Available online at www.sciencedirect.com ScienceDirect JGG

Journal of Genetics and Genomics 43 (2016) 369e379

ORIGINAL RESEARCH

Parkin Somatic Mutations Link Melanoma and Parkinson’s Disease

Lotan Levin a, Shani Srour a, Jared Gartner b, Oxana Kapitansky a, Nouar Qutob c, Shani Dror a, Tamar Golan a, Roy Dayan a, Ronen Brener d, Tamar Ziv e, Mehdi Khaled f, Ora Schueler-Furman g, Yardena Samuels c, Carmit Levy a,*

a Department of Human Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel b Surgery Branch, National Cancer Institute, NIH, Bethesda, MD 20892, USA c Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel d Oncology Institute, Edith Wolfson Medical Center, Holon 5822012, Israel e Technion Smoler Proteomics Center, Faculty of Biology, Technion e Israel Institute of Technology, Haifa 32000, Israel f Institut Gustave Roussy, INSERM U753, Villejuif 94805, France g Department of Microbiology and Molecular Genetics, Institute for Medical Research Israel-Canada, Faculty of Medicine, Hebrew University, Jerusalem 91120, Israel

Received 30 October 2015; revised 11 May 2016; accepted 12 May 2016 Available online 13 May 2016

ABSTRACT

Epidemiological studies suggest a direct link between melanoma and Parkinson’s disease (PD); however, the underlying molecular basis is unknown. Since mutations in are the major driver of early-onset PD and Parkin was recently reported to play a role in cancer development, we hypothesized that Parkin links melanoma and PD. By analyzing whole exome/genome sequencing of Parkin from 246 melanoma patients, we identified five non-synonymous mutations, three synonymous mutations, and one splice region variant in Parkin in 3.6% of the samples. In vitro analysis showed that wild-type Parkin plays a tumor suppressive role in melanoma development resulting in cell-cycle arrest, reduction of metabolic activity, and apoptosis. Using a mass spectrometry-based analysis, we identified potential Parkin substrates in melanoma and generated a functional association network. The activity of mutated Parkin was assessed by protein structure modeling and examination of Parkin E3 ligase activity. The Parkin-E28K mutation impairs Parkin ubiquitination activity and abolishes its tumor suppressive effect. Taken together, our analysis of genomic sequence and in vitro data indicate that Parkin is a potential link between melanoma and Parkinson’s disease. Our findings suggest new approaches for early diagnosis and treatment against both diseases.

