Truncating PREX2 Mutations Activate Its GEF Activity and Alter Gene Expression Regulation in NRAS-Mutant Melanoma

Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma

Item Type Article

Authors Lissanu Deribe, Yonathan; Shi, Yanxia; Rai, Kunal; Nezi, Luigi; Amin, Samir B.; Wu, Chia-Chin; Akdemir, Kadir C.; Mahdavi, Mozhdeh; Peng, Qian; Chang, Qing Edward; Hornigold, Kirsti; Arold, Stefan T.; Welch, Heidi C. E.; Garraway, Levi A.; Chin, Lynda

Citation Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma 2016, 113 (9):E1296 Proceedings of the National Academy of Sciences

Eprint version Post-print

DOI 10.1073/pnas.1513801113

Publisher Proceedings of the National Academy of Sciences

Journal Proceedings of the National Academy of Sciences

Rights Archived with thanks to Proceedings of the National Academy of Sciences

Download date 25/09/2021 13:20:05

Link to Item http://hdl.handle.net/10754/600889 Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma

Yonathan Lissanu Deribe1#*, Yanxia Shi1,2#, Kunal Rai1, Luigi Nezi1, Samir B. Amin1,

Chia-Chin Wu1, Kadir C. Akdemir1, Mozhdeh Mahdavi1, Qian Peng1,Q. Edward

Chang3 ,Kirsti Hornigold4, Stefan T. Arold5, Heidi Welch4,

Levi Garraway6, and Lynda Chin1,3*

1. Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA 2. Sun Yat-Sen University Cancer Center; State Key Laboratory of Oncology in South China; Collaborate Center for Cancer Medicine, Guangzhou 510060, China

3. Institute for Applied Cancer Science (IACS), The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA

4. The Babraham Institute, Babraham Research Campus, Cambridge, UK

5. King Abdullah University of Science and Technology (KAUST), Division of Biological and Environmental Sciences and Engineering, Thuwal, 23955-6900 Saudi Arabia

6. Broad Institute of MIT and Harvard, Boston, MA 02141, USA *Corresponding authors: Email: [email protected] and [email protected] # These authors contributed equally to this work. Classification: BIOLOGICAL SCIENCES; Medical Sciences Short title: PREX2 melanoma mutations activate its GEF activity

Keywords: melanoma, PREX2, GEF, Akt, mouse models of cancer, p57, Rac1

1 Supplementary Materials and Methods:

Supplementary Methods Cell culture and generation of mouse embryonic fibroblasts

The generation of human primary melanocytes

(PMEL/hTERT/CDK4(R24C)/P53DD with either NRASG12D or BRAFV600E have been described previously (1). These cells were grown in Ham’s F10 medium (Invitrogen), containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. 293FT cells were grown in DMEM (Invitrogen) containing 10% FBS. We generated MEFs from embryonic day 13.5 (E13.5) embryos using standard methods following PTENL/L,

Rosa26-CreERT2 mouse intercrosses. MEFs were grown in DMEM (Invitrogen) containing 10% FBS and 1% penicillin/streptomycin. All cells were grown at 37oC in a humidified 5% CO2 incubator. The Rac1 inhibitor EHT1864 was purchased from

Selleckchem and dissolved to 10mM in DMSO.

In vitro GEF assay

PREX2 GEF assays were conducted as previously described in detail (2, 3).

Briefly, assays were performed for 10 min at 300C, liposomes consisting of PC, PS and

PI, cold and radioactive GTPgammaS, and purified proteins (no PIP3 or Gbetagamma in any of these assays). As substrate, we used 100 nM GDP-loaded human recombinant

Rac protein, purified from Sf9 cells, throughout. Recombinant human P-Rex proteins

(full length and iDHPH2 M1 to R363) were purified from Sf9-cells and used at 50 nM in the assay.

Rac1 activation assay

Rac1 activation assays were performed using Rac1 pull-down activation assay biochemical kit according to the manufacturer’s detailed protocol (Cytoskeleton Inc,

Denver, CO, USA).

