Mutations in the gene encoding the protein kinase LRRK2 (leucine rich repeat kinase 2) cause autosomal dominant Parkinson's disease [1, 2]. The most common mutation replaces glycine 2019 with a serine within the magnesium-binding DFG motif of the kinase domain thereby increasing LRRK2 kinase activity 3-fold [3, 4]. This indicates that inhibitors of LRRK2 kinase activity might be of therapeutic benefit for the treatment of Parkinson's disease and has stimulated much activity in this field of research.
Recent work has revealed that LRRK2 interacts with14-3-3 phospho-binding adaptor isoforms that is mediated by phosphorylation of Ser910 and Ser935 located prior to the leucine rich repeat domain mediates [5]. Interestingly, 14-3-3 binding has been linked to Parkinson's disease as Ser910 as well as Ser935 and interaction with the 14-3-3 is inhibited by five of the six validated LRRK2 pathogenic mutations (R1441C, R1441G, R1441H, Y1699C and I2020T) [5, 6].
Intriguingly, treatment of cell and animal models with structurally unrelated LRRK2 kinase inhibitors also results in dephosphorylation of Ser910 and Ser935 that is accompanied by loss of 14-3-3 binding [7, 8]. The kinase(s) and phosphatase(s) that act on Ser910, Ser935, Ser955 and Ser973 are unknown, but the evidence points towards these residues not comprising autophosphorylation sites [7].
Nic Dzamko a Postdoc in Dario Alessi's lab who is now a CJ Martin Fellow at the Neuroscience Research Institute in Sydney Australia generated and characterised state of the art hugely sensitive and specific Total and Ser910 and Ser935 phospho specific antibodies in close collaboration with the Michael J Fox foundation for Parkinson's Research in New York (http://www.michaeljfox.org/), one of the world greatest and most innovative supporters of medical research in the area of understanding and treating Parkinson's disease. These antibodies enable for the first time the assessment of LRRK2 phosphorylation state by immunoblot analysis in very small amounts of cell extract (20 micrograms). We expect that these will be usable in ELISA and Immunohistochemical applications as well.
We hope that these reagents will have tremendous benefit in helping the pharmaceutical industry as well as academic researchers assess the efficacy of LRRK2 inhibitors that are being developed. We also think that they will also help in further understanding the link between 14-3-3 binding to LRRK2 and Parkinson's Disease.
To learn more about these antibodies and to order these please click here for total antibody; click here for the Phospho935 antibody and click here for the phosphSer910 antibody.
We would like to thank the Michael J Fox foundation and especially Sonal Das an associate Director of the Foundation for their support for this project.
References
1 Zimprich, A., Biskup, S., Leitner, P., Lichtner, P., Farrer, M., Lincoln, S., Kachergus, J., Hulihan, M., Uitti, R. J., Calne, D. B., Stoessl, A. J., Pfeiffer, R. F., Patenge, N., Carbajal, I. C., Vieregge, P., Asmus, F., Muller-Myhsok, B., Dickson, D. W., Meitinger, T., Strom, T. M., Wszolek, Z. K. and Gasser, T. (2004) Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology. Neuron. 44, 601-607
2 Paisan-Ruiz, C., Jain, S., Evans, E. W., Gilks, W. P., Simon, J., van der Brug, M., Lopez de Munain, A., Aparicio, S., Gil, A. M., Khan, N., Johnson, J., Martinez, J. R., Nicholl, D., Carrera, I. M., Pena, A. S., de Silva, R., Lees, A., Marti-Masso, J. F., Perez-Tur, J., Wood, N. W. and Singleton, A. B. (2004) Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease. Neuron. 44, 595-600
3 West, A. B., Moore, D. J., Biskup, S., Bugayenko, A., Smith, W. W., Ross, C. A., Dawson, V. L. and Dawson, T. M. (2005) Parkinson's disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity. Proc Natl Acad Sci U S A. 102, 16842-16847
4 Jaleel, M., McBride, A., Lizcano, J. M., Deak, M., Toth, R., Morrice, N. A. and Alessi, D. R. (2005) Identification of the sucrose non-fermenting related kinase SNRK, as a novel LKB1 substrate. FEBS Lett. 579, 1417-1423
5 Nichols, R. J., Dzamko, N., Morrice, N. A., Campbell, D. G., Deak, M., Ordureau, A., Macartney, T., Tong, Y., Shen, J., Prescott, A. R. and Alessi, D. R. (2010) 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization. Biochem J. 430, 393-404
6 Li, X., Wang, Q. J., Pan, N., Lee, S., Zhao, Y., Chait, B. T. and Yue, Z. (2011) Phosphorylation-dependent 14-3-3 binding to LRRK2 is impaired by common mutations of familial Parkinson's disease. PLoS One. 6, e17153
7 Dzamko, N., Deak, M., Hentati, F., Reith, A. D., Prescott, A. R., Alessi, D. R. and Nichols, R. J. (2010
) Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization. Biochem J. 430, 405-413
8 Deng, X., Dzamko, N., Prescott, A., Davies, P., Liu, Q., Yang, Q., Lee, J. D., Patricelli, M. P., Nomanbhoy, T. K., Alessi, D. R. and Gray, N. S. (2011) Characterization of a selective inhibitor of the Parkinson's disease kinase LRRK2. Nat Chem Biol. 7, 203-205
Recent work has revealed that LRRK2 interacts with14-3-3 phospho-binding adaptor isoforms that is mediated by phosphorylation of Ser910 and Ser935 located prior to the leucine rich repeat domain mediates [5]. Interestingly, 14-3-3 binding has been linked to Parkinson's disease as Ser910 as well as Ser935 and interaction with the 14-3-3 is inhibited by five of the six validated LRRK2 pathogenic mutations (R1441C, R1441G, R1441H, Y1699C and I2020T) [5, 6].
Intriguingly, treatment of cell and animal models with structurally unrelated LRRK2 kinase inhibitors also results in dephosphorylation of Ser910 and Ser935 that is accompanied by loss of 14-3-3 binding [7, 8]. The kinase(s) and phosphatase(s) that act on Ser910, Ser935, Ser955 and Ser973 are unknown, but the evidence points towards these residues not comprising autophosphorylation sites [7].
Nic Dzamko a Postdoc in Dario Alessi's lab who is now a CJ Martin Fellow at the Neuroscience Research Institute in Sydney Australia generated and characterised state of the art hugely sensitive and specific Total and Ser910 and Ser935 phospho specific antibodies in close collaboration with the Michael J Fox foundation for Parkinson's Research in New York (http://www.michaeljfox.org/), one of the world greatest and most innovative supporters of medical research in the area of understanding and treating Parkinson's disease. These antibodies enable for the first time the assessment of LRRK2 phosphorylation state by immunoblot analysis in very small amounts of cell extract (20 micrograms). We expect that these will be usable in ELISA and Immunohistochemical applications as well.
We hope that these reagents will have tremendous benefit in helping the pharmaceutical industry as well as academic researchers assess the efficacy of LRRK2 inhibitors that are being developed. We also think that they will also help in further understanding the link between 14-3-3 binding to LRRK2 and Parkinson's Disease.
To learn more about these antibodies and to order these please click here for total antibody; click here for the Phospho935 antibody and click here for the phosphSer910 antibody.
We would like to thank the Michael J Fox foundation and especially Sonal Das an associate Director of the Foundation for their support for this project.
References
1 Zimprich, A., Biskup, S., Leitner, P., Lichtner, P., Farrer, M., Lincoln, S., Kachergus, J., Hulihan, M., Uitti, R. J., Calne, D. B., Stoessl, A. J., Pfeiffer, R. F., Patenge, N., Carbajal, I. C., Vieregge, P., Asmus, F., Muller-Myhsok, B., Dickson, D. W., Meitinger, T., Strom, T. M., Wszolek, Z. K. and Gasser, T. (2004) Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology. Neuron. 44, 601-607
2 Paisan-Ruiz, C., Jain, S., Evans, E. W., Gilks, W. P., Simon, J., van der Brug, M., Lopez de Munain, A., Aparicio, S., Gil, A. M., Khan, N., Johnson, J., Martinez, J. R., Nicholl, D., Carrera, I. M., Pena, A. S., de Silva, R., Lees, A., Marti-Masso, J. F., Perez-Tur, J., Wood, N. W. and Singleton, A. B. (2004) Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease. Neuron. 44, 595-600
3 West, A. B., Moore, D. J., Biskup, S., Bugayenko, A., Smith, W. W., Ross, C. A., Dawson, V. L. and Dawson, T. M. (2005) Parkinson's disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity. Proc Natl Acad Sci U S A. 102, 16842-16847
4 Jaleel, M., McBride, A., Lizcano, J. M., Deak, M., Toth, R., Morrice, N. A. and Alessi, D. R. (2005) Identification of the sucrose non-fermenting related kinase SNRK, as a novel LKB1 substrate. FEBS Lett. 579, 1417-1423
5 Nichols, R. J., Dzamko, N., Morrice, N. A., Campbell, D. G., Deak, M., Ordureau, A., Macartney, T., Tong, Y., Shen, J., Prescott, A. R. and Alessi, D. R. (2010) 14-3-3 binding to LRRK2 is disrupted by multiple Parkinson's disease-associated mutations and regulates cytoplasmic localization. Biochem J. 430, 393-404
6 Li, X., Wang, Q. J., Pan, N., Lee, S., Zhao, Y., Chait, B. T. and Yue, Z. (2011) Phosphorylation-dependent 14-3-3 binding to LRRK2 is impaired by common mutations of familial Parkinson's disease. PLoS One. 6, e17153
7 Dzamko, N., Deak, M., Hentati, F., Reith, A. D., Prescott, A. R., Alessi, D. R. and Nichols, R. J. (2010
) Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization. Biochem J. 430, 405-413
8 Deng, X., Dzamko, N., Prescott, A., Davies, P., Liu, Q., Yang, Q., Lee, J. D., Patricelli, M. P., Nomanbhoy, T. K., Alessi, D. R. and Gray, N. S. (2011) Characterization of a selective inhibitor of the Parkinson's disease kinase LRRK2. Nat Chem Biol. 7, 203-205