(3), 633–646.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Anja Büchner
    • 1
    • 5
  • Petranka Krumova
    • 1
    • 2
  • Sundar Ganesan
    • 3
  • Mathias Bähr
    • 1
  • Katrin Eckermann
    • 1
    • 5
  • Jochen H. Weishaupt
    • 1
    • 4
  1. 1.Department of NeurologyUniversity Medical Center GöttingenGöttingenGermany
  2. 2.Novartis Institutes for Biomedical ResearchBaselSwitzerland
  3. 3.National Institutes of Health (NIH)Rockville, BethesdaUSA
  4. 4.Neurology DepartmentUlm UniversityUlmGermany
  5. 5.Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB)GöttingenGermany

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