Plasmids, lentiviral and adenoviral transduction

PREX2 plasmids have been previously described (4). Lentiviral stocks were prepared by co-transfecting 293FT cells with pLenti6.3-PREX2 expression constructs and standard viral packaging systems. 48 and 72 hours later viral supernatants were collected and used to transduce PMEL cells. All the PTEN expression constructs were in pSG5L vector [described previously (5)] and received from Addgene. PTEN plasmids were transfected into 293T cells using Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. Rac1 wt or Q61L mutant were cloned into the pLenti6.3 vector system, lentivirus prepared and used to transduce PMEL cells.

Xenograft studies

All animal studies were approved by MD Anderson Cancer Center Internal

Animal Care and Use Committee (IACUC). Cells were subcutaneously implanted

(1106 cells) in female NCR-NUDE mice (Taconic). Animals were monitored for tumors formation and were sacrificed when tumor diameter approached 1.5 cm. Log- rank statistical test was performed using Prism 5 (Graphpad).

Tumors were dissected and snap frozen in liquid Nitrogen for protein, RNA and other molecular profiling experiments.

Immunoprecipitation, western blotting and antibodies

Cells were lysed in lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA,

1% NP40) containing protease and phosphatase inhibitor cocktail tablets (Roche) on ice. For phosphorylation of Akt we lysed cells in RIPA buffer. Lysates were subjected to immunoprecipitation using the respective antibodies (usually 1:100 dilution) for 1 to

3 several hours. In case of V5, we used V5-coupled beads (Sigma-Aldrich). An equal mixture of Protein A and G beads were then added and incubated for an additional hour rotating in a cold room. Beads were washed in lysis buffer at least three times and bound proteins were eluted by boiling in SDS-loading buffer (BioRad). Lysates or IPs were then loaded on SDS-PAGE gels (BioRad) and transferred using BioRad Turbo semidry transfer system to nitrocellulose membranes which were probed with respective antibodies: V5 (1:5000), PREX2 antibodies (1:1000, Sigma-Aldrich or monoclonal antibody from Abcam), HA (1:1000, Santa-Cruz), PTEN (1:1000), phospho-Akt S475

(1:1000, Cell Signaling), phospho-Akt T308 (1:1000 Cell Signaling).

Immunohistochemistry and Ki67 quantification

Tumors were fixed in formalin for 24 hours, paraffin embedded and sectioned. After antigen retrieval, slides were stained using Ki67 antibody (1:200, Dako) using standard methods. NuclearQuant 1.15.1 software (3DHISTECH) was used for Ki67 quantitation.

Experienced pathologist set up the algorithm. Three non-necrotic tumor areas were selected for analysis on each slide. The software automatically generated quantitative results based on H-Score formula.

Reverse phase protein array (RPPA)

Cellular proteins were denatured by 1% SDS (with beta-mercaptoethanol) and diluted in five 2-fold serial dilutions in dilution buffer (lysis buffer containing 1% SDS). Serial diluted lysates were arrayed on nitrocellulose-coated slides (Grace Biolab) by Aushon

2470 Arrayer (Aushon BioSystems). Total 5808 array spots were arranged on each slide including the spots corresponding to positive and negative controls prepared from mixed cell lysates or dilution buffer, respectively.

Each slide was probed with a validated primary antibody plus a biotin-conjugated secondary antibody. Only antibodies with a Pearson correlation coefficient between

RPPA and western blotting of greater than 0.7 were used in reverse phase protein array study. Antibodies with a single or dominant band on western blotting were further assessed by direct comparison to RPPA using cell lines with differential protein expression or modulated with ligands/inhibitors or siRNA for phospho- or structural proteins, respectively.

The signal obtained was amplified using a Dako Cytomation–catalyzed system (Dako) and visualized by DAB colorimetric reaction. The slides were scanned, analyzed, and quantified using a customized-software, Microvigene (VigeneTech Inc.), to generate spot intensity.

Each dilution curve was fitted with a logistic model (“Supercurve Fitting” developed by the Department of Bioinformatics and Computational Biology in MD Anderson

Cancer Center, “http://bioinformatics.mdanderson.org/OOMPA”). This fits a single curve using all the samples (i.e., dilution series) on a slide with the signal intensity as the response variable and the dilution steps as the independent variable. The fitted curve is plotted with the signal intensities – both observed and fitted - on the y-axis and the log2-concentration of proteins on the x-axis for diagnostic purposes. The protein concentrations of each set of slides were then normalized by median polish, which was corrected across samples by the linear expression values using the median expression levels of all antibody experiments to calculate a loading correction factor for each sample.

