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Cdk5 is essential for synaptic vesicle endocytosis

Nature Cell Biology volume 5, pages 701–710 (2003)Cite this article

Abstract

Synaptic vesicle endocytosis (SVE) is triggered by calcineurin-mediated dephosphorylation of the dephosphin proteins. SVE is maintained by the subsequent rephosphorylation of the dephosphins by unidentified protein kinases. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates dynamin I on Ser 774 and Ser 778 in vitro, which are identical to its endogenous phosphorylation sites in vivo. Cdk5 antagonists and expression of dominant-negative Cdk5 block phosphorylation of dynamin I, but not of amphiphysin or AP180, in nerve terminals and inhibit SVE. Thus Cdk5 has an essential role in SVE and is the first dephosphin kinase identified in nerve terminals.

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Figure 1: Dynamin I is an in vitro substrate for Cdk5.
Figure 2: In vitro functional effects of Cdk5 phosphorylation of dynamin I.
Figure 3: Dynamin phosphorylation sites in vitro and in vivo.
Figure 4: Roscovitine inhibits rephosphorylation of dynamin I in intact synaptosomes.
Figure 5: Dominant-negative Cdk5 inhibits dynamin phosphorylation in neurites.
Figure 6: Cdk5 activity is required for SVE in nerve terminals.
Figure 7: Inhibition of Cdk5 produces a morphologically distinct block in SVE.
Figure 8: Cdk5DN blocks SVE in cerebellar granule neurons.

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References

  1. Liu, J.P., Sim, A.T.R. & Robinson, P.J. Calcineurin inhibition of dynamin I GTPase activity coupled to nerve terminal depolarization. Science 265, 970–973 (1994).

    Article  CAS  Google Scholar 

  2. Marks, B. & McMahon, H.T. Calcium triggers calcineurin-dependent synaptic vesicle recycling in mammalian nerve terminals. Curr.Biol. 8, 740–749 (1998).

    Article  CAS  Google Scholar 

  3. Cousin, M.A., Tan, T.C. & Robinson, P.J. Protein phosphorylation is required for endocytosis in nerve terminals. Potential role for the dephosphins dynamin I and synaptojanin, but not AP180 or amphiphysin. J. Neurochem. 76, 105–116 (2001).

    Article  CAS  Google Scholar 

  4. Cousin, M.A. Synaptic vesicle endocytosis: calcium works overtime in the nerve terminal. Mol. Neurobiol. 22, 115–128 (2000).

    Article  CAS  Google Scholar 

  5. Cousin, M.A. & Robinson, P.J. The dephosphins: Dephosphorylation by calcineurin triggers synaptic vesicle endocytosis. Trends Neurosci. 24, 659–665 (2001).

    Article  CAS  Google Scholar 

  6. Wenk, M.R. et al. PIP Kinase Iγ is the major PI(4,5)P2 synthesizing enzyme at the synapse. Neuron 32, 79–88 (2001).

    Article  CAS  Google Scholar 

  7. Powell, K.A. et al. Phosphorylation of dynamin I on Ser-795 by protein kinase C blocks its association with phospholipids. J. Biol. Chem. 275, 11610–11617 (2000).

    Article  CAS  Google Scholar 

  8. Marks, B. et al. GTPase activity of dynamin and resulting conformation change are essential for endocytosis. Nature 410, 231–235 (2001).

    Article  CAS  Google Scholar 

  9. Shupliakov, O. et al. Synaptic vesicle endocytosis impaired by disruption of dynamin-SH3 domain interactions. Science 276, 259–263 (1997).

    Article  CAS  Google Scholar 

  10. Gad, H. et al. Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin. Neuron 27, 301–312 (2000).

    Article  CAS  Google Scholar 

  11. Robinson, P.J. Dephosphin, a 96,000 dalton substrate of protein kinase C in synaptosomal cytosol is phosphorylated in intact synaptosomes. FEBS Lett. 282, 388–392 (1991).

    Article  CAS  Google Scholar 

  12. Robinson, P.J. et al. Dynamin GTPase regulated by protein kinase C phosphorylation in nerve terminals. Nature 365, 163–166 (1993).

