GnRH Receptors

Therefore, the T304/377D mutant may have only exhibited a partial effect on LRRK2-mediated p53 phosphorylation, although we used the mutant as a phosphomimetic protein in this study

Therefore, the T304/377D mutant may have only exhibited a partial effect on LRRK2-mediated p53 phosphorylation, although we used the mutant as a phosphomimetic protein in this study. 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. Conclusion p53 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 material The online version of this article (doi:10.1186/s13041-015-0145-7) contains supplementary material, which is available to authorized users. Background Leucine-rich repeat kinase 2 ((-synuclein), (Parkin), and [1, 2]. LRRK2 contains both kinase and GTPase domains [3C5] whose activities are critical for signal transduction. Moreover, the G2019S pathogenic mutation [6] increases kinase activity [7, 8] and affects various pathogenic phenotypes such as increased neuronal cytotoxicity and protein aggregation [7, 9, 10], decreased neurite length [11, 12] and changes in the autophagy rate [12, 13]. In addition, pharmacological inhibition of kinase activity rescues pathogenic phenotypes, such as neurocytotoxicity and defective neurite outgrowth [14]. Because of the relevance of LRRK2 kinase activity to PD pathogenesis, LRRK2 has emerged as a therapeutic target of PD, and the effort to identify LRRK2 kinase substrates and chemical inhibitors has intensified [15C21]. To date, several diverse proteins have been reported as potential LRRK2 kinase substrates. Some examples are -tubulin [22], ArfGAP1 [23, 24], tau [25], members of the mitogen-activated protein kinase kinase family [26], eukaryotic initiation factor 4E-binding protein (4E-BP; [27]), Akt1 [28], ribosomal protein s15 [19], endophilin A [18], Rab5 [21], Bcl-2 [29] and Snapin [20]. It is unclear whether these proteins are actual physiological substrates. Some studies suggest that LRRK2 plays roles in 360A vesicle trafficking, autophagy, mitochondrial dysfunction, and inflammation [13, 18, 30C32]. In addition, one study identified several ribosomal proteins as LRRK2 kinase substrates using an unbiased proteomics suggesting that LRRK2 is a translational regulator [19] along with a previous 4E-BP study [27]. p53 is encoded by kinase assay. The GST-N LRRK2 wild type (WT) phosphorylated p53 significantly and the corresponding G2019S (GS) and 360A kinase-dead D1994A (DA) mutant proteins enhanced and nullified p53 phosphorylation, respectively, as expected (Fig.?1A-a). The 360A GST-N LRRK2 was used instead of the full length WT because the former exhibited stronger kinase activity than the latter (data not shown). Open in a separate 360A window Fig. 1 LRRK2 phosphorylates p53 at T304 and T377 in an kinase assay. A. Recombinant GST-N LRRK2 phosphorylates recombinant human p53 WT (A) or mutant proteins B. The recombinant p53 protein was subjected to the kinase IL22 antibody assay with cold ATP (b???d) or 32P-ATP (a) and analyzed by Western blot with the indicated antibodies (b, p-TXR: phospho-TXR; c, LRRK2: MJFF2; d, p53:DO1) or autoradiography (a). WT: wild type, GS: G2019S, DA: D1994A. C. A schematic diagram of the p53 functional domains and location of the human p53 TXR motifs. Thr 387 which was used as a control was also shown. Numbers below the gel figures are the densitometric results for each band, [radiolabeled p53(a)]/[total p53(d)] Because the TXR motif is suggested as a conserved LRRK2 phosphorylation site [20, 40C42], we checked whether such sites are present in p53. We found three sites: 211TFR, 304TKR, and 377TSR (numbers indicate position of threonine in human p53 protein, Fig.?1c). To investigate whether p53 is phosphorylated on these sites, we tested to see if phosphorylated p53 was detectable by Western blot with anti-phospho-TXR antibody (p-TXR). p-TXR is a commercial phospho-specific antibody that recognizes a phosphorylated threonine residue in the TXR motif. This antibody was shown to detect a specific phosphorylated threonine (T1410) in the LRRK2 TXR motif (TQR) after autophosphorylation of LRRK2 [41]. The Western blot result using the p-TXR antibody after the kinase assay detected phosphorylated p53 proteins after incubation with LRRK2 WT or GS, but not with DA. This is similar to the autoradiogram shown in Fig.?1A-a, suggesting that p53 was phosphorylated at threonines in the TXR sites (Fig.?1A-b). The kinase assay was carried out with p53 WT and the mutant proteins to detect the p53 phosphorylation sites. We first used the 1C293 deletion mutant of p53, in which the C-terminus of 100 amino acids has been.