A neo-substrate that amplifies catalytic activity of parkinson's-disease-related kinase PINK1

Abstract

Mitochondria have long been implicated in the pathogenesis of Parkinson's disease (PD). Mutations in the mitochondrial kinase PINK1 that reduce kinase activity are associated with mitochondrial defects and result in an autosomal-recessive form of early-onset PD. Therapeutic approaches for enhancing the activity of PINK1 have not been considered because no allosteric regulatory sites for PINK1 are known. Here, we show that an alternative strategy, a neo-substrate approach involving the ATP analog kinetin triphosphate (KTP), can be used to increase the activity of both PD-related mutant PINK1(G309D) and PINK1(WT). Moreover, we show that application of the KTP precursor kinetin to cells results in biologically significant increases in PINK1 activity, manifest as higher levels of Parkin recruitment to depolarized mitochondria, reduced mitochondrial motility in axons, and lower levels of apoptosis. Discovery of neo-substrates for kinases could provide a heretofore-unappreciated modality for regulating kinase activity.

Figure 1. Neo-substrate Kinetin Triphosphate (KTP) amplifies PINK1 kinase activity in-vitro

(A) Chemical structure of kinase substrate adenosine triphosphate with gamma thiophosphate (ATPγS) and neo-substrate kinetin triphosphate gamma thiophosphate (KTPγS). (B–D) PINK1 kinase assay (B), with substrate _60–704TRAP1 (1 mg/ml decreasing by 1/3) and 500 μM indicated nucleotide (C) or (D), PINK1 alone (4.3 μM) 100, 200, 400 μM nucleotide analyzed by immunoblotting for thiophospho labeled TRAP1 and PINK1. (E) PINK1 autophosphorylation site identified by specific peptide capture and LCMSMS found only with PINK1^wt and KTPγS indicating this nucleotide is utilized as a bona fide substrate (F) SF21 produced PINK1 was incubated with _60–704TRAP1 and the indicated nucleotide (structure shown in panel C) and analyzed as in (B–D) (G) PINK1 kinase assay with indicated nucleotide (250 to 1250 μM in increments of 250 μM) analyzed as in (B–D) reveals much reduced phosphorylation activity with PINK1^KDDD (lanes 11–20), increased autophosphorylation was seen with neo-substrate KTPγS over endogenous substrate ATPγS. See also Figure S1 and S2

Figure 2. Neo-substrate precursor kinetin leads to KTP in human cells and activates PINK1 dependent phosphorylation of Bcl-xL

(A) Schematic of the ribosylation reaction of kinetin to KMP via APRT and PRPP followed by cellular conversion to KTP. (B) LCMS analysis of production of either AMP (adenine) or KMP (kinetin and 9MK) by LCMS analysis reveals that kinetin can undergo APRT mediated ribosylation to KMP, but N⁹ methyl-kinetin cannot. (C) LCMS analysis of kinetin reaction; major peak represents KMP+H peak at 428.3 m/z. (D) HPLC analysis of standards (ADP, GTP, ATP, KDP, KTP, BTP) or cellular lysate of DMSO or kinetin treated HeLa cells with 250 μM BTP* addition reveals a novel peak present in kinetin treated cells at 31.39 minutes. (E) Absorbance spectrum of peak at 31.39 minutes in kinetin treated cells compared to absorbance spectrum of KTP in standard. (F) Zoom of HPLC analysis of DMSO or kinetin treated cell lysate with BTP addition, or kinetin cell lysate with BTP addition and KTP addition reveals an increase in the peak that co-elutes with KTP (186%) where BTP decreases to 76% of original area suggesting the peak is KTP. (G) Immunoblot analysis with the indicated antibodies for phosphorylation on serine 62 of Bcl-xL reveals an increase following kinetin treatment. (H) Quantitative analysis of data shown in G where a significant increase in the phosphorylation of Bcl-xL on serine 62 with pre-treatment with kinetin versus DMSO or adenine (p=0.01, p=0.04;t-test) in SH-Control expressing cells not in SH-PINK1 cells. (P values shown are the result of two-tailed student’s t-test, *P<0.05 **P≤0.01) (all values shown are mean ± sem) See also Table 1 and Figure S3