KEYWORDS: Melanoma; Parkinson’s disease; Parkin; Mutation

INTRODUCTION seem to have very different, even opposite, phenotypes at the cellular level, as melanoma results from an uncontrolled, Melanoma, a melanocytic neoplasm, is responsible for 80% of extensive proliferation of cells, whereas PD is characterized by skin cancer mortality (Flaherty et al., 2012). Parkinson’s dis- degeneration and death of dopaminergic neurons. Intriguingly, ease (PD) is the most common neurodegenerative movement however, epidemiological studies indicate a potential rela- disorder; PD affects 1% of the population above 60 years of tionship between melanoma and PD (Herrero Hernandez, age (Veeriah et al., 2010; Pan et al., 2011). Melanoma and PD 2009; Bertoni et al., 2010; Inzelberg et al., 2011; Liu et al., 2011; Kareus et al., 2012), demonstrating that PD patients * Corresponding author. Tel: þ972 3 640 9900, fax: þ972 3 640 5168. and their relatives have a higher frequency of melanoma than E-mail address: [email protected] (C. Levy). http://dx.doi.org/10.1016/j.jgg.2016.05.005 1673-8527/Copyright Ó 2016, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved. 370 L. Levin et al. / Journal of Genetics and Genomics 43 (2016) 369e379 the general population, and vice versa (Herrero Hernandez, RESULTS 2009; Gao et al., 2011; Liu et al., 2011). Recently, Parkin was also suggested as a genetic link between melanoma and Somatic mutations of the Parkin in human PD (Hu et al., 2016). However, the molecular mechanism melanoma patients behind this link is still unknown. Loss-of-function mutations in the Parkin gene (also To determine whether mutations in Parkin gene are present in named PARK2, PRKN, MIM#602544) are responsible for melanoma, we compiled somatic mutation data from whole nearly 50% of early onset PD cases (Kahle and Haass, 2004; exome/genome sequencing sources (Berger et al., 2012; Hodis Guo et al., 2008, 2010). Somatic mutations in the Parkin et al., 2012; Krauthammer et al., 2012; Nikolaev et al., 2012). gene are a known cause of autosomal recessive juvenile PD These data and the analysis methods were previously (MIM#600116), in which a progressive death of neurons in described (Dutton-Regester et al., 2014). Our analysis revealed thesubstantianigratakesplace(Romani-Aumedes et al., mutations in 3.6% of the 246 samples analyzed. There were 2014). Alterations in the Parkin gene are also associated six non-synonymous mutations, three synonymous mutations, with other diseases as demonstrated by MalaCards (Table and one splice region variant (Table 1). The Parkin protein has S1), such as lung cancer (MIM#211980), ovarian cancer an N-terminal ubiquitin-like (UBL) domain, which is (MIM#167000) and susceptibility to leprosy (MIM#607572). responsible for the recognition of destined for ubiq- Parkin is an E3 ubiquitin ligase responsible for substrate uitination. The C-terminal RING box includes two zinc fingers recognition in the ubiquitination process (Hershko and separated by an in-between RING (IBR) domain that is Ciechanover, 1998) that leads to substrate degradation in responsible for specific interaction with E2 enzymes (Imai the proteasome (Hampe et al., 2006). The known substrates et al., 2000; Shimura et al., 2000). A linker region connects of Parkin include cell cycle regulators, e.g., Cyclin D1 (Yeo the UBL and RING domains; this linker contains cleavage et al., 2012) and Cyclin E (Veeriah et al., 2010); apoptosis sites for caspases 1 and 8 (Shimura et al., 2000; Kahle and mediators, e.g., Bax (Johnson et al., 2012)andBcl2(Chen Haass, 2004)(Fig. 1A). The melanoma-specific mutations et al., 2010); proteins involved in synaptic transmission, are shown in Fig. 1A in relation to the protein domains. e.g., Synaptotagmin 11 (Kahle and Haass, 2004). Importantly, our analysis was only performed on point Abnormal Parkin function may underlie certain cancers mutations and small indels, and did not include large indels or (Veeriah et al., 2010). The Parkin gene is located on chro- mutations of exon rearrangements. Therefore, it is possible mosome 6q in the fragile site FRA6E (Wang et al., 2009), that Parkin mutational frequency in melanoma may be higher which is altered in a variety of human cancers (Letessier et al., than 3.6%. 2007). In the absence of Parkin expression, cancer cell lines The Parkin mutations commonly carried by PD patients exhibit extensive proliferation and cell cycle abnormalities (Hedrich et al., 2004; Kay et al., 2010) were indexed by exon (Veeriah et al., 2010; Yeo et al., 2012), while re-expression of and by type of mutation (i.e., point mutations, exon rear- wild-type Parkin slows the growth of colorectal carcinoma rangements, small deletions, and amplifications) and are rep- (Poulogiannis et al., 2010), breast cancer (Letessier et al., resented in proportions to their amount in each exon (Fig. 1A, 2007; Wang et al., 2009), and lung cancer (Poulogiannis upper panel). Known Parkin mutations span the entire gene. et al., 2010; Veeriah et al., 2010; Yeo et al., 2012) through Although mutations in different regions alter different an unknown mechanism (Liu et al., 2011). biochemical properties of the Parkin protein, apparently all Since mutations in Parkin arethemajordriverofearly- mutations disrupt the ability of Parkin to degrade substrates, onset PD (Lu¨cking et al., 2000; Kahle and Haass, 2004) and thus manifest as loss-of-function mutations (Sriram et al., and there is evidence that Parkin is a key player in cancer 2005). Interestingly, the most unstable region of the fragile site progression (Veeriah et al., 2010), we compiled and on 6q, FRA6E, is found in the region that con- analyzed sequences of Parkin from 246 melanoma patients. tains exons 2 through 8 of the Parkin gene (Letessier et al., We identified five melanoma-specific non-synonymous mu- 2007), and this is the region where most melanoma and PD tations in Parkin in exons known to carry mutations in PD mutations are observed (Fig. 1A). Exons 2 and 7 of the gene patients. We showed that one of these mutations, E28K, were previously identified as mutational hot spots (Hedrich abolished the ubiquitination activity of Parkin. We further et al., 2004). Remarkably, melanoma-specific somatic muta- demonstrated that Parkin is an inhibitor of cell cycle and an tions in Parkin appear in the same domains as the most apoptotic cell death driver in melanoma cells, whereas common germline PD mutations (Fig. 1A), supporting the Parkin-E28K abolishes Parkin tumor suppressive effects in hypothesis of a genetic link between the two diseases. melanoma. Finally, in our attempt to uncover the role of The structure of Parkin has been determined by crystal- Parkin in melanoma, we identified potential Parkin sub- lography ( ID 4k95) (Trempe et al., 2013). strates in melanoma using immunoprecipitation followed by We used this structure to assess possible effects of the muta- mass spectrometry, and clustered the identified substrates by tions observed in melanoma patients on Parkin activity functionality analysis. Our data suggest that Parkin links (Fig. 1B). The E28K mutation is located in the helix of the between PD and melanoma and that Parkin may serve as a UBL domain. Based on a corresponding residue in NEDD8 diagnostic marker. protein, which is conserved and crucial for interaction with its L. Levin et al. / Journal of Genetics and Genomics 43 (2016) 369e379 371

partner NAE1 (Nedd8-activating enzyme E1, Protein Data Bank ID 3gzn), the E28K mutation will lead to charge reversal and, therefore, will likely disrupt binding of Parkin to sub- strates (Fig. 1B, bottom-right enlargement square). The L112F BRAF NRAS status mutation is located in the linker between the UBL domain and the first RING0 domain; this region is not resolved in the crystal structure. The R275W mutation is located at the end of the first helix in the second RING1 domain; R275 contacts a