mRNA expression profiling

Total RNA isolation was performed with the Qiagen RNeasy kit, according to the manufacturer’s instructions. Affmetrix GeneChip Mouse genome 430 2.0 or Human

Genome U133 plus 2 arrays containing probe sets for >45,000 characterized genes and expressed sequence tags, were used. Sample labeling and processing, hybridization, and scanning were performed according to Affymetrix protocols. Briefly, double- stranded cDNA was synthesized from total RNA with GeneChip 3’ IVT express kit

(Affmetrix), with a T7 RNA polymerase promoter site added to its 3′ end (Genset, La

Jolla, CA). Biotinylated cRNAs were generated from cDNAs in vitro and amplified by using GeneChip 3’ IVT express kit. After purification of cRNAs by the RNeasy mini kit (Qiagen, Hilden, Germany), 15 μg of cRNA was fragmented at 94°C for 35 min.

Approximately 12.5 μg of fragmented cRNA was used in a 250-μl hybridization mixture containing herring-sperm DNA (0.1 mg/ml; Promega), plus bacterial and phage cRNA controls (1.5 pM BioB, 5 pM BioC, 25 pM BioD, and 100 pM Cre) to serve as internal controls for hybridization efficiency. Aliquots (200 μl) of the mixture were hybridized to arrays for 16 h at 45°C in a GeneChip Hybridization Oven 640

(Affymetrix). Each array was washed and stained with streptavidin–phycoerythrin

(Life Technologies) and amplified with biotinylated anti-streptavidin antibody (Vector

Laboratories) on the GeneChip Fluidics Station 450 (Affymetrix). Arrays were scanned with the GeneArray G7 scanner (Affymetrix) to obtain image and signal intensities.

For qRT-PCR, cDNA was derived from mRNA using SuperscriptIII

(Invitrogen) and used in quantitative PCR reactions using SybrGreen method

(Invitrogen) and normalized to GAPDH and/or beta-actin expression values.

Cross-species integrative gene expression analysis

Gene expression analysis in human and mouse samples was performed respectively using Affymetrix Human U133 plus 2 arrays and Affymetrix Mouse 430

2.0 arrays. The Limma package from R Bioconductor(6) was used to do quantile normalization of expression arrays and analyze differentially expressed genes of

PREX2 E824 mutant versus wild type in human and mouse samples. We have 2,138 and 7,827 differentially expressed genes in human and mouse data, respectively, with the criteria, p-value<0.05. We mapped mouse genes to their human orthologs using

HomoloGene (http://www.ncbi.nlm.nih.gov/homologene) data and have 6,044 mapped genes. The Hypergeometric test was then used to determine the overlapping significance of these two gene sets. We find these two gene sets are significantly overlapped (p-value= 6.08e-09). Gene ontology/pathway analyses of overlapped differentially expressed genes in Human and Mouse data are performed using the web- accessible program DAVID (Database for Annotation, Visualization and Integrated

Discovery)(7). Pathways that enrich differentially expressed genes are selected

(p<0.05).

Except for the overlap analysis of gene sets selected by a criteria (i.e., p- value<0.05), we also used meta-analysis to combine results of human and mouse data.

All mouse genes in the Mouse 430 2.0 arrays were first mapped to their human orthologs. Fisher's combined probability test was used to combine p-values of each gene from Human and Mouse differential statistical analysis. All genes were then ranked based on the combined p-values. The gene set enrichment analysis (GSEA)(8) was applied to identify pathways and biological processes that enrich genes with significant p-values. All microarray data is deposited in GEO public gene expression

7 repository and can be accessed through the following link. http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=ifedkiqynbonxah&acc=GSE57

311

Homology Modeling

Homology modeling of the complex between PREX2 DH-PH and Rac1 was carried out using SwissModel (9) based on the 71% identical structure of the PREX1 DH-PH domain bound to Rac1 [PDB accession 4YON (10)]. Structures were visualized using PyMOL (www.pymol.org).

