Mutations in MAP 3 K 1 tilt the balance from SOX 9 / FGF 9 to WNT / b-catenin signaling

In-frame missense and splicing mutations (resulting in a 2 amino acid insertion or a 34 amino acid deletion) dispersed through the MAP3K1 gene tilt the balance from the male to female sex-determining pathway, resulting in 46,XY disorder of sex development. These MAP3K1 mutations mediate this balance by enhancing WNT/b-catenin/ FOXL2expressionandb-cateninactivityandbyreducingSOX9/FGF9/FGFR2/SRYexpression. Theseeffectsare mediated at multiple levels involving MAP3K1 interaction with protein co-factors and phosphorylation of downstream targets. In transformed B-lymphoblastoid cell lines and NT2/D1 cells transfected with wild-type or mutant MAP3K1 cDNAs under control of the constitutive CMV promoter, these mutations increased binding of RHOA, MAP3K4, FRAT1 and AXIN1 and increased phosphorylation of p38 and ERK1/2. Overexpressing RHOA or reducing expression of MAP3K4 in NT2/D1 cells produced phenocopies of the MAP3K1 mutations. Using siRNA knockdown of RHOA or overexpressing MAP3K4 in NT2/D1 cells produced anti-phenocopies. Interestingly, the effects of the MAP3K1 mutations were rescued by co-transfection with wild-type MAP3K4. Although MAP3K1 is not usually required for testis determination, mutations in this gene can disrupt normal development through the gains of function demonstrated in this study.


INTRODUCTION
Sex determination in mammals is a genetically encoded process that mediates the balance between testis and ovary developmental pathways. To date, much work has focused on the roles of transcription factors (SRY, SOX9, NR0B1), growth factors (FGF9, PDGF) and signaling molecules (WNT4, RSPO1, b-catenin) that regulate these pathways (1 -3). Knockout of these genes in the permissive developing gonad or overexpression in the non-permissive developing gonad lead to genetic sex reversal. Examples of these effects include homozygous loss-of-function alleles in RSPO1 and ectopic expression of SRY and SOX9 all leading to 46,XX testicular disorder of sex development as well as knockout of SRY and SOX9 and overexpression of WNT4 and stabilization of b-catenin leading to ovarian development or gonadal dysgenesis (4 -11). Despite prior observations that signal transduction molecules in the MAP kinase pathway play a role in mediating the expression of these genes and their products, especially in chondrocyte development, their roles in mediating the balance between SOX9/ FGF9 expression for testicular determination and WNT/ b-catenin expression for ovarian determination is poorly understood (12)(13)(14).
Previously, we showed that missense mutations at wellconserved sites or in-frame splicing variants with in-frame insertion in MAP3K1 resulted in 46,XY gonadal dysgenesis and milder forms of this phenotype based on co-inheritance in multiple families (15). We have also demonstrated missense mutations in MAP3K1 in several cases of sporadic 46,XY gonadal dysgenesis. In turn, these mutations altered phosphorylation of the downstream targets, p38 and ERK1/2, and increased binding of the co-factors, RHOA and MAP3K4 as shown in our previous studies (16). Yet, knockout of the MAP3K1 gene itself led to only minor testicular abnormalities in the developing mouse * To whom correspondence should be addressed at: Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Ullman 817, Bronx, NY 10461, USA. Tel: +1 7184308605; Email: harry.ostrer@einstein.yu.edu gonad, suggesting that it is not necessary for testicular development (17). Our previous studies showed a series of N-terminal mutations through exon 10; here, we extend the repertoire of mutations that cause 46,XY gonadal dysgenesis further downstream to exon 13 and 14, spreading across multiple functional domains of MAP3K1 (15). We observed that these mutations tilt the balance in the sex-determining pathways not only by upregulating b-catenin expression and activity, but also at multiple levels by downregulating SOX9, SRY, FGF9 and FGFR2 expression. The effects of these mutations in MAP3K1 were rescued by co-transfection with wild-type MAP3K4 in NT2/D1 cells.