status residue located between helices 3 and 4. This mutation will probably abolish this stabilizing contact and destabilize this domain. The E300K mutation is located in RING1 domain and is exposed to solvent in the crystal structure. This mutation will likely lead to a steric clash with L187 in the RING0 domain. Finally, the C360S mutation is located in the IBR domain and is involved in the binding of zinc. This mutation

amino acid sequence, and a splice region variant is defined will probably abolish zinc binding and significantly destabilize the IBR structure. The finding that melanoma-specific muta- Patient sample number Heterozygosity tions in Parkin occur in similar regions to those in PD patients, gene. The coordinates were mapped and annotated using Ensembl6. A MEL-JWCI-WGS-7-Tumor NA Wild type A C077A C077 Homozygous Wild type Homozygous Wild type A C084 Heterozygous Wild type A MEL-JWCI-WGS-1-Tumor NA Wild type A MEL-Ma-Mel-94-Tumor NA RAS Q61R A MEL-13463-Tumor NA RAS Q61R A YULAN Heterozygous Wild type A C067 Heterozygous Wild type T MEL-JWCI-WGS-12-Tumor NA BRAF V600E suggest that mutations in this E3 ubiquitin ligase might lead to > > > > > > > > > > both diseases. Parkin T/G T/G T/G T/G T/G T/G T/G T/G T/G A/A > > > > > > > > > > 8 bases of the intron. Reference and variable alleles are given on the plus strand.

type Parkin reduces melanoma proliferation and promotes e apoptosis

In order to determine the biological role of Parkin in mela- Codon Mutation noma, we first examined the effect of Parkin overexpression on melanoma clonogenic propensity. WM3314 and A375

Amino acid change melanoma cell lines were transiently transfected with a vector

3 bases of the exon or 3 for Parkin expression or an empty vector as a control and were e ) were analyzed for mutations in subjected to a colony formation assay. In both melanoma cell Protein position lines, Parkin expression significantly decreased the number of detected colonies (Fig. 2A). Further, Parkin overexpression significantly attenuated metabolic activity of WM3314 and CDS position A375 cells (Fig. 2B) and growth rate of these cells (Fig. 2C). Moreover, overexpression of Parkin decreased cell cycle pro- gression of WM3314 and A375 cells (Fig. 2D). We found that cDNA position

Dutton-Regester et al., 2014 Parkin induces apoptosis, since overexpression in both WM3314 and A375 melanoma cells increased the percentage of apoptotic and TUNEL-positive cells compared to cells transfected with empty vector (Fig. 2E and F). These results clearly demonstrate that Parkin reduces proliferation and promotes apoptosis in melanoma cells, suggesting that loss-of-

Mutational consequence function mutations in Parkin enable melanoma cells to avoid cell cycle arrest and proliferate.

Variable allele Parkin-E28K melanoma somatic mutation perturbs Parkin biological activity in melanoma gene in human melanoma patients

allele To determine the effects of the identified melanoma-specific

Parkin mutations on Parkin function, we generated expression vec- tors of each melanoma-specific Parkin mutant. We first examined the ubiquitin ligase activity of these mutants by testing and comparing the ubiquitination capacity of wild-type (WT) Parkin and Parkin mutants C360S, E300K, L112F, R275W, and E28K in the presence or absence of a proteasome inhibitor (MG132). In the absence of MG132, less ubiquiti- Table 1 Somatic mutations of the Chromosome Position Reference Data from whole exome and whole genome samples described previously ( Chr. 6Chr. 6 161969891 AMissense variants are defined 161781243 as mutations that cause G a change in amino T acid identity, synonymous variants are defined as mutations that do not alter the A Missense variant 1181 Splice region variant 1078 360 C/S Tgt/Agt T C Chr. 6Chr. 6 161969928 C 161781169 C Tas a sequence variant in which a change has occurred within the region of T the splice Synonymous site, variant either within 1 1144 Synonymous variant 1041 1339 347 1236 Q 412 caG/caA K C aaG/aaA C Chr. 6 161990422 C T Missense variant 1001 898 300 E/K Gag/Aag C Chr. 6 162206852 G A Missense variant 926 823 275 R/W Cgg/Tgg C Chr. 6 162683635 G A Missense variant 437 334 112 L/F Ctc/Ttc C Chr. 6 162683635 G A Missense variant 437 334 112 L/F Ctc/Ttc C Chr. 6 162864431 C T Missense variant 185 82 28 E/K Gag/Aag C Chr. 6 162864468 C T Synonymous variant 148 45 15 Vnation gtG/gtA C activity was detected upon expression of WT Parkin. 372 L. Levin et al. / Journal of Genetics and Genomics 43 (2016) 369e379

A PD mutations exons 12 3456789 10 11 12

V17VE28K L112F R275W E300K Q347QC360S K412K

Protein NH2 COOH domains UBL RING IBR RING 1-76 238-293 313-377 418-449

B R275W

E300K

C360S

E28K

Parkin

Fig. 1. Somatic mutations of the Parkin gene in human melanoma patients. A: Schematic diagram of Parkin protein with boundaries of the 12 exons and protein domains indicated. Locations of mutations and the corresponding amino acid changes are shown; missense mutations are shown in purple, synonymous mutations in black. The sizes of purple spheres represent the frequency of mutations in Parkin exons in PD patients. The numbers 1e12 indicate the exon number. The dashed lines mark the borders of each exon in relation to the protein domains. B: Melanoma mutations in the UBL, RING and IBR domains of Parkin, shown as spheres on cartoon depictions of the different domains (colored as in panel A). Gray spheres represent zinc atoms. The framed diagrams show enlargements of the regions of different mutated positions. The enlargment at the lower right shows the structure of a homolog, NEDD8, bound to its partner NAE1, highlighting the potential importance of E28 for proper interactions with partners. Structures were rendered using Pymol (http://www.pymol.org).

This was likely due to higher levels of degradation of the cell cycle progression of both WM3314 and A375 cells ubiquitinated substrates by the proteasome. Of the Parkin (Fig. 3C and S1). Finally, the E28K mutation impaired Parkin mutants evaluated, only Parkin-E28K had significantly lower biological activity as an apoptosis inducer in WM3314 mel- ubiquitination capacity than WT Parkin (Fig. 3A). Since only anoma cells (Fig. 3D and S1). These data demonstrate that in E28K in the UBL domain abolished Parkin ubiquitination melanoma, a glutamic acid located at the consensus position activity (Fig. 3A), we proceeded with this mutation for further 28 of Parkin is essential for the E3 ligase activity of Parkin and functional characterization of Parkin. Structural analysis of a thus for its biological function as an inhibitor of proliferation homolog of Parkin ubiquitin-like domain, NEDD8, bound to and an apoptosis inducer. NAE1, shows that this residue is conserved, and that it forms a salt-bridge with NAE1 residue R251. Therefore, we speculate Parkin interactors in melanoma that E28 is important for the interaction with a partner during the ubiquitination process. In order to further explore the molecular mechanism of Parkin To further evaluate the biological activity of Parkin-E28K, activity in melanoma, we identified Parkin substrates using WM3314 melanoma cells were transfected with vectors for the mass spectrometry analysis. WM3314 melanoma cells were expression of WT Parkin or Parkin-E28K, and proliferation transfected with an expression vector of GFP-tagged-Parkin or rates were measured relative to cells transfected with a control with a GFP control vector. Following immunoprecipitation of vector. Cells that expressed WT Parkin grew more slowly than GFP-Parkin with anti-GFP antibody (Fig. 4A, left panel), control cells, and cells that expressed Parkin-E28K prolifer- associated proteins were identified using mass spectrometry ated significantly more rapidly than the control cells (Fig. 3B). analysis. The experiment was repeated twice to increase the The overexpression of Parkin-E28K resulted in an enhanced significance of the results. Parkin interacting proteins that L. Levin et al. / Journal of Genetics and Genomics 43 (2016) 369e379 373

ABC 40 4.0 * 4.0 3.5 3.5 30 * 3.0 3.0 * * 20 2.5 2.5 A375 2.0 2.0 * 10 1.5 1.5 0 1.0 1.0

2.0 3.5 * 300 * 3.0 Number of colonies * Relative cell number 200 2.5 * 1.5 Relative mitochondrial activity 2.0 WM3314 100 1.5

0 1.0 1.0 1 23 0 1 23 Control Parkin Day Day

DEF

1.6 1.6 1.5 1.5 TUNEL * 1.4 * 1.4 1.3

A375 1.3 1.2 1.2

1.1 1.1 DAPI 1 1

G1/(G2-M) 1.6 1.6 * 1.5 1.5 * TUNEL 1.4 1.4 1.3 1.3 1.2 1.2 Relative apoptotic cell number

WM3314 1.1 1.1 DAPI 1 1

Control Parkin Control Parkin Control Parkin

Fig. 2. Parkin reduces melanoma proliferation and promotes apoptosis. WM3314 and A375 cells were transiently or stably transfected with a vector for Parkin expression or a vector control, respectively. A: Colony formation assay. Number of colonies is plotted; error bars represent SEM, and * indicates P < 0.05 (n ¼ 3). B: XTT assay for mitochondrial activity. Data are relative to color levels on day 1. Error bars represent SEM, and * indicates P < 0.05 (n ¼ 3). C: Crystal violet staining to determine cell number. Data are relative to color levels on day 0. Error bars represent SEM, and * indicates P < 0.05 (n ¼ 3). D: Cell cycle analysis. Data are presented as fraction of cells in G1 phase relative to G2/M phase. Error bars represent SEM, and * indicates P < 0.05 (n ¼ 3). E: Annexin-PI staining to quantify apoptotic cells. Cells were subjected to serum-free medium and stained with Annexin V and PI. Apoptotic cells were measured by FACS Calibur flow cytometer. Error bars represent SEM, and * indicates P < 0.05 (n ¼ 3). F: TUNEL labeling anaysis using fluorescence microscope. Cells were subjected to serum-free medium and were labeled with TUNEL (red) and DAPI (blue). were identified in two independent experiments (Table S2), are mass spectrometry analysis, HSP70 (Moore et al., 2008), alpha represented in a functional protein association network con- and beta Tubulin (Ren et al., 2003) and VDAC1 (Narendra structed using STRING in Fig. S2. As expected, since Parkin et al., 2010). Importantly, this is the first time that these sub- is a known E3 ubiquitin ligase (Moore et al., 2008), the ma- strates are identified as Parkin interactors in melanoma. In line jority of identified proteins are associated with the ubiquiti- with our experimental results, analysis revealed nation process and are components of the 26S proteasome a significant enrichment of cell cycle regulators and mito- system, which strengthen the validity of our analysis. Addi- chondrial proteins (Fig. 4B and Table S3), emphasizing the tionally, four known Parkin substrates were identified in our role of Parkin-E3 ligase in melanoma cell cycle regulation. 374 L. Levin et al. / Journal of Genetics and Genomics 43 (2016) 369e379

A IP: Myc Input E28K + Ub E28K + Ub L112F + Ub L112F L112F + Ub L112F Parkin + Ub Parkin + Ub Parkin + Ub Parkin + Ub E300K + Ub E300K + Ub C360S + Ub C360S + Ub R275W + Ub R275W R275W + Ub R275W pcDNA3.1 + Ub pcDNA3.1 + Ub pcDNA3.1 + Parkin pcDNA3.1 + Parkin

MG132 ++– ++++++ ++ –+ +++++ (N) (N) Ub WB: α-HA Ub

WB: α-Parkin

WB: α-β-actin

BCD 1.3 * 2.2 Control * 1.2 2.0 WT E28K 1.8 1.1 TUNEL 1.6 1.0 G1/(G2–M) 1.4 * 0.9 1.2 DAPI Relative cell number 1.0 0.8 012 Control WT E28K Control WT E28K Day

Fig. 3. Parkin-E28K melanoma somatic mutation perturbs Parkin biological activity in melanoma. A: Ubiquitination assay. HEK293 cells were co-transfected with vectors for expression of myc-tagged Parkin or mutant Parkin and HA-tagged ubiquitin in the presence or absence of MG132. Parkin was immunoprecipitated, and ubiquitination was detected by anti-HA (a-HA). Staining with anti-b-actin was used as a loading control. B: Crystal violet staining. WM3314 were transfected with WT Parkin, Parkin-E28K, or control vectors. Cells were stained with crystal violet, and the color intensity was measured. Data are relative to color levels on day 0. Error bars represent SEM, * indicates P < 0.05 (n ¼ 3). C: Cell cycle analysis. Data are represented as G1 phase relative to G2/M phase. Error bars represent SEM, * indicates P < 0.05 (n ¼ 3). D: TUNEL labeling analysis using a fluorescence microscope. Cells were subjected to serum-free medium and were labeled with TUNEL (red) and DAPI (blue).

DISCUSSION increase the risk of melanoma (Fiala et al., 2003), and the risk for melanoma is increased before PD is diagnosed (Olsen The co-occurrence of PD and melanoma has been reported in et al., 2006). Thus, it is reasonable that the joint susceptibil- several epidemiological studies (Olsen et al., 2006; Gao et al., ity for both diseases is based on genetic components. Here we 2009), and recently in a genetic study (Hu et al., 2016); focus on Parkin as a plausible candidate underlying the PD- however, the molecular link is still unknown. To the best of melanoma link. Germline mutations in the Parkin gene are our knowledge, no study has stratified PD patients by common major cause of the familial forms of PD (Lu¨cking et al., 2000), PD mutations (Parkin, DJ-1, SNCA, etc.) and looked for and mutations or deletions of Parkin promote various types of melanoma appearance in each group. Epidemiologically, only cancers development (Veeriah et al., 2010). We discovered five a positive correlation between the two diseases has been re- somatic missense mutations in the Parkin gene in human ported. For many years, melanoma appearance in PD patients melanoma samples; these mutations map to regions where the was thought to be related to levodopa treatment (Fiala et al., majority of Parkin mutations were located in PD patients. In 2003). It was shown that levodopa treatment does not our analysis, one of the mutations, Parkin-E28K, nearly L. Levin et al. / Journal of Genetics and Genomics 43 (2016) 369e379 375

A 2011). This may indicate that cell proliferative state dictates

GFP Parkin the opposite outcomes of Parkin function. In accordance with IP: GFP previous works demonstrating that Parkin functions as a tumor WM3314 WB: α-Parkin suppressor (Veeriah et al., 2010; Yeo et al., 2012), we showed here that Parkin expression decreased the ability of melanoma B cells to progress through cell cycle and inhibited proliferation. In addition, the mutant Parkin-E28K could not suppress, and GO term % P-Value possibly promote melanoma cell cycle progression. As Nucleotide binding 43.75 2.31 E-07 differentiated neurons that receive signals for proliferation die Ribonucleotide binding 39.0625 3.49 E-07 (Sumrejkanchanakij et al., 2006), it will be interesting to Purine ribonucleotide binding 39.0625 3.49 E-07 examine whether Parkin functions as a kind of a cell cycle Mitotic cell cycle 17.1875 2.03 E-06 guardian to prevent neuronal cells from entering cell cycle, and its absence causes re-entrance to cell cycle leading to Mitochondrial part 20.3125 4.28 E-06 apoptotic cell death. 9.375 5.04 E-06 Proteasome complex In melanoma, Parkin expression results in increased apoptotic cell death; this was not observed when the Parkin- E28K mutant was expressed. These results are contradictory to numerous previous reports that intact Parkin is required for neuronal survival and health, and that dysfunctional Parkin leads to neuronal death (Shen and Cookson, 2004), like in PD. Taken together, however, these results strongly support the fact that mutated Parkin leads to opposite phenotypes in different lineages. Fig. 4. Parkin interactors in melanoma cells. In our study, we focus on the effect of the E28K variant in A: WM3314 cells were transfected with GFP-tagged Parkin or with control melanoma cells, since it is the only mutation that perturbed vector. Cells were treated with MG132 for 4 h, followed by immunoprecipi- Parkin E3-ligase ubiquitination activity in our prior tation with anti-GFP. Protein complexes were detected with anti-Parkin and were subjected to mass spectrometry analysis. B: GO terms enriched in the biochemical assays. However, other mutations that were identified Parkin interacting proteins. identified here, including the R275W variant, may also impair Parkin activity and induce changes of cellular phenotypes. For example, Sriram et al. (2005) have previously demonstrated abolished Parkin ubiquitination activity. This mutation is that the R275W variant does not impair Parkin E3-ligase located in the UBL domain, and thus probably disrupts sub- ubiquitination activity. Additionally, Xiong et al. (2015) re- strate binding. Another UBL located mutation R42P was re- ported on the presence of germline R275W mutation in fa- ported previously to be involved in PD, and was shown to milial lung cancer patients. It will be very interesting to further potentially disrupt binding to the proteasome (Sriram et al., investigate the effect of each mutation on intracellular local- 2005). ization, substrate binding and degradation, solubility, and In PD, there is massive loss of dopaminergic neurons in the enzymatic activity. substantia nigra (Lim et al., 2005; Hampe et al., 2006; Oyama Interestingly, the majority of Parkin mutations we identified et al., 2010), whereas cancer cells are characterized by un- did not decrease, and even elevated, the ubiquitination activity controlled cell division and growth (Hanahan and Weinberg, of Parkin (Fig. 3A and 3A). Additionally, differences in 2011). Parkin is expressed in a wide variety of tissues and is extracting expressed mutants from lysed cells were observed. well conserved from nematodes to humans (Xu et al., 2014). Specifically, Parkin mutants do not appear equally in the Although Parkin inactivation has mostly been shown to cause Triton X-100 soluble fraction, whereas roughly similar neuronal dysfunction, it is reasonable that it will also affect amounts of each mutant were detected in the sodium dodecyl other tissues. Indeed, Parkin alterations are involved in various sulfate lysis buffer (Fig. S3B). The lysate generated by SDS types of cancer (Veeriah et al., 2010), supporting a central role buffer includes whole cell content, whereas the lysate gener- for Parkin in various tissues. It appears, therefore, that muta- ated by Triton X-100-based buffer includes only the soluble tions in Parkin cause different phenotypes in different cell fraction, as the insoluble fraction (including cell membranes) lineages. This contradiction might be due to various Parkin are excluded from the final analysis. Specifically Parkin- regulators and substrates in different cell types, in accordance R275W, E300K, and C360S were not soluble in Triton X- with the different cellular roles, function, differentiation state, 100 when compared with WT Parkin (Fig. S3B, lower panel). proliferative capacity and other unique features of each cell Importantly, all Parkin mutant mRNAs were expressed at type. One prominent difference between cancer cells and similar levels (Fig. S3C). These observations suggest that neurons is their ability to progress through cell cycle. Neurons some mutations of Parkin may change the protein cell local- are fully differentiated post mitotic cells that do not prolifer- ization (Sriram et al., 2005); alternatively, mutation might lead ate, whereas cancer cells partly lose their differentiation state to a conformational change, thus affecting protein solubility, and retain high proliferative capacity (Hanahan and Weinberg, and possibly protein activity. 376 L. Levin et al. / Journal of Genetics and Genomics 43 (2016) 369e379

Here we identify a possible genetic association between PD purchased from Clontech (USA). The pRK5-Parkin-myc and melanoma by using somatic mutations data of melanoma expression vector is a gift from Dr. Simone Englender (The patients. Of note, Parkin mutations in PD patients are germline Rapport Institute for Research, Faculty of Medicine, Technion (Kahle and Haass, 2004; Guo et al., 2008, 2010). Recently, Hu e Israel Institute of Technology, Haifa, Israel). The et al. (2016) published a work in which they identified Parkin pcDNA3.1-Parkin-FLAG expression vector was kindly pro- germline mutations in melanoma patients; the results are in vided by Dr. Luc Morris (Memorial Sloan-Kettering Cancer line with what we propose here, and further demonstrate the Center, New York, NY, USA). To tag Parkin with GFP, a genetic connection between melanoma and PD. It is highly fragment spanning the WT or mutant Parkin coding region likely that germline PD mutations in Parkin play a role in was amplified from RSK-Parkin constructs, digested with melanoma predisposition, either in the initial development of EcoRI-SAL I, and cloned into pEGFP-C2. the tumor or its progression. Interestingly, most of the Parkin somatic mutations found in variety of cancers (Veeriah et al., Colony-forming assay 2010) occur at the same domains and residues as the germ- line Parkin mutations implicated in PD. In addition, although Cells were selected using G418 until visible separated col- Parkin KO mice do not recapitulate signs central to Parkin- onies were formed. Cells were then fixed with 10% acetic acid sonism, these mice are prone to cancer development (Fujiwara and 10% ethanol for 40 min. Colonies were stained with 0.2% et al., 2008). Mice with homozygote Parkin exon 3 deletions (w/v) crystal violet (SigmaeAldrich) in 10% acetic acid and spontaneously develop hepatic tumors with characteristics of 10% ethanol for 1 h, washed, and counted. hepatocellular carcinoma (HCC). Moreover, Parkin heterozy- gous deletion accompanied by APC heterozygous deletion Crystal violet staining promotes intestinal adenoma development in mice. As HCC appears in the mice at advanced age, and there is a dependency Cells were washed with PBS and fixed with 10% acetic acid between Parkin and APC mutations in adenoma development, and 10% ethanol solution. Cells were then stained using 0.2% it is possible that a mutation in Parkin alone is not sufficient to (w/v) crystal violet in 10% acetic acid and 10% ethanol for initiate development of some types of cancer, and it may 1 h. For quantification of the crystal violet staining, the dye require additional risk factors. Moreover, we identified Parkin was re-dissolved in 10% acetic acid solution. Color intensity interacting proteins in melanoma cells by immunoprecipita- was measured using a UVevis plate reader at 595 nm. tion followed by mass spectrometry. The proteins detected are mainly connected to cell cycle, mitochondrial function, and Mitochondrial activity assay proteasome function; this result is consistent with the study of Sarraf et al. (2013). In conclusion, our data indicate that Parkin Cells were treated with activated XTT solution (Biotium, has a tumor suppressive role in melanoma. Our findings open USA). The signal was measured according to the manufac- new possible methods for early diagnosis and treatments turer’s protocol at 490 nm and normalized to signal at 690 nm. against both diseases. Cell cycle analysis MATERIAL AND METHODS Cells were synchronized in G1 phase by treatment with Cell culture and transfection 2 mmol/L thymidine (SigmaeAldrich) for 22 h. Cells were then washed twice with PBS and incubated for 18 h in DMEM WM3314 and A375 melanoma cells were generously provided with 10% serum. Cells pellets were fixed with 70% ice cold by Dr. Levi A. Garraway (Department of Medical Oncology ethanol and resuspended in propidium iodide (PI) solution and Center for Cancer Genome Discovery, Dana-Farber Can- containing 0.05% Triton X-100, 0.05 mg/mL PI (Sigma- cer Institute, Boston, MA, USA). Cells were cultured in eAldrich), and 0.1 mg/mL of RNase A. Cell cycle phases DMEM medium supplemented with 10% fetal bovine serum were determined using a BD FACSCalibur flow cytometer and (FBS, Sigma-Aldrich, USA) and 1% penicillin/streptomycin/ analyzed by Kaluza analysis software. glutamine (Invitrogen, USA). For establishment of stable cell lines, A375 cells were transfected with a vector for expression Annexin-PI staining of WT Parkin-FLAG or control vector, and were selected by culture with 100 mg/mL G418 (SigmaeAldrich). Constructs Stress was induced by growing cells in serum-free medium for were transfected using jetPEIÔ according to the manufac- 48 h. The cell pellets were suspended in binding buffer con- turer’s instructions. taining 0.1 mol/L HEPES, pH 7.4, 1.4 mol/L NaCl, and 5 25 mmol/L CaCl2.To10 cells, 1 mg/mL of Annexin V Plasmids and cloning (Biolegend, USA) and 0.05 mg/ml PI were added. Samples were incubated in the dark at room temperature for 15 min Site-directed mutagenesis was performed using the Stratagene then analyzed on a BD FACSCalibur flow cytometer with Quick Change method (Stratagene, USA) according to man- Flowjo analysis software. Samples treated with Annexin V ufacturer’s protocol. pEGFP-N1 and pEGFP-C2 vectors were only and PI only were used as controls. L. Levin et al. / Journal of Genetics and Genomics 43 (2016) 369e379 377