Methylated DNA Immunoprecipitation (MeDIP)

Genomic DNA was prepared using DNeasy Kit (Qiagen). Five micrograms of DNA was subjected to sonication using Bioruptor (Diagenode) to achieve shear length of

300-500bp. Four micrograms of denatured DNA was then incubated overnight with

5MeC antibody (Eurogentec, 10ug) conjugated Dynabeads (Invitrogen) in MeDIP buffer (20 mM Tris (pH 7.5), 140 mM NaCl, 0.05% Triton X-100). Next day, beads were washed with MeDIP buffer three times and precipitated DNA was then eluted from beads by digestion with Proteinase K. DNA was then purified using PCR purification kit (Qiagen). Quantitative PCR was performed for CDKN1C DMR and a negative control lacking CpG sites within 1kb proximity each side.

Melanoma TCGA data mining

Publicly available melanoma TCGA data from https://confluence.broadinstitute.org/display/GDAC/Home were examined for expression of CDKN1C and methylation of the imprinting regulatory DMR in intron

10 of KCNQ1. Box plots were generated showing CDKN1C expression in samples grouped according to PTEN deletion status. Expression signal is a normalized RSEM value from level 3 TCGA data. For each box plot, upper and lower boundary of box

8 represent 75th and 25th percentile, respectively. Median expression is shown as a band inside box. Upper and lower whiskers represent maximum and minimum expression values within 1.5 times the inter-quartile range (IQR). Value outside 1.5xIQR is outlier value and shown as a hollow circle. Significant calculated by one-tailed Mann-Whitney

U (MWU) test. Similarly box plots of methylation beta values were generated for probes within CDKN1C DMR in TCGA SKCM samples grouped according to PTEN deletion status. Increase in beta value indicates higher methylation, and vice versa.

Again, for each box plot, upper and lower boundary of box represent 75th and 25th percentile, respectively. Median expression is shown as a band inside box. Upper and lower whiskers represent maximum and minimum expression values within 1.5 times the inter-quartile range (IQR). Value outside 1.5xIQR are outlier values and shown as hollow circles. Differential CDKN1C DMR methylation between homozygous PTEN deleted group and WT PTEN group is highly significant based on one-tailed Mann-

Whitney U (MWU) test.

Supplementary Fig. S1-8

Supplementary Table S1

Supplementary Materials

Supplementary Figure 1. PREX2 truncation E824* cooperates with NRAS mutations but do not cooperate with BRAF V600E mutation to accelerate

melanoma xenograft formation.

(A) Tumor growth. Immortalized primary melanocytes (PMELs) expressing NRAS

Q61K were stably transduced with lentivirus encoding control GFP, PREX2 wt, or three different truncating PREX2 mutants (K278*, E824*, Q1430*). Cells (1x106) were then injected subcutaneously in nude mice and tumor development monitored. Kaplan-

Meier curve already published (4).

(B) Immortalized primary melanocytes (PMELs) expressing BRAF V600E (B) were stably transduced with lentivirus encoding control GFP, PREX2 wt, or three different truncating PREX2 mutants (K278*, E824*, Q1430*). Cells (1x106) were then injected subcutaneously in nude mice and tumor development monitored. Kaplan-Meier curve shows no difference between control and PREX2 mutants, demonstrating no cooperativity between BRAF mutation and PREX2 mutations.

Supplementary Figure 2. Gene set enrichment analysis (GSEA) in TetO-LSL-

PREX2 E824* transgenic mouse tumors and xenografts expressing truncating

PREX2

Genes which were dysregulated in both TetO-LSL-PREX2 E824* transgenic and xenografts expressing truncating PREX2 (E824*) were compiled and subjected to gene

10 set enrichment analysis. Indicated are enrichment plots for two of the top significantly enriched pathways.

Supplementary Figure 3. Decreased expression of p21 and p27 increased expression of IGF2 in PREX2 mutant tumors.

(A) Three independent tumors from iNRAS mice and three independent tumors from iNRAS + TetO-LSL-PREX2 E824* mice were lysed and immunoblotted with the indicated antibodies.

(B) Quantitative RT-PCR (left panel) and immunoblotting (right panel) assays were done to assess mRNA and protein expression levels of IGF2 gene in RNA isolated from

3 xenograft tumors of each experimental (PREX2 wt and mutants) and control group

(GFP). **statistical significance of p<0.05 between each experimental group and the control GFP group.

