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• W2912706099 abstract "•STK19 phosphorylates and activates oncogenic NRAS to promote melanomagenesis•We developed a novel potent and selective STK19 inhibitor, ZT-12-037-01 (1a)•ZT-12-037-01 (1a) inhibits NRAS-driven melanomagenesis and melanoma growth Activating mutations in NRAS account for 20%–30% of melanoma, but despite decades of research and in contrast to BRAF, no effective anti-NRAS therapies have been forthcoming. Here, we identify a previously uncharacterized serine/threonine kinase STK19 as a novel NRAS activator. STK19 phosphorylates NRAS to enhance its binding to its downstream effectors and promotes oncogenic NRAS-mediated melanocyte malignant transformation. A recurrent D89N substitution in STK19 whose alterations were identified in 25% of human melanomas represents a gain-of-function mutation that interacts better with NRAS to enhance melanocyte transformation. STK19D89N knockin leads to skin hyperpigmentation and promotes NRASQ61R-driven melanomagenesis in vivo. Finally, we developed ZT-12-037-01 (1a) as a specific STK19-targeted inhibitor and showed that it effectively blocks oncogenic NRAS-driven melanocyte malignant transformation and melanoma growth in vitro and in vivo. Together, our findings provide a new and viable therapeutic strategy for melanomas harboring NRAS mutations. Activating mutations in NRAS account for 20%–30% of melanoma, but despite decades of research and in contrast to BRAF, no effective anti-NRAS therapies have been forthcoming. Here, we identify a previously uncharacterized serine/threonine kinase STK19 as a novel NRAS activator. STK19 phosphorylates NRAS to enhance its binding to its downstream effectors and promotes oncogenic NRAS-mediated melanocyte malignant transformation. A recurrent D89N substitution in STK19 whose alterations were identified in 25% of human melanomas represents a gain-of-function mutation that interacts better with NRAS to enhance melanocyte transformation. STK19D89N knockin leads to skin hyperpigmentation and promotes NRASQ61R-driven melanomagenesis in vivo. Finally, we developed ZT-12-037-01 (1a) as a specific STK19-targeted inhibitor and showed that it effectively blocks oncogenic NRAS-driven melanocyte malignant transformation and melanoma growth in vitro and in vivo. Together, our findings provide a new and viable therapeutic strategy for melanomas harboring NRAS mutations. RAS proteins are small membrane-bound guanine nucleotide-binding GTPases, acting as molecular switches by converting between GDP-bound inactive state and GTP-bound active state (Bos, 1989Bos J.L. ras oncogenes in human cancer: a review.Cancer Res. 1989; 49: 4682-4689PubMed Google Scholar, Downward, 2003Downward J. Targeting RAS signalling pathways in cancer therapy.Nat. Rev. Cancer. 2003; 3: 11-22Crossref PubMed Scopus (2520) Google Scholar, Milburn et al., 1990Milburn M.V. Tong L. deVos A.M. Brünger A. Yamaizumi Z. Nishimura S. Kim S.H. Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins.Science. 1990; 247: 939-945Crossref PubMed Scopus (846) Google Scholar, Pylayeva-Gupta et al., 2011Pylayeva-Gupta Y. Grabocka E. Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web.Nat. Rev. Cancer. 2011; 11: 761-774Crossref PubMed Scopus (1221) Google Scholar). They play a central role in the regulation of cell proliferation, differentiation, and survival by activating different downstream signaling pathways including RAF-MEK-ERK and PI3K-AKT pathways (Downward, 2003Downward J. Targeting RAS signalling pathways in cancