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LRRK2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation site status

Missense mutations in the Leucine-Rich Repeat protein Kinase 2 (LRRK2) gene are the most common genetic predisposition to develop Parkinson’s disease (PD) (Farrer et al., 2005; Skipper et al., 2005; Di Fonzo et al., 2006; Healy et al., 2008; Paisan-Ruiz et al., 2008; Lesage et al., 2010). LRRK2 is a...

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Autores principales: Reynolds, April, Doggett, Elizabeth A., Riddle, Steve M., Lebakken, Connie S., Nichols, R. Jeremy
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4068021/
https://www.ncbi.nlm.nih.gov/pubmed/25009464
http://dx.doi.org/10.3389/fnmol.2014.00054
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author Reynolds, April
Doggett, Elizabeth A.
Riddle, Steve M.
Lebakken, Connie S.
Nichols, R. Jeremy
author_facet Reynolds, April
Doggett, Elizabeth A.
Riddle, Steve M.
Lebakken, Connie S.
Nichols, R. Jeremy
author_sort Reynolds, April
collection PubMed
description Missense mutations in the Leucine-Rich Repeat protein Kinase 2 (LRRK2) gene are the most common genetic predisposition to develop Parkinson’s disease (PD) (Farrer et al., 2005; Skipper et al., 2005; Di Fonzo et al., 2006; Healy et al., 2008; Paisan-Ruiz et al., 2008; Lesage et al., 2010). LRRK2 is a large multi-domain phosphoprotein with a GTPase domain and a serine/threonine protein kinase domain whose activity is implicated in neuronal toxicity; however the precise mechanism is unknown. LRRK2 autophosphorylates on several serine/threonine residues across the enzyme and is found constitutively phosphorylated on Ser910, Ser935, Ser955, and Ser973, which are proposed to be regulated by upstream kinases. Here we investigate the phosphoregulation at these sites by analyzing the effects of disease-associated mutations Arg1441Cys, Arg1441Gly, Ala1442Pro, Tyr1699Cys, Ile2012Thr, Gly2019Ser, and Ile2020Thr. We also studied alanine substitutions of phosphosite serines 910, 935, 955, and 973 and specific LRRK2 inhibition on autophosphorylation of LRRK2 Ser1292, Thr1491, Thr2483 and phosphorylation at the cellular sites. We found that mutants in the Roc-COR domains, including Arg1441Cys, Arg1441His, Ala1442Pro, and Tyr1699Cys, can positively enhance LRRK2 kinase activity, while concomitantly inducing the dephosphorylation of the cellular sites. Mutation of the cellular sites individually did not affect LRRK2 intrinsic kinase activity; however, Ser910/935/955/973Ala mutations trended toward increased kinase activity of LRRK2. Increased cAMP levels did not lead to increased LRRK2 cellular site phosphorylation, 14-3-3 binding or kinase activity. In cells, inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser1292 by Calyculin A and Okadaic acid sensitive phosphatases, while the cellular sites are dephosphorylated by Calyculin A sensitive phosphatases. These findings indicate that comparative analysis of both Ser1292 and Ser910/935/955/973 phosphorylation sites will provide important and distinct measures of LRRK2 kinase and biological activity in vitro and in vivo.
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spelling pubmed-40680212014-07-09 LRRK2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation site status Reynolds, April Doggett, Elizabeth A. Riddle, Steve M. Lebakken, Connie S. Nichols, R. Jeremy Front Mol Neurosci Neuroscience Missense mutations in the Leucine-Rich Repeat protein Kinase 2 (LRRK2) gene are the most common genetic predisposition to develop Parkinson’s disease (PD) (Farrer et al., 2005; Skipper et al., 2005; Di Fonzo et al., 2006; Healy et al., 2008; Paisan-Ruiz et al., 2008; Lesage et al., 2010). LRRK2 is a large multi-domain phosphoprotein with a GTPase domain and a serine/threonine protein kinase domain whose activity is implicated in neuronal toxicity; however the precise mechanism is unknown. LRRK2 autophosphorylates on several serine/threonine residues across the enzyme and is found constitutively phosphorylated on Ser910, Ser935, Ser955, and Ser973, which are proposed to be regulated by upstream kinases. Here we investigate the phosphoregulation at these sites by analyzing the effects of disease-associated mutations Arg1441Cys, Arg1441Gly, Ala1442Pro, Tyr1699Cys, Ile2012Thr, Gly2019Ser, and Ile2020Thr. We also studied alanine substitutions of phosphosite serines 910, 935, 955, and 973 and specific LRRK2 inhibition on autophosphorylation of LRRK2 Ser1292, Thr1491, Thr2483 and phosphorylation at the cellular sites. We found that mutants in the Roc-COR domains, including Arg1441Cys, Arg1441His, Ala1442Pro, and Tyr1699Cys, can positively enhance LRRK2 kinase activity, while concomitantly inducing the dephosphorylation of the cellular sites. Mutation of the cellular sites individually did not affect LRRK2 intrinsic kinase activity; however, Ser910/935/955/973Ala mutations trended toward increased kinase activity of LRRK2. Increased cAMP levels did not lead to increased LRRK2 cellular site phosphorylation, 14-3-3 binding or kinase activity. In cells, inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser1292 by Calyculin A and Okadaic acid sensitive phosphatases, while the cellular sites are dephosphorylated by Calyculin A sensitive phosphatases. These findings indicate that comparative analysis of both Ser1292 and Ser910/935/955/973 phosphorylation sites will provide important and distinct measures of LRRK2 kinase and biological activity in vitro and in vivo. Frontiers Media S.A. 2014-06-24 /pmc/articles/PMC4068021/ /pubmed/25009464 http://dx.doi.org/10.3389/fnmol.2014.00054 Text en Copyright © 2014 Reynolds, Doggett, Riddle, Lebakken and Nichols. http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Neuroscience
Reynolds, April
Doggett, Elizabeth A.
Riddle, Steve M.
Lebakken, Connie S.
Nichols, R. Jeremy
LRRK2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation site status
title LRRK2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation site status
title_full LRRK2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation site status
title_fullStr LRRK2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation site status
title_full_unstemmed LRRK2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation site status
title_short LRRK2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation site status
title_sort lrrk2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation site status
topic Neuroscience
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4068021/
https://www.ncbi.nlm.nih.gov/pubmed/25009464
http://dx.doi.org/10.3389/fnmol.2014.00054
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