Supplementary Figure 4. Truncating PREX2 mutations in melanoma abolish its interaction with PTEN. (A) Schematic representation of truncating mutations originally identified(4) and used in the study; (B) schematic representation of additional truncating mutations observed in melanoma by recent genome sequencing projects(11)(Green font) and Melanoma TCGA (Black font); (C-E) Western blot analysis of co-immunoprecipitation (Co-IP) between stable expression of control GFP, wild type (wt) PREX2 or three truncating mutations in (C) PMEL-NRASQ61K and (D)

PMEL-BRAFV600E primary melanocytes and (E) Immunoprecipitation of V5-tagged

GFP, wt, or various PREX2 melanoma mutants after transient overexpression in 293T

11 cells was done by V5 antibody and followed by immunoblotting against endogenous

PTEN showing selective loss of interaction only with the truncating PREX2 mutations.

Supplementary Figure 5. Domains implicated in the binding of PTEN to PREX2

Co-IP between transient expression of indicated HA-tagged PTEN and V5-tagged wt

PREX2 constructs in 293T cells. Immunoprecipitation (IP) done using V5 antibody and probing done using HA antibody. Input represents 5% of whole cell extract used in IPs.

Lower panel shows schematic representation of PTEN domain structure and location of the relevant amino acids for the deletion constructs.

Supplementary Figure 6. Expression of V5-tagged PREX2 constructs

PMEL-NRAS cells expressing GFP or wt PREX2 as controls or the indicated PREX2 truncating constructs were analysed by western blotting using V5-antibody to show expression levels of the various target proteins (same cells as in Fig 4a). Arrow indicates a non-specific band.

Supplementary Figure 7. Effect of IGF2 on Akt activation and expression of

CDKN1C (p57)

(A) PMEL cells were grown to approximately 30-40% confluency. Cells were then serum deprived for 2 hours and stimulated with recombinant human IGF2 (100ng/ml) for indicated times. Cells were subsequently lysed in RIPA buffer and immunoblotted with the respective antibodies. (B) PMEL cells were grown to approximately 30-40% confluency. Cells were then serum deprived for 16 hours and stimulated with increasing

12 concentrations of recombinant human IGF2 (0-200ng/ml) for 24 hours. RNA was isolated and qRT-PCR performed to quantify expression of CDKN1C (p57).

Supplementary Figure 8. Imprinting-based gene expression regulation of

CDKN1C.

(A) In wild type conditions, CKDN1c is expressed only from the maternal allele (filled boxes). In the paternal allele, promoter of KCNQ1OT1 in the intron of KCNQ1 (also known as KvDMR1) is hypomethylated leading to expression of KCNQ1OT1, which suppresses the surrounding imprinted genes. However, in the maternal allele, methylation of KvDMR1 (red circles) decreases expression of KCNQ1OT1 and relieves the repression, thus activating expression of CKDN1c.

(B) In case of KvDMR1 hypomethylation (e.g., as in Beckwith-Wiedemann syndrome patients), KCNQ1OT1 is expressed and inhibits the surrounding gene expression from both alleles.

Supplementary Table 1. Reverse Phase Protein Array (RPPA). Xenograft tumors derived from PMEL-NRAS cells with expression of GFP, wt PREX2 or PREX2 mutants were lysed and RPPA analysis performed as detailed in methods section. Table depicts the median centered values of all the antibodies used in the analysis and 15 samples (three independent tumors per experimental group).

References

1. Garraway LA, Widlund HR, Rubin MA, Getz G, Berger AJ, Ramaswamy S, et al. Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma. Nature. 2005;436:117-22. 2. Hill K, Welch HC. Purification of P-Rex1 from neutrophils and nucleotide exchange assay. Methods Enzymol. 2006;406:26-41. 3. Welch HC, Coadwell WJ, Ellson CD, Ferguson GJ, Andrews SR, Erdjument- Bromage H, et al. P-Rex1, a PtdIns(3,4,5)P3- and Gbetagamma-regulated guanine- nucleotide exchange factor for Rac. Cell. 2002;108:809-21. 4. Berger MF, Hodis E, Heffernan TP, Deribe YL, Lawrence MS, Protopopov A, et al. Melanoma genome sequencing reveals frequent PREX2 mutations. Nature. 2012;485:502-6. 5. Ramaswamy S, Nakamura N, Vazquez F, Batt DB, Perera S, Roberts TM, et al. Regulation of G1 progression by the PTEN tumor suppressor protein is linked to inhibition of the phosphatidylinositol 3-kinase/Akt pathway. Proceedings of the National Academy of Sciences of the United States of America. 1999;96:2110-5. 6. Smyth GK, Michaud J, Scott HS. Use of within-array replicate spots for assessing differential expression in microarray experiments. Bioinformatics. 2005;21:2067-75. 7. Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature protocols. 2009;4:44- 57. 8. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proceedings of the National Academy of Sciences of the United States of America. 2005;102:15545-50. 9. Arnold K, Bordoli L, Kopp J, Schwede T. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics. 2006;22:195-201. 10. Lucato CM, Halls ML, Ooms LM, Liu HJ, Mitchell CA, Whisstock JC, et al. The Phosphatidylinositol (3,4,5)-Trisphosphate-dependent Rac Exchanger 1.Ras- related C3 Botulinum Toxin Substrate 1 (P-Rex1.Rac1) Complex Reveals the Basis of Rac1 Activation in Breast Cancer Cells. The Journal of biological chemistry. 2015;290:20827-40. 11. Hodis E, Watson IR, Kryukov GV, Arold ST, Imielinski M, Theurillat JP, et al. A landscape of driver mutations in melanoma. Cell. 2012;150:251-63.

