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Leucine-rich repeat kinase 2 modulates retinoic acid-induced neuronal differentiation of murine embryonic stem cells.

Schulz C, Paus M, Frey K, Schmid R, Kohl Z, Mennerich D, Winkler J, Gillardon F - PLoS ONE (2011)

Bottom Line: LRRK2 is expressed in neural precursor cells suggesting a role in neurodevelopment.By contrast, expression of neurotransmitter receptors and neurotransmitter release was increased in LRRK2+/- cultures indicating that LRRK2 promotes neuronal differentiation.Alterations in phosphorylation of the putative LRRK2 substrates, translation initiation factor 4E binding protein 1 and moesin, do not appear to be involved in altered differentiation, rather there is indirect evidence that a regulatory signaling network comprising retinoic acid receptors, let-7 miRNA and downstream target genes/mRNAs may be affected in LRRK2-deficient stem cells in culture.

View Article: PubMed Central - PubMed

Affiliation: Boehringer Ingelheim Pharma GmbH & Co KG, CNS Research, Biberach an der Riss, Germany.

ABSTRACT

Background: Dominant mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent cause of Parkinson's disease, however, little is known about the biological function of LRRK2 protein. LRRK2 is expressed in neural precursor cells suggesting a role in neurodevelopment.

Methodology/principal findings: In the present study, differential gene expression profiling revealed a faster silencing of pluripotency-associated genes, like Nanog, Oct4, and Lin28, during retinoic acid-induced neuronal differentiation of LRRK2-deficient mouse embryonic stem cells compared to wildtype cultures. By contrast, expression of neurotransmitter receptors and neurotransmitter release was increased in LRRK2+/- cultures indicating that LRRK2 promotes neuronal differentiation. Consistently, the number of neural progenitor cells was higher in the hippocampal dentate gyrus of adult LRRK2-deficient mice. Alterations in phosphorylation of the putative LRRK2 substrates, translation initiation factor 4E binding protein 1 and moesin, do not appear to be involved in altered differentiation, rather there is indirect evidence that a regulatory signaling network comprising retinoic acid receptors, let-7 miRNA and downstream target genes/mRNAs may be affected in LRRK2-deficient stem cells in culture.

Conclusion/significance: Parkinson's disease-linked LRRK2 mutations that associated with enhanced kinase activity may affect retinoic acid receptor signaling during neurodevelopment and/or neuronal maintenance as has been shown in other mouse models of chronic neurodegenerative diseases.

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Retinoic acid receptor alpha is efficiently phosphorylated by recombinant LRRK2.(A) Autoradiogram demonstrating phosphorylation of recombinant tubulin-beta 2C (lane 1–3), heat-treated meosin (lane 4–6), and retinoic acid receptor alpha (lane 7–9) by recombinant LRRK2(G2019S) (amino acids 970-2527). Three-fold dilutions of each recombinant protein were incubated with kinase and [γ-33P]ATP for 45 min. (B) Coomassie Blue protein post-staining of the gel. (C) (Left panel) Kinase-dead LRRK2(D1994A) does not autophosphorylate or phosphorylate retinoic acid receptor alpha (lane 2) compared to active LRRK2(G2019S) (lane 1). (Right panel) Similar protein amounts are detected by Coomassie Blue protein staining.
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pone-0020820-g007: Retinoic acid receptor alpha is efficiently phosphorylated by recombinant LRRK2.(A) Autoradiogram demonstrating phosphorylation of recombinant tubulin-beta 2C (lane 1–3), heat-treated meosin (lane 4–6), and retinoic acid receptor alpha (lane 7–9) by recombinant LRRK2(G2019S) (amino acids 970-2527). Three-fold dilutions of each recombinant protein were incubated with kinase and [γ-33P]ATP for 45 min. (B) Coomassie Blue protein post-staining of the gel. (C) (Left panel) Kinase-dead LRRK2(D1994A) does not autophosphorylate or phosphorylate retinoic acid receptor alpha (lane 2) compared to active LRRK2(G2019S) (lane 1). (Right panel) Similar protein amounts are detected by Coomassie Blue protein staining.

Mentions: Gene expression profiling also provided a first hint that retinoic acid signaling may be altered in LRRK2+/− neurons during differentiation from ES cells. As shown in Table 1, several retinoic acid receptor target genes (e.g. retinoic acid induced 14, stimulated by retinoic acid gene 8, cellular retinoic acid binding protein 2) were differentially expressed, while mRNA levels of retinoic acid receptors alpha and beta were unaltered. Transcriptional regulation by retinoic acid receptors is modulated by various kinases (e.g. MAP kinases) through receptor phosphorylation thereby modulating binding to coactivators, repressors, or DNA [25]. This led us to investigate whether LRRK2 is able to phosphorylate retinoic acid receptors in vitro and to compare efficiency of phosphorylation of retinoic acid receptor to moesin and tubulin-beta 2C which are potently phosphorylated by recombinant LRRK2 [18]. Using threefold dilutions of each recombinant protein substrate we observed that retinoic acid receptors alpha (Figure 7 A, B) and beta (not shown) become phosphorylated by LRRK2 as efficiently as moesin and tubulin-beta 2C, respectively. As a negative control, kinase-dead LRRK2(D1994A) did not phosphorylate either protein under the same assay conditions (Figure 7C). By tandem mass spectrometry one phosphorylated peptide (DGGGLAPPPGSCSPSLSPSSNR) was detected in a tryptic digest of retinoic acid receptor alpha (63% sequence coverage), and serine-445 was identified as phosphorylation site.


