Abstract
BackgroundLeucine-rich repeat kinase 2 (LRRK2) is a gene in which a mutation causes Parkinson’s disease (PD), and p53 is a prototype tumor suppressor. In addition, activation of p53 in patient with PD has been reported by several studies. Because phosphorylation of p53 is critical for regulating its activity and LRRK2 is a kinase, we tested whether p53 is phosphorylated by LRRK2.ResultsLRRK2 phosphorylates threonine (Thr) at TXR sites in an in vitro kinase assay, and the T304 and T377 were identified as putative phosphorylated residues. An increase of phospho-Thr in the p53 TXR motif was confirmed in the cells overexpressing G2019S, and human induced pluripotent stem (iPS) cells of a G2019S carrier. Interactions between LRRK2 and p53 were confirmed by co-immunoprecipitation of lysates of differentiated SH-SY5Y cells. LRRK2 mediated p53 phosphorylation translocalizes p53 predominantly to nucleus and increases p21WAF1/CIP1 expression in SH-SY5Y cells based on reverse transcription-polymerase chain reaction and Western blot assay results. The luciferase assay using the p21WAF1/CIP1 promoter-reporter also confirmed that LRRK2 kinase activity increases p21 expression. Exogenous expression of G2019S and the phosphomimetic p53 T304/377D mutants increased expression of p21WAF1/CIP1 and cleaved PARP, and cytotoxicity in the same cells. We also observed increase of p21 expression in rat primary neuron cells after transient expression of p53 T304/377D mutants and the mid-brain lysates of the G2019S transgenic mice.Conclusionp53 is a LRRK2 kinase substrate. Phosphorylation of p53 by LRRK2 induces p21WAF1/CIP1 expression and apoptosis in differentiated SH-SY5Y cells and rat primary neurons.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-015-0145-7) contains supplementary material, which is available to authorized users.
Highlights
Leucine-rich repeat kinase 2 (LRRK2) is a gene in which a mutation causes Parkinson’s disease (PD), and p53 is a prototype tumor suppressor
P53 is encoded by TP53, and is a well-known tumor suppressor that is mutated in numerous types of tumor cells [33]. p53 is a transcription factor that is degraded through interacting with MDM2 under normal condition, but p53 is phosphorylated at specific residues, become stabilized and resistant to its own degradation during exposure to genomic or other stressors [34, 35]
Because of p53 activation in PD brains, we tested whether LRRK2 phosphorylates p53 using E. coliexpressed recombinant p53 protein and [γ-32P]ATP in an in vitro kinase assay
Summary
LRRK2 phosphorylates p53 in vitro We were interested in identifying novel LRRK2 kinase substrates. The Western blot result using the p-TXR antibody after the in vitro kinase assay detected phosphorylated p53 proteins after incubation with LRRK2 WT or GS, but not with DA Treating SHSY5Y cells with a LRRK2 kinase inhibitor, LRRK2-IN-1, exhibited the similar results- decrease of p21 mRNA and protein levels based on the RT-PCR and Western blot assays (Additional file 4: Figure S4C-D). The Western blot with the G2019S TG mice mid-brain lysates revealed that p21 expression in the TG mice significantly increased compared to that in the non TG littermates (Fig. 6d) These data suggested the activation of p21 and apoptotic cell death via specific p53 phosphorylation by LRRK2 kinase activity
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