Figure 3. PINK1 neo-substrate kinetin accelerates PINK1 dependent Parkin recruitment in cells

(A) Chemical structure of adenine or kinase neo-substrate precursor kinetin. (B) Schematic depicting HeLa cell drug treatment. (C–D) HeLa cells treated with indicated drug, co-transfected with mitoGFP, mCherryParkin, and indicated PINK1 construct imaged at either 10 or 15 minutes after 5 μM CCCP addition. (E–F) Kinetin treated cells reached R₅₀ significantly faster than with adenine or DMSO (all data shown is mean ± sem) by two-way ANOVA analysis kinetin has an effect in both cases when compared to adenine (wt; F=25.41 p<0.0001, G309D; F=31.89, p<0.0001) and DMSO (wt; F=21.94 p<0.0001, G309D; F=12.79, p<0.0011), no significant difference for DMSO adenine in either case (at least 150 cells/experiment n=3 experiments-all values are mean ± sem). See also Table 2 and Figure S4

Figure 4. Kinetin halts axonal mitochondrial motility in a PINK1 dependent manner

(A) Chemical structure of negative control non-metabolizable kinetin analog 9-methyl-kinetin (B–E) Kymograph for analysis of mitochondrial movement in representative PINK1^wt expressing rat derived hippocampal axons transfected with mitoGFP. Scale bar represents 10 μm. (F) The percentage of time each mitochondrion was in motion was determined and averaged. Kinetin significantly blocks mitochondrial motility whereas 9MK has no effect (DMSO-kinetin, P=0.0005; DMSO-9MK, P=0.86) (G) Kinetin induces a small decrease in velocity (DMSO-kinetin, P=0.03; DMSO-9MK, P=0.24). (H) Kymograph for analysis of mitochondrial movement in C57BL/6 shows a response to kinetin (kinetin 9MK, P<0.0001), whereas in PINK1 knockout derived hippocampal axons kinetin has no effect (kinetin 9MK, P=0.64). (I) Both kinetin and 9MK have no effect on mitochondrial velocity of moving mitochondrion (C57BL/6 kinetin-9MK, P=0.64; PINK1 KO, P=0.074) (all values are mean ± sem, analysis was two tailed students t-test) See also Figure S5

Figure 5. Kinetin inhibits oxidative stress induced apoptosis in human cells in a PINK1 dependent manner

(A) Caspase 3/7 cleavage activity assay following pre-treatment with DMSO, adenine or kinetin for 48 hours followed by MG132 treatment for 12 hours compared to no MG132 treated cells reveals reduced Caspase 3/7 activity (p=0.005, p=0.004;t-test) only in sh-control expressing cells not in cells expressing shRNA against PINK1. (B) Caspase 3/7 cleavage activity of SH-SY5Y cells pre-treated with indicated concentration of adenine or kinetin for 96 hours followed by MG132 treatment for 12 hours in all conditions. Two-way ANOVA analysis revealed that kinetin has an effect when compared to DMSO or adenine (Figure 5B) (DMSO; F=34.95 p<0.0001;adenine; F=38.37 p<0.0001) but no statistically significant effect (DMSO; F=3.552 p=0.084;adenine; F=1.7 p=0.215) in cells expressing an shRNA against PINK1 (C) SH-SY5Y cells pre-treated as above, were stained with FITC conjugated Annexin V and propidium iodide and analyzed by FACS. (D) Quantification of (C) shows kinetin treated cells have significantly lower induction of apoptosis (DMSO-kinetin, P=0.0023) but adenine had no effect (DMSO-adenine, P=0.09). (E) Indicated SH-SY5Y cell lines were treated as is (A). Kinetin treated cells had significantly lower induction of apoptosis only when PINK1 was present (normalized to DMSO control untreated cells)(sh-control DMSO-kinetin, P=0.008) (sh-PINK1 DMSO-kinetin, P=0.23)(sh-Control DMSO-adenine, P=0.48; sh-PINK1 DMSO-adenine, P=0.23) (all values are mean ± sem, analysis was Wilcoxon t-test) See also Figure S6