Leucine-Rich Repeat Kinase 2 (LRRK2) phosphorylates p53 and induces p21WAF1/CIP1 expression

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.

LRRK2 impairs autophagy by mediating phosphorylation of leucyl‐tRNA synthetase

“…Phosphorylated LRS was detected by isotope labelling and pTXR antibody, which specifically recognized phosphorylated threonine in the TXR site (Figure A). TXR is a putative phosphorylation site of LRRK2 . LRS can bind to ATP for its aminoacylation activity.…”

“…Commercial N‐terminal truncation fused to GST (GST‐ΔN, Invitrogen) was used for the in vitro kinase assay with [γ‐ 32 P]‐ATP, as described previously . Either His‐tagged or GST‐fused recombinant LRS WT or mutant protein was induced, purified from E coli BL21 (DE3) strain and used as kinase substrate.…”

“…Coimmunoprecipitation and Western blot analysis were carried out as described previously . Immunoprecipitates or supernatants of cell lysates were analysed with indicated antibodies.…”

“…The amount of bound LRS was compared after Western blot analysis with LRS antibody. Immunostaining was carried out as described previously …”

“…Normal human fibroblast (IMR‐90, ATCC) and PD patient fibroblasts with LRRK2‐G2019S mutation (#ND29492, Coriell Cell Repositories) were reprogrammed to induced pluripotent stem cells (iPSCs) as described in previous reports . They were used to differentiate to dopaminergic neurons . Lysates of differentiated neurons were subjected to Western blot analysis using antibodies indicated.…”