    Article  CAS  Google Scholar 

  13. Hosoya, H. et al. Phosphorylation of dynamin by cdc2 kinase. Biochem. Biophys. Res. Commun. 202, 1127–1133 (1994).

    Article  CAS  Google Scholar 

  14. Earnest, S., Khokhlatchev, A., Albanesi, J.P. & Barylko, B. Phosphorylation of dynamin by ERK2 inhibits the dynamin–microtubule interaction. FEBS Lett. 396, 62–66 (1996).

    Article  CAS  Google Scholar 

  15. Chen-Hwang, M.-C., Chen, H.-R., Elzinga, M. & Hwang, Y.-W. Dynamin is a minibrain kinase/Dyrk1A substrate. J. Biol. Chem. 277, 17597–17604 (2002).

    Article  CAS  Google Scholar 

  16. Songyang, Z. et al. A structural basis for substrate specificities of protein Ser/Thr kinases: primary sequence preference of casein kinases I and II, NIMA, phosphorylase kinase, calmodulin-dependent kinase II, CDK5, and Erk1. Mol. Cell. Biol. 16, 6486–6493 (1996).

    Article  CAS  Google Scholar 

  17. Tomizawa, K. et al. Cdk5/p35 regulates neurotransmitter release through phosphorylation and downregulation of P/Q-type voltage-dependent calcium channel activity. J. Neurosci. 22, 2590–2597 (2002).

    Article  CAS  Google Scholar 

  18. Matsubara, M. et al. Site-specific phosphorylation of synapsin I by mitogen-activated protein kinase and Cdk5 and its effects on physiological functions. J. Biol. Chem. 271, 21108–21113 (1996).

    Article  CAS  Google Scholar 

  19. Shuang, R.Q. et al. Regulation of Munc-18 syntaxin 1A interaction by cyclin-dependent kinase 5 in nerve endings. J. Biol. Chem. 273, 4957–4966 (1998).

    Article  CAS  Google Scholar 

  20. Floyd, S.R. et al. Amphiphysin binds the cdk5 regulatory subunit p35 and is phosphorylated by cdk5 and cdc2. J. Biol. Chem. 276, 8104–8110 (2001).

    Article  CAS  Google Scholar 

  21. Hill, E., van der, K.J., Downes, C.P. & Smythe, E. The role of dynamin and its binding partners in coated pit invagination and scission. J. Cell Biol. 152, 309–324 (2001).

    Article  CAS  Google Scholar 

  22. Grabs, D. et al. The SH3 domain of amphiphysin binds the proline-rich domain of dynamin at a single site that defines a new SH3 binding consensus sequence. J. Biol. Chem. 272, 13419–13425 (1997).

    Article  CAS  Google Scholar 

  23. Meijer, L. et al. Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. Eur. J. Biochem. 243, 527–536 (1997).

    Article  CAS  Google Scholar 

  24. Davies, S.P., Reddy, H., Caivano, M. & Cohen, P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351, 95–105 (2000).

    Article  CAS  Google Scholar 

  25. Cousin, M.A. et al. Synapsin I-associated phosphatidylinositol 3-kinase mediates synaptic vesicle delivery to the readily releasable pool. J. Biol. Chem. (cited 16 May 2003) DOI: 10.1074/jbc.M302386200 (2003).

  26. Rosales, J.L., Nodwell, M.J., Johnston, R.N. & Lee, K.Y. Cdk5/p25(nck5a) interaction with synaptic proteins in bovine brain. J. Cell. Biochem. 78, 151–159 (2000).

    Article  CAS  Google Scholar 

  27. Cousin, M.A. & Robinson, P.J. Ca2+ inhibition of dynamin arrests synaptic vesicle recycling at the active zone. J. Neurosci. 20, 949–957 (2000).

    Article  CAS  Google Scholar 

  28. Koenig, J.H. & Ikeda, K. Disappearance and reformation of synaptic vesicle membrane upon transmitter release observed under reversible blockage of membrane retrieval. J. Neurosci. 9, 3844–3860 (1989).