RESULTS
Mutations in MAP3K1 increase phosphorylation of downstream targets and binding of associated proteins. In the current study, we examined six different mutations in the MAP3K1 gene, five of which caused abnormal developmental phenotypes (Fig. 1, Table 1). These mutations have the characteristic of being in-frame alterations, either non-conservative single-nucleotide variants (p.P153L, p.L189R, p.L189P, p.K246E) or familial splice acceptor site variant (c.634-8T.A and c.2180-2A.G) (Fig. 1C). Previously, we showed that the c.634-8T.A mutation created a novel splice acceptor site that results in insertion of two amino acids residues in-frame between codons 211 and 212 (15). The c.2180-2A.G mutation results in skipping exon 13 or use of a cryptic acceptor at c.2283_2284; chr5:56177013-5617714 (UCSC hg19) with loss of 34 amino acid residues in-frame between codons 727 and 761 (Fig. 1C). Thus, these mutations occurred in exons 2, 3, 13 and 14 of this 20 exon gene. Transformed B-cell lymphoblastoid cell lines (LCLs) were available for all of these mutations. These were used for analysis of phosphorylation of downstream targets, interactions with MAP3K1binding proteins, or relative abundance of b-catenin.
As we have reported previously for the p.L189P, p.L189R and c.634-8A mutations that were associated with gonadal dysgenesis, analysis of the LCLs for the newly identified mutations demonstrated varying increases in phosphorylation of the downstream targets, p38 and ERK1/2, and increases in binding of The c.634-8T.A mutation creates a novel splice acceptor site that results in the insertion of two amino acids residues in-frame between codons 211 and 212 (15). The c.2180-2A.G mutation results in use of a cryptic acceptor at c.2283_2284; chr5:56177013-5617714 (UCSC hg19) with loss of 34 amino acid residues in-frame between codons 727 and 761. (B) Pedigree of multiple individuals with biopsy-proven 46,XY gonadal dysgenesis and MAP3K1 c.2180-2A.G mutation (II-3, II-4 and III-1). All three individuals with 46,XY mutations had an unvirilized female phenotype and dysgenetic gonads at histology. The uterus of Subject II-3 was observed at laparoscopy and Subjects II-3 and II-4 had elevated LH and FSH. Individual II-2 was an unaffected 46,XX carrier. Sanger sequencing demonstrated the heterozygous splicing site mutation in Subject II-3. (C) The mutation was predicted to affect splicing in one of three ways: (1) skipping of exon 13 (causing a frameshift insertion of a STOP codon after seven amino acids, resulting in complete loss of the kinase domain (c.2180_2369del; p.Ser728Ilefs * 8), (2) use of a cryptic acceptor site within exon 13 (c.2283_2284 AG;g.56177013-5617714AG). In this case, the mutated mRNA would lose the first 105 nucleotides of exon 13 (c.2180_2284del), preserving the open reading frame and resulting in a protein with the first 35 amino acids of exon 13 deleted (p.Gly727_Ile761del), or (3) a normal transcript. RT-PCR with primers external to the exons involved in the mutations results in two bands corresponding by size to the wild type and to the form that uses the cryptic acceptor splice site internal to exon 13. Cloning of the RT-PCR product and sequencing of colonies showed the presence of both variant splicing forms (using the cryptic acceptor site internal to exon 13 and exon 13 skipping) along with the wild-type form.  Table 1) (15,16). The phosphorylation of ERK1/2 and the binding of RHOA were measured by immunoprecipitation western blot analysis and confirmed by the flow-variant analysis (FVA) method that uses modified immunoprecipitation with flow cytometry to measure the binding of specific proteins to fluorochrome-coupled antibodies (16). The p38 phosphorylation and RHOA binding in the non-pathogenic hypomorphic p.K246E variant LCL were only slightly increased compared with the wild-type male LCL control and significantly less than in the other mutation-bearing LCLs. We have examined a total of 11 wild types: seven normal males and four normal females in triplicates of three biological repeats to establish the normal baseline control. The phosphorylation of ERK1/2 and the binding of MAP3K4 to MAP3K1 in the p.K246E-bearing LCL were not increased compared with the control.