Supplementary Figure 1 PREX2 truncation E824* cooperates with NRAS mutations but do not cooperate with BRAF V600E mutation to accelerate melanoma xenograft formation.

Melanocytes with NRAS Q61K background

B Melanocytes with BRAF V600E background

n.s Percent surviving Supplementary Figure 2 Gene set enrichment analysis (GSEA) in TetO-LSL-PREX2 E824* transgenic mouse tumors and xenografts expressing truncating PREX2 Supplementary Figure 3 Decreased expression of p21 and p27 and increased expression of IGF2 in PREX2 mutant tumors.

GEM model Tumor lysates

PREX2 wt PREX2 E824*

p21

p27

Vinculin

B Relative mRNA level Relative mRNA Supplementary Figure 4 Truncating PREX2 mutations abolish its interaction with PTEN.

A B Q261* P321* E730* Q1321* Q1430* K278* Q961* E824*

C D Input IP V5 Input IP V5 PREX2 PREX2 PREX2 PREX2 wt wt GFP K278* GFP K278* E824* Q1430* E824* Q1430* wt K278* E824* Q1430* wt GFP GFP K278* E824*

Q1430* PTEN PTEN PTEN

V5 V5 V5

Vinculin Vinculin

E Input IP V5 PREX2 PREX2 V1441 F GFP GFP wt P948S wt T868I V1441F P948S P1145S G844D G844D T994A G106E P1145S T868I T994A G106E A1376V A1376V E824* K278* E824* K278* Q1430* Q1430* PTEN

V5 (longer exposure)

Total AKT (loading control) Supplementary Figure 5 Domains implicated in the binding of PTEN to PREX2

Input IP:V5

+ + HA-PTEN wt + + Lipid phosphatase dead HA-PTEN C124S + + Protein phosphatase dead HA-PTEN G129E + + HA-PTEN 1-274 + + Lack C-tail and PDZ-BD + HA-PTEN 1-336 + Lack part of C2 and is not + HA-PTEN ∆274-342 + Recruited to membrane PREX2-V5 - + + + + + + + - + + + + + + +

HA-PTEN

V5- PREX2

Vinculin

274 336 342 PDZ‐ PTEN C‐Tail PBD Phosphatase C2 BD 1 15 185 351 401 403 Supplementary Figure 6 Expression of V5-tagged PREX2 constructs

PREX2 wt K278* E824* Q1430* GFP

* *

* CDKN1C (p57) Effect ofIGF2 SupplementaryFigure 7 (100ng/ml) IGF2 A B Relative mRNA level of CDKN1C - Immortalized melanocytelysate 5’ 10’ 15’ 30’ on activation andexpressionAkt of 60’ AtT308 pAkt AtS473 pAkt Vinculin Akt Total Supplementary Figure 8 Imprinting-based gene expression regulation of CDKN1C.

CDKN1C KCNQ1 PATERNAL KCNQ1OT1 CKDN1C OFF

CDKN1C KCNQ1 CKDN1C expressed MATERNAL KCNQ1OT1

PATERNAL CDKN1C KCNQ1 KCNQ1OT1 CKDN1C OFF

CDKN1C KCNQ1 MATERNAL CKDN1C OFF KCNQ1OT1