TUNEL assay Mass spectrometry analysis

Stress was induced by growing cells in serum free medium The tryptic peptides were desalted over C18 resin, dried, and for 48 h. Cells were fixed in 4% PFA for 1 h at room tem- resuspended in 0.1% formic acid. The peptides were separated peratureandthenincubatedonicefor2mininper- by reversed-phase chromatography on 0.075 180-mm fused meabilization solution containing 0.1% Triton X-100 in 0.1% silica capillaries (Agilent J&W, USA) packed with Reprosil sodium citrate. Cells were then incubated with TUNEL re- reversed-phase material (Dr. Maisch GmbH, Germany). The action mixture (in situ cell death detection kit, Roche, USA) peptides were eluted using a linear 60-min gradient of 5%e28% for1handthenstainedwith406-diamidino-2-phenylindole solvent A, 15-min gradient of 28%e95% solvent A, and 15 min (DAPI) (SigmaeAldrich). Cells were analyzed by fluores- solvent B. Solvent A was 95% acetonitrile with 0.1% formic cence microscopy. acid in water and solvent B was water. The flow rate was 0.15 mL/min. Mass spectrometry was performed by Q Exactive Gel electrophoresis, immunoblotting, and plus mass spectrometer (Thermo Scientific, USA) in a positive immunoprecipitation mode using repetitively full MS scan followed by collision induced dissociation of the 10 most dominant ions selected Cells were lysed 48 h after transfection in buffer containing from the first MS scan. The mass spectrometry data from two 50 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 0.5% Triton X- biological repeats was analyzed using Proteome Discoverer 1.4 100, 1 mmol/L EDTA, and protease inhibitor cocktail (Sig- software with Sequest (Thermo Scientific, USA) and Mascot maeAldrich). For Input, cells were resuspended in 4% SDS, (Matrix Science, UK) algorithms against Human Uniprot 0.1 mol/L Tris pH 7.5. Once protein concentration was database with 1% FDR. Semi-quantitative analysis was done by determined, 0.1 mol/L DTT was added to the lysate. Samples calculating the peak area of each peptide based its extracted ion were resolved by SDS-PAGE, transferred to nitrocellulose currents, and the area of the protein was calculated as the membranes, and exposed to the appropriate antibodies: mouse average of the three most intense peptides from each protein. anti-hParkin (MAB14381; R&D Systems, USA), mouse anti- b-actin (AC-74; SigmaeAldrich), and rat anti-HA (3F10; ACKNOWLEDGMENTS Roche, Switzerland). Proteins were visualized with Super- SignalChemiluminescent Substrates (Pierce, USA) using We would like to thank Drs. Meenhard Herlyn, Levi Garraway, horseradish peroxidase-conjugated anti-mouse secondary and Carl Novina for supplying melanoma cultures and the antibody (7076; Cell Signaling, USA) or goat anti-rat (sc- Smoler Proteomics Center at the Technion for mass spec- 2006; Santa Cruz Biotechnology, USA). troscopy analyses. C.L. gratefully acknowledges grant support from I-CORE (grant number 41/11), Mr. David Brown through Ubiquitination assay the Israel Cancer Association (ICA) (grant number 20150101), Israel Cancer Research Fund (ICRF) (grant number RCBA-11- For ubiquitination assay, HEK293 cells were co-transfected 706), Fritz Thyssen Stiftung (grant number AZ.10.12.1.188), with constructs for expression of WT or mutant myc-tagged- Marie Curie Career Integration Grants (CIG) (grant number Parkin or control vector and with HA-tagged ubiquitin, fol- 293594), Dalya Gridinger Fund, Fingerhot Carol and Lionara lowed by immunoprecipitation of Parkin with anti-myc. Fund, and the Shtacher Family award. Ubiquitination was detected by anti-HA. MG132 (Sigma- eAldrich) treatment was done at 10 mmol/L for 4 h before harvest. SUPPLEMENTARY DATA

Sample preparation for mass spectrometry analysis Fig. S1. Parkin reduces melanoma proliferation and promotes apoptosis. WM3314 cells were transfected with GFP-tagged-Parkin or Fig. S2. Parkin interactors in melanoma. with control vector. After 48 h, cells were incubated for 4 h Fig. S3. Mutations in Parkin results in altered solubility. with 20 mmol/L MG132 and then washed twice with PBS. Table S1. Parkin-related diseases. Cells were lysed in lysis buffer containing 150 mmol/L NaCl, Table S2. Mass spectrometry analysis of PARK2 potential 50 mmol/L Tris pH 7.5, 1% NP-40, 1 mmol/L Na3VO4, substrates in melanoma. 1 mmol/L NaF, 0.1% b-mercaptoethanol, and protease in- Table S3. Significant functional annotations of PARK2 po- hibitor. Proteins were incubated with mouse anti-GFP anti- tential substrates in melanoma. body (SC-9996; Santa Cruz Biotechnology, USA) for 18 h at Supplementary data related to this article can be found at 4 C and then incubated with protein A/G agarose beads for http://dx.doi.org/10.1016/j.jgg.2016.05.005. 2hat4C. Proteins were eluted in 50 mmol/L Tris, pH 8.5, 8 mol/L urea, and 1 mmol/L DTT for 2 h and in 50 mmol/L REFERENCES Tris, pH 8.5, 8 mol/L urea, and 5 mmol/L IAA for 5 min, followed by overnight incubation at room temperature in the Berger, M.F., Hodis, E., Heffernan, T.P., Deribe, Y.L., Lawrence, M.S., dark. Protopopov, A., Ivanova, E., Watson, I.R., Nickerson, E., Ghosh, P., 378 L. Levin et al. / Journal of Genetics and Genomics 43 (2016) 369e379

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