This increased binding was not confined to RHOA and MAP3K4, previously MAP3K1 has been shown to bind AXIN1 in various truncated deletion models, which in turn, binds to FRAT1 (18)(19)(20). FVA performed on wild-type or mutant LCLs using MAP3K1 as bait showed increased binding to FRAT1 and AXIN1 for those bearing the p.L189P and p.153L mutations, but not the p.K246E variant. The increased binding of AXIN1 to MAP3K1 is independent of the binding of FRAT1 to MAP3K1 (Fig. 2D).
The effects of MAP3K1 mutations on phosphorylation of downstream targets and binding of associated proteins can be recapitulated in NT2/D1 cells. Human teratocarcinoma cell line NT2/D1 has been shown previously to express the repertoire of genes observed in testis determination and has been used to examine the effects of mutations in SF1 and upregulation of b-catenin on the expression of SOX9 (21,22). Transfection of mutant and wild-type cDNAs with CMV-driven expression plasmids led to efficient expression of MAP3K1 (Supplementary Material, Fig. S2A). The p.L189P, p.L189R and c.634-8A mutations increased phosphorylation of p38 and ERK1/2, as had been observed in LCLs previously (15). These increases in phosphorylation were detected using standard western blots (Fig. 3A, Supplementary Material, Fig. 1S) and phosphorylated digital cell western (DCW) where expression of multiple target proteins (total and phosphorylated) are measured in large numbers of intact fixed cells simultaneously (Fig. 3B). Using the co-immunoprecipitation method of FVA, the NT2/D1 cells transfected with the p.L189P, p.L189R and c.634-8A mutant cDNAs showed increased RHOA binding to MAP3K1 bait on the epoxy-coated beads (Y-axis) and forward scatter (FSC-A-X-axis), as has been observed previously in LCLs (Fig. 3C) (15). FSC is the light scatter fluorescence measured at low-angle forward proportional to the diameter of the bead or cell. FSC provides a suitable method for detecting particles greater than a given size, independent of their fluorescence. Although overexpression of the wild-type MAP3K1 cDNA increased both RHOA binding and p38 and ERK1/2 phosphorylation, these effects were 2.5-fold increased when the mutant cDNAs were transfected (Fig. 3D).
Mutations in MAP3K1 tilt the balance of gene expression in the testis-determining pathway. Three mutations, p.L189P, p.L189R and c.634-8A, were studied in greater detail to understand their effects on the testis-determining pathway. Transfection of these mutant cDNAs decreased expression of SOX9   Co-immunoprecipitation of these lysates, where MAP3K1 was the bait, showed increased affinity to RHOA and FRAT1 (probe antibodies). The p.K246E variant did not have an effect that varied from wild-type LCLs. Histone as loading control, and MAP3K1 as input control. (B) FVA using primary wild-type LCLs or those bearing p.L189P and p.153L mutations or p.K246E variant was performed using MAP3K1 as bait and showed increased binding to RHOA (Y-axis, and as shown in A) and MAP3K4 (X-axis). Pseudo primary colors were assigned using FLOWJO 6.0 for each target, MAP3K4-green, RHOA-red and MAP3K1-blue. If all three targets are present on the target bead in equal ratio, a white pixel is generated against the black background. (C) Quantification of FVA fluorescence intensity for MAP3K4 (solid black bar) and RHOA (gray bar) binding to MAP3K1 and normalized to MAP3K1 input fluorescence (Y-axis is normalized fluorescent events) and compared in a pairwise fashion by Student's t-test shown as * P , 0.05 and * * P , 0.005. The binding of MAP3K4 and RHOA was increased for all three variants/mutations, yet significantly higher for the p.L189P and p.P153L mutations. (D) FVA using primary wild-type or mutant LCLs was performed using MAP3K1 as bait and showed increased binding to FRAT1 (Y-axis, and as shown in A) and AXIN1 (X-axis) for those bearing the p.L189P and p.153L mutations, but not the p.K246E variant. Pseudo primary colors were assigned using FLOWJO 6.0 for each target, FRAT1-green, AXIN1-red and MAP3K1-blue. If all three targets are present on the target bead in equal ratio, a white pixel is generated against the black background. If only AXIN1 and MAP3K1 are present on the target bead a pink pixel is generated.

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Human Molecular Genetics, 2014, Vol. 23, No. 4 60.5-fold, respectively ( Fig. 6A and B), whereas knockdown of RHOA by siRNA and overexpression of MAP3K4 led to the opposite effects-increased expression of SOX9 levels by 508-and 171-fold, respectively, and dramatically decreased expression of b-catenin levels ( Fig. 6A and B). Thus, overexpression of RHOA and knockdown of MAP3K4 provided phenocopies of the MAP3K1 mutations in the MAP kinase signaling pathway, suggesting that overexpression of wild-type MAP3K4 could rescue the effects of MAP3K1 mutations. The effects of MAP3K1 mutations were rescued by co-transfection with wild-type MAP3K4. To test for MAP3K4 rescue of MAP3K1 mutations, co-transfection experiments were performed using a wild-type MAP3K4 plasmid and wild type or mutant MAP3K1 plasmids efficient transfection into NT2/D1 cells (Supplementary Material, Fig. S2B). In separate experiments, the NT2/D1 cells transfected with only mutant MAP3K1 cDNAs (p.L189P, p.L189R and c.634-8A) were compared with cotransfected mutant cDNAs with MAP3K4 showed restoration of the mRNA and protein expression of SOX9 in mutants ( Fig. 4A and B) and, for b-catenin levels, MAP3K4 restored the mutants to wild-type levels ( Fig. 4C and D), that is, the expression of SOX9 was increased and the expression of b-catenin was reduced in mutants when MAP3K4 was introduced by transfection. Moreover, the reduction in b-catenin expression was confirmed by TCF dual-luciferase reporter showing similar reduction in b-catenin activity in mutant cotransfected with MAP3K4 (Fig. 5D). Thus, MAP3K4 rescued the effects of MAP3K1 mutations. This rescue of the MAP3K1 mutations and of the RHOA overexpression phenocopy appeared to be mediated by increasing SRY expression. Similarly, Taqman qPCR on SRY mRNA expression levels showed marked reduction compared to wild type when the NT2/D1 cells were transfected with mutant MAP3K1 cDNAs or RHOA CMV-driven expression plasmids. Co-transfection with a MAP3K4 expression construct, rescued the SRY mRNA expression in these mutant transfected cells by restoring SRY to that of wild-type cells (Fig. 6C).

DISCUSSION
The development of the embryonic bipotential gonad is genetically controlled. The somatic cell progenitors express both testicular (SOX9/FGF9) and ovarian (WNT4/ b-catenin) factors in a controlled spatial pattern. If SRY is expressed, SOX9 and FGF9 are upregulated via a mutual feedforward loop and the somatic cells adopt a Sertoli fate (1). In turn, SOX9 employs two distinct mechanisms to inhibit WNT/b-catenin signaling. The N-terminus of SOX9 promotes b-catenin degradation, whereas the C-terminus inhibits b-catenin transcriptional activity without affecting its stability (23). Thus, the normal role of SRY in XY gonads is to tip the balance toward the testis-specific pathway (4,6). This pathway can be disrupted by mutations in SRY, SOX9 and SF1, all transcription factors that bind to the SOX9 TESCO enhancer (24). In mice, the pathway can be overridden by a dominant stabilizing mutation in b-catenin (11). Here, we show that the pathway can also be overridden by a series of five different in-frame mutations in MAP3K1 that were identified in individuals with abnormal gonadal development or by RHOA and MAP3K4 modulations. The effect of these mutations was observed to decrease SOX9 expression and increase b-catenin expression and activity through multiple effects in the MAP kinase pathway (Fig. 7).