    Article  CAS  Google Scholar 

  29. Liu, J.P., Powell, K.A., Südhof, T.C. & Robinson, P.J. Dynamin I is a Ca2+-sensitive phospholipid-binding protein with very high affinity for protein kinase C. J. Biol. Chem. 269, 21043–21050 (1994).

    CAS  PubMed  Google Scholar 

  30. Robinson, P.J. Differential stimulation of protein kinase C activity by phorbol ester or calcium/phosphatidylserine in vitro and in intact synaptosomes. J. Biol. Chem. 267, 21637–21644 (1992).

    CAS  PubMed  Google Scholar 

  31. Robinson, P.J., Liu, J.P., Powell, K.A., Fykse, E.M. & Südhof, T.C. Phosphorylation of dynamin I and synaptic vesicle recycling. Trends Neurosci. 17, 348–353 (1994).

    Article  CAS  Google Scholar 

  32. Dhavan, R., Tsai, L.H. & Tsai, L.H. A decade of cdk5. Nature Rev. Mol. Cell Biol. 2, 749–759 (2001).

    Article  CAS  Google Scholar 

  33. Ohshima, T. et al. Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death. Proc. Natl Acad. Sci. USA 93, 11173–11178 (1996).

    Article  CAS  Google Scholar 

  34. Kwon, Y.T., Tsai, L.H. & Crandall, J.E. Callosal axon guidance defects in p35(−/−) mice. J. Comp. Neurol. 415, 218–229 (1999).

    Article  CAS  Google Scholar 

  35. Tsai, L.H., Takahashi, T., Caviness, V.S.J. & Harlow, E. Activity and expression pattern of cyclin-dependent kinase 5 in the embryonic mouse nervous system. Development 119, 1029–1040 (1993).

    CAS  PubMed  Google Scholar 

  36. Bibb, J.A. et al. Effects of chronic exposure to cocaine are regulated by the neuronal protein Cdk5. Nature 410, 376–380 (2001).

    Article  CAS  Google Scholar 

  37. Fletcher, A.I. et al. Regulation of exocytosis by cyclin-dependent kinase 5 via phosphorylation of munc18. J. Biol. Chem. 274, 4027–4035 (1999).

    Article  CAS  Google Scholar 

  38. Li, B.S. et al. Regulation of NMDA receptors by cyclin-dependent kinase-5. Proc. Natl Acad. Sci. USA 98, 12742–12747 (2001).

    Article  CAS  Google Scholar 

  39. Yan, Z., Chi, P., Bibb, J.A., Ryan, T.A. & Greengard, P. Roscovitine: a novel regulator of P/Q-type calcium channels and transmitter release in central neurons. J. Physiol. 540, 761–770 (2002).

    Article  CAS  Google Scholar 

  40. Nicholls, D.G. The glutamatergic nerve terminal. Eur. J. Biochem. 212, 613–631 (1993).

    Article  CAS  Google Scholar 

  41. Wilde, A. & Brodsky, F.M. In vivo phosphorylation of adaptors regulates their interaction with clathrin. J. Cell Biol. 135, 635–645 (1996).

    Article  CAS  Google Scholar 

  42. Kariya, K. et al. Regulation of complex formation of POB1/epsin/adaptor protein complex 2 by mitotic phosphorylation. J. Biol. Chem. 275, 18399–18406 (2000).

    Article  CAS  Google Scholar 

  43. Slepnev, V.I., Ochoa, G.C., Butler, M.H., Grabs, D. & DeCamilli, P. Role of phosphorylation in regulation of the assembly of endocytic coat complexes. Science 281, 821–824 (1998).

    Article  CAS  Google Scholar 

  44. Rasmussen, R.K., Rusak, J., Price, G., Robinson, P.J. & Dorow, D.S. Mixed lineage kinase 2–SH3 domain binds dynamin and greatly enhances GTPase activation by phospholipid. Biochem. J. 335, 119–124 (1998).

    Article  CAS  Google Scholar 

  45. Negash, S., Wang, H.S., Gao, C., Ledee, D. & Zelenka, P. Cdk5 regulates cell-matrix and cell-cell adhesion in lens epithelial cells. J. Cell Sci. 115, 2109–2117 (2002).

    CAS  PubMed  Google Scholar 

  46. van den Heuvel, S. & Harlow, E. Distinct roles for cyclin-dependent kinases in cell cycle control. Science 262, 2050–2054 (1993).

    Article  CAS  Google Scholar 

  47. Stowell, M.H., Marks, B., Wigge, P. & McMahon, H.T. Nucleotide-dependent conformational changes in dynamin: evidence for a mechanochemical molecular spring. Nature Cell Biol. 1, 27–32 (1999).

    Article  CAS  Google Scholar 

  48. Wang, L.-H., Südhof, T.C. & Anderson, R.G. The appendage domain of α-adaptin is a high-affinity binding site for dynamin. J. Biol. Chem. 270, 10079–10083 (1995).

    Article  CAS  Google Scholar 

  49. Micheva, K.D., Ramjaun, A.R., Kay, B.K. & McPherson, P.S. SH3 domain-dependent interactions of endophilin with amphiphysin. FEBS Lett. 414, 308–312 (1997).

    Article  CAS  Google Scholar 

  50. Zhou, W., Merrick, B.A., Khaledi, M.G. & Tomer, K.B. Detection and sequencing of phosphopeptides affinity bound to immobilized metal ion beads by matrix-assisted laser desorption/ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 11, 273–282 (2000).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by grants from the Australian National Health and Medical Research Council (NHMRC to P.J.R.), the Cunningham Trust and Wellcome Trust (M.A.C.) and the Danish Natural Science Research Council (M.R.L.). We thank P. Rowe for critical reading of the manuscript. We also thank A. Quan (Children's Medical Research Institute), B. Hanna (Children's Medical Research Institute) and L. Deer (Institute of Clinical Pathology and Medical Research, Westmead Hospital) for valuable technical assistance. In addition, we also thank our colleagues listed in the constructs section for the plasmids required for this project.

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Authors and Affiliations

  1. Cell Signalling Unit, Children's Medical Research Institute, Locked Bag 23, Wentworthville, 2145, NSW, Australia

    Timothy C. Tan, Valentina A. Valova, Chandra S. Malladi, Mark E. Graham, Leise A. Berven, Gurdip Hansra & Phillip J. Robinson

  2. Royal Hospital for Women in Randwick, Barker St, Randwick, 2031, NSW, Australia

    Sonya J. McClure

  3. Cancer Research Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, 2010, NSW, Australia

    Boris Sarcevic

  4. Electron Microscope Laboratory, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead, 2145, NSW, Australia

    Ross A. Boadle

  5. Australian Proteomic Analysis Facility, Macquarie University, Balaclava Road, North Ryde, 2109, NSW, Australia

    Martin R. Larsen

  6. Division of Biomedical Sciences, Membrane Biology Group, George Square, University of Edinburgh, EH8 9XD, Scotland, UK

    Orla J. Jupp & Michael A. Cousin

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Supplementary information

Supplementary Information Fig. S1-S6

Supplementary Information Fig. S1a: Effect of cdk5 phosphorylation on dynamin's GTPase activity. (PDF 1528 kb)

Supplementary Information Fig. S2: Roscovitine inhibits dynamin I phosphorylation by cdk5 but not by PKC-α in vitro. is presented. Arrows indicate either dynamin I (Dyn) or synapsin I (Syn, doublet).

Supplementary Information Fig. S3: Lack of effect of PD 98059 on dynamin I phosphorylation and SVE.

Supplementary Information Fig. S4: Double pull-down of 4 dephosphins and synapsin from synaptosomes.

Supplementary Information Fig. S5; Morphology of synaptosomes at rest or after two cycles of stimulus- recovery.

Supplementary Information Fig. S6: Morphology of synaptosomes during S2 in the presence of roscovitine.

Supplementary Information Methods

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Tan, T., Valova, V., Malladi, C. et al. Cdk5 is essential for synaptic vesicle endocytosis. Nat Cell Biol 5, 701–710 (2003). https://doi.org/10.1038/ncb1020

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