LRRK2 Kinase Activity Is Dependent on LRRK2 GTP Binding Capacity but Independent of LRRK2 GTP Binding
Leucine rich repeat kinase 2 (LRRK2) is a Parkinson's disease (PD) gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound “...
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| Format: | Online |
| Language: | English |
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Public Library of Science
2011
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| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3155532/ |
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pubmed-3155532 |
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pubmed-31555322011-08-19 LRRK2 Kinase Activity Is Dependent on LRRK2 GTP Binding Capacity but Independent of LRRK2 GTP Binding Taymans, Jean-Marc Vancraenenbroeck, Renée Ollikainen, Petri Beilina, Alexandra Lobbestael, Evy De Maeyer, Marc Baekelandt, Veerle Cookson, Mark R. Research Article Leucine rich repeat kinase 2 (LRRK2) is a Parkinson's disease (PD) gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound “on” state and a GDP bound “off” state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC) GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC. Public Library of Science 2011-08-12 /pmc/articles/PMC3155532/ /pubmed/21858031 http://dx.doi.org/10.1371/journal.pone.0023207 Text en Taymans et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
| repository_type |
Open Access Journal |
| institution_category |
Foreign Institution |
| institution |
US National Center for Biotechnology Information |
| building |
NCBI PubMed |
| collection |
Online Access |
| language |
English |
| format |
Online |
| author |
Taymans, Jean-Marc Vancraenenbroeck, Renée Ollikainen, Petri Beilina, Alexandra Lobbestael, Evy De Maeyer, Marc Baekelandt, Veerle Cookson, Mark R. |
| spellingShingle |
Taymans, Jean-Marc Vancraenenbroeck, Renée Ollikainen, Petri Beilina, Alexandra Lobbestael, Evy De Maeyer, Marc Baekelandt, Veerle Cookson, Mark R. LRRK2 Kinase Activity Is Dependent on LRRK2 GTP Binding Capacity but Independent of LRRK2 GTP Binding |
| author_facet |
Taymans, Jean-Marc Vancraenenbroeck, Renée Ollikainen, Petri Beilina, Alexandra Lobbestael, Evy De Maeyer, Marc Baekelandt, Veerle Cookson, Mark R. |
| author_sort |
Taymans, Jean-Marc |
| title |
LRRK2 Kinase Activity Is Dependent on LRRK2 GTP Binding Capacity but Independent of LRRK2 GTP Binding |
| title_short |
LRRK2 Kinase Activity Is Dependent on LRRK2 GTP Binding Capacity but Independent of LRRK2 GTP Binding |
| title_full |
LRRK2 Kinase Activity Is Dependent on LRRK2 GTP Binding Capacity but Independent of LRRK2 GTP Binding |
| title_fullStr |
LRRK2 Kinase Activity Is Dependent on LRRK2 GTP Binding Capacity but Independent of LRRK2 GTP Binding |
| title_full_unstemmed |
LRRK2 Kinase Activity Is Dependent on LRRK2 GTP Binding Capacity but Independent of LRRK2 GTP Binding |
| title_sort |
lrrk2 kinase activity is dependent on lrrk2 gtp binding capacity but independent of lrrk2 gtp binding |
| description |
Leucine rich repeat kinase 2 (LRRK2) is a Parkinson's disease (PD) gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound “on” state and a GDP bound “off” state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC) GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC. |
| publisher |
Public Library of Science |
| publishDate |
2011 |
| url |
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3155532/ |
| _version_ |
1611470770896109568 |