The mutations spanned exons 2, 3, 13 and 14 and affected both coding (missense) and splicing. The splicing mutations resulted in insertion of 2 amino acids or deletion of 34 amino acids. Yet, all of the mutations resulting in gonadal dysgenesis caused increased phosphorylation of ERK1/2 and p38 and increased binding of RHOA and MAP3K4 and the representative mutations studied increased binding of AXIN1 and FRAT1 proteins. Therefore, these were a range of sites N-terminal to the MAP3K1 kinase domain that could influence binding of cofactors and increase kinase activity. The increased phosphorylation of ERK1/2 and p38 is known to mediate both inactivation of GSK3b, which, in turn, leads to stabilization, and upregulation of b-catenin (25)(26)(27). AXIN1 and FRAT1 also mediate inactivation of GSK3b, AXIN1 interacts with GSK3b to reduce b-catenin abundance, whereas FRAT1 inhibits this process (18 -20). Indeed, previous work in b-catenin signaling demonstrated that transfection of siRNA to GSK3b increased expression and activity of b-catenin, similar to our observation of NT2/D1 cell phenocopies of the MAP3K1 mutations. A previous study showed that AXIN1 association to the MAP3K1 N-terminal region of 'KGANLLIDSTGORL' acted as an activation complex. Upon MAP3K1 depletion in the HEK293T cotransfected with TCF reporter constructs, there was a reduction in TOPFlash activity, suggesting that MAP3K1 is an integral part of AXIN1 sequestration (18). It is possible that FRAT1 and AXIN1 association with MAP3K1 is acting as a sink to promote b-catenin stabilization, but such an observation warrants an independent investigation into the WNT signaling cascade mechanisms.
All of the mutations enhanced binding to RHOA, a known positive regulator of MAP3K1 kinase activity (28). In chondrocytes For Taqman qPCR, results were normalized to housekeeping gene, GAPDH, and compared with control NT2/D1 cells transfected with empty plasmids by Student's t-test, * P , 0.05 and * * P , 0.005. (A) Quantification of normalized SOX9 mRNA expression by Taqman qPCR (Y-axis is fold change relative to empty plasmids) and compared in a pairwise fashion. The mRNA expression of SOX9 was decreased for all three mutations and was rescued by co-transfection with wild-type MAP3K4 (P ¼ 0.0002 for the mutant group versus wild type). (B) Quantification of normalized SOX9 protein expression (Y-axis is fold change relative to empty plasmids) and compared in a pairwise fashion. The protein expression of SOX9 was decreased when either three mutations were transfected and was rescued by co-transfection with wild-type MAP3K4 (P ¼ 0.0003 for the mutant group versus wild type). (C) Quantification of normalized b-catenin mRNA expression (Y-axis is fold change relative to empty plasmids) and compared in a pairwise fashion. The mRNA expression of b-catenin was increased for all three mutations and was rescued by co-transfection with wild-type MAP3K4 (P ¼ 1.9 × 10 216 for the mutant group versus wild type). (D) Quantification of normalized b-catenin protein expression (Y-axis is fold change relative to empty plasmids) and compared in a pairwise fashion. The protein expression of b-catenin was increased for all three mutant transfected and was rescued by co-transfection with wild-type MAP3K4 (P ¼ 0.005 for the mutant group versus wild type).

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Human Molecular Genetics, 2014, Vol. 23,No. 4 development, RHOA regulates the transcriptional activity of SOX9 and its feedback loop (14,29,30). Likewise, we generated phenocopies of the MAP3K1 mutants by overexpressing RHOA or anti-phenocopies by downregulating expression of RHOA by siRNA transfection. Furthermore, all of the mutations examined here enhanced binding of MAP3K4 to MAP3K1 protein complex. This might have arisen through interactions with their shared binding partner, AXIN1 (31,32). Both MAP kinases compete for AXIN binding, albeit at different sites (31). We have suggested previously that the presence of these MAP3K1 mutations may alleviate this competition (16). Unlike MAP3K1, MAP3K4 is an essential testis-determining gene. In mice, homozygous loss-of-function alleles in MAP3K4 lead to disrupted testis development from failure to support cord development (33). This failure of testicular development results from failure to upregulate Sry, an effect mediated by the Map3k4 binding partner, Gadd45g. In the current experiments, knocking down the expression of MAP3K4-produced molecular phenocopies of the MAP3K1 mutations and overexpressing MAP3K4produced anti-phenocopies. This overexpression of MAP3K4 corrected the expression patterns of SOX9 and b-catenin and normalized b-catenin activity in co-transfection experiments with MAP3K1 mutations. Although bone abnormalities have not been reported in individuals harboring MAP3K1 mutations, these may nonetheless be present and may represent an ascertainment bias. Heterozygous mutations in the SOX9 gene with resulting haploinsufficiency cause campomelic dysplasia in humans and hypoplasia of endochondral bones in mice (9,34). This phenotype has also been produced by stabilization of b-catenin in chondrocytes (12).
These experiments demonstrate that mutations in MAP3K1 caused abnormal testicular development by downregulating SOX9 expression mediated by RHOA and by the b-catenin negative feedback loop. Furthermore, the mutated MAP3K1 Figure 5. Overexpression of mutant MAP3K1 increases FOXL2 and decreases FGF9 and FGFR2 mRNA levels and increases of TCF dual-luciferase reporter activity relative to wild type. qPCR analysis of mRNA at 24 h of NT2/D1 cells transfected with expression plasmids bearing wild-type or either mutants (p.L189R, p.L189P, and c.634-8A) cDNAs, a rescue co-transfection is performed alongside with MAP3K4 for each mutant. Taqman qPCR results were normalized to housekeeping gene, GAPDH, and compared with control NT2/D1 cells transfected with empty plasmids by Student's t-test, * P , 0.05 and * * P , 0.005. (A) Quantification of normalized TCF/LEF dual reporter luciferase activity (Y-axis is normalized relative luciferase units) and compared in a pairwise fashion (mutant versus rescued mutant). Control-3 is the triplicate pool of untreated NT2/D1 cells. The luciferase reporter activity assayed at 48 h following transfection showed marked increase in all three mutants. Overexpression of RHOA similarly increased reporter activity. Knockdown of RHOA or MAP3K1 by either siRNAs abrogated the reporter activity. In separate experiments, co-transfection of any of the three mutants with MAP3K4 rescued their phenotypes, observed as marked reduction of the reporter activity found in mutants. Co-transfection of TCF luciferase reporter with Stealth siRNA to RHOA or MAP3K1 abrogated the reporter activities. (B) Quantification of normalized FGFR2 expression (Y-axis is fold change relative to empty plasmids) and compared in a pairwise fashion. The expression of FGFR2 was decreased for all three mutations and FGFR2 expression was rescued by co-transfection with wild-type MAP3K4 into mutants. (C) Quantification of normalized FGF9 expression (Y-axis is fold change relative to empty plasmids) and compared in a pairwise fashion. The expression of FGF9 was dramatically decreased when NT2/D1 cells were transfected with any of the three mutants. FGF9 expression levels were restored in all mutants' transfected cells when MAP3K4 was added in separate co-transfection experiments. (D) Quantification of normalized FOXL2 mRNA expression (Y-axis is fold change relative to empty plasmids) and compared in a pairwise fashion. The expression of FOXL2 was increased for all three mutant transfected NT2/D1 cells, with the highest marked increase observed in C634-8A mutant. MAP3K4 was cotransfected with each mutant in separate experiments showing phenotype rescue similar to wild type.
Human Molecular Genetics, 2014, Vol. 23, No. 4 1079 proteins upregulated b-catenin expression and activity through increased phosphorylation of p38 and ERK1/2 and increased binding of AXIN1 and FRAT1 proteins. Seemingly, a threshold of cofactor binding and kinase activity must be exceeded to affect gonadal development. The p.K246E mutation reported here increased p38 kinase activity at modest level, yet had no effect on RHOA binding, ERK1/2 phosphorylation, nor testicular development. This mutation is representative of a series of 23 rare missense variants in the MAP3K1 gene that have been reported in dbSNP, each with an individual allele frequency ,1% and collectively with an allele frequency of 1.56% (http:// www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?locusId=4214).
Some of these may alter the cofactor binding and kinase activity above the threshold in aggregate to cause abnormal testicular development and explain the seeming prevalence of MAP3K1 mutations that we have observed as a cause for 46,XY gonadal dysgenesis (15).

Cell culture
This study was approved by the institutional review boards of Albert Einstein College of Medicine and the collaboration   into 1 × 10 5 NT2/D1 cells using the Lipofectamine transfection reagents (Life Technologies). Gene expression of the mRNA and protein were examined 48 h after transfection by qPCR, western blot and DCW analysis. For DCW, 1 × 10 6 cells were counted for each sample and 16% formaldehyde was added directly into the culture medium to a final concentration of 1.5%). The cells were incubated for 10 min at 258C or room temperature, then pelleted by low-speed centrifugation at 2000 g at 48C. The cell pellet was resuspended by vortexing in 500 ml ice-cold methanol and incubated on ice for 5 min. Cells were stored at -808C with minimum degradation. Prior to DCW analysis, 50-100 mL of cells or 500 million cells were stained with fluorescently labeled antibodies at 1:100.

Reporter gene assays and transient transfection
The TCF/LEF Cignal luciferase reporter assay is a preformulated mix of Renilla with a transfection-ready TCF/LEF reporter construct, negative control or positive control (Qiagen, Valencia, CA, USA). The transcription factor reporters and controls were transfected in parallel with identical experimental parameters. Dual-luciferase results were calculated for each transfectant by normalizing to internal fluorescence of Renilla and then calculating the change in the activity by comparing the normalized luciferase activities of the reporter in treated versus untreated transfectants. The identically treated negative control transfectants served as specificity controls. Transfection efficiency was determined from the activity of green fluorescent protein positive control as well as a positive control for both the firefly and Renilla luciferase assays. The cells were lysed after 24 and 48 h using luciferase lysis buffer (Promega Corp., Madison, WI, USA), and luciferase activities were measured using the dual-luciferase reporter assay system on dual injector Biotek Synergy H1 reader (Biotek, Winooski, VT, USA). All transfection experiments were performed in triplicates.
In the siRNA knockdown co-transfection experiments, NT2/ D1 cells were seeded at a density of 1 × 10 5 cells/6-well plate in triplicates and siRNA targeting the human RHOA or MAP3K4 gene or control non-targeting medium GC content siRNA was delivered to NT2/D1 cells the following day after overnight serum deprivation in DMEM-only media. After 24 h, cells were transfected with Cignal TCF/LEF luciferase reporter mix constructs according to manufacturer's protocol using Lipofectamine 2000 (Life Technologies).
In shRNA knockdown co-transfection experiments using luciferase assays to examine the effect of wild-type MAP3K1 and point mutations in MAP3K1, NT2/D1 cells were plated in 40 -50% density in 24-well plates and 2.5 ng/well MAP3K1 constructs or control DNA were transfected together with reporter plasmid and internal control plasmid several hours after seeding.

Quantitative RT -PCR
Quantitative RT -PCR (qRT -PCR) experiments were analyzed in a VIAA7 real-time PCR detection system (Life Technologies) using Taqman gene expression master mix (Life Technologies). Values were normalized using b-actin as a control. The following Taqman assays were used for examining the effect of MAP3K1, RHOA and MAP3K4 after transfections: Site-directed mutagenesis-MAP3K1 mutants were generated using the Quick Change Site-Directed Mutagenesis II kit (Stratagene, Cedar Creek, TX, USA). Wild-type full-length MAP3K1 expression vector (a kind gift from Dr. Michael Karin) was used as a template for PCR based mutagenesis with primer arms flanking 22 bases on each side of the mutation site.

Data analysis
Comparison of differences between Cignal luciferase activity assay and qRT -PCR expression was performed using the twotailed Student's t-test (where equal variance between groups was assumed). A P-value of ,0.05 was considered statistically significant.