Leucine-rich repeat kinase 2 modulates retinoic acid-induced neuronal differentiation of murine embryonic stem cells.

Schulz C, Paus M, Frey K, Schmid R, Kohl Z, Mennerich D, Winkler J, Gillardon F - PLoS ONE (2011)

Retinoic acid receptor alpha is efficiently phosphorylated by recombinant LRRK2.(A) Autoradiogram demonstrating phosphorylation of recombinant tubulin-beta 2C (lane 1–3), heat-treated meosin (lane 4–6), and retinoic acid receptor alpha (lane 7–9) by recombinant LRRK2(G2019S) (amino acids 970-2527). Three-fold dilutions of each recombinant protein were incubated with kinase and [γ-33P]ATP for 45 min. (B) Coomassie Blue protein post-staining of the gel. (C) (Left panel) Kinase-dead LRRK2(D1994A) does not autophosphorylate or phosphorylate retinoic acid receptor alpha (lane 2) compared to active LRRK2(G2019S) (lane 1). (Right panel) Similar protein amounts are detected by Coomassie Blue protein staining.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3111438&req=5

pone-0020820-g007: Retinoic acid receptor alpha is efficiently phosphorylated by recombinant LRRK2.(A) Autoradiogram demonstrating phosphorylation of recombinant tubulin-beta 2C (lane 1–3), heat-treated meosin (lane 4–6), and retinoic acid receptor alpha (lane 7–9) by recombinant LRRK2(G2019S) (amino acids 970-2527). Three-fold dilutions of each recombinant protein were incubated with kinase and [γ-33P]ATP for 45 min. (B) Coomassie Blue protein post-staining of the gel. (C) (Left panel) Kinase-dead LRRK2(D1994A) does not autophosphorylate or phosphorylate retinoic acid receptor alpha (lane 2) compared to active LRRK2(G2019S) (lane 1). (Right panel) Similar protein amounts are detected by Coomassie Blue protein staining.
Mentions: Gene expression profiling also provided a first hint that retinoic acid signaling may be altered in LRRK2+/− neurons during differentiation from ES cells. As shown in Table 1, several retinoic acid receptor target genes (e.g. retinoic acid induced 14, stimulated by retinoic acid gene 8, cellular retinoic acid binding protein 2) were differentially expressed, while mRNA levels of retinoic acid receptors alpha and beta were unaltered. Transcriptional regulation by retinoic acid receptors is modulated by various kinases (e.g. MAP kinases) through receptor phosphorylation thereby modulating binding to coactivators, repressors, or DNA [25]. This led us to investigate whether LRRK2 is able to phosphorylate retinoic acid receptors in vitro and to compare efficiency of phosphorylation of retinoic acid receptor to moesin and tubulin-beta 2C which are potently phosphorylated by recombinant LRRK2 [18]. Using threefold dilutions of each recombinant protein substrate we observed that retinoic acid receptors alpha (Figure 7 A, B) and beta (not shown) become phosphorylated by LRRK2 as efficiently as moesin and tubulin-beta 2C, respectively. As a negative control, kinase-dead LRRK2(D1994A) did not phosphorylate either protein under the same assay conditions (Figure 7C). By tandem mass spectrometry one phosphorylated peptide (DGGGLAPPPGSCSPSLSPSSNR) was detected in a tryptic digest of retinoic acid receptor alpha (63% sequence coverage), and serine-445 was identified as phosphorylation site.

Bottom Line: LRRK2 is expressed in neural precursor cells suggesting a role in neurodevelopment.By contrast, expression of neurotransmitter receptors and neurotransmitter release was increased in LRRK2+/- cultures indicating that LRRK2 promotes neuronal differentiation.Alterations in phosphorylation of the putative LRRK2 substrates, translation initiation factor 4E binding protein 1 and moesin, do not appear to be involved in altered differentiation, rather there is indirect evidence that a regulatory signaling network comprising retinoic acid receptors, let-7 miRNA and downstream target genes/mRNAs may be affected in LRRK2-deficient stem cells in culture.

View Article: PubMed Central - PubMed

Affiliation: Boehringer Ingelheim Pharma GmbH & Co KG, CNS Research, Biberach an der Riss, Germany.

ABSTRACT

Background: Dominant mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most prevalent cause of Parkinson's disease, however, little is known about the biological function of LRRK2 protein. LRRK2 is expressed in neural precursor cells suggesting a role in neurodevelopment.

Methodology/principal findings: In the present study, differential gene expression profiling revealed a faster silencing of pluripotency-associated genes, like Nanog, Oct4, and Lin28, during retinoic acid-induced neuronal differentiation of LRRK2-deficient mouse embryonic stem cells compared to wildtype cultures. By contrast, expression of neurotransmitter receptors and neurotransmitter release was increased in LRRK2+/- cultures indicating that LRRK2 promotes neuronal differentiation. Consistently, the number of neural progenitor cells was higher in the hippocampal dentate gyrus of adult LRRK2-deficient mice. Alterations in phosphorylation of the putative LRRK2 substrates, translation initiation factor 4E binding protein 1 and moesin, do not appear to be involved in altered differentiation, rather there is indirect evidence that a regulatory signaling network comprising retinoic acid receptors, let-7 miRNA and downstream target genes/mRNAs may be affected in LRRK2-deficient stem cells in culture.

Conclusion/significance: Parkinson's disease-linked LRRK2 mutations that associated with enhanced kinase activity may affect retinoic acid receptor signaling during neurodevelopment and/or neuronal maintenance as has been shown in other mouse models of chronic neurodegenerative diseases.

Show MeSH
Related in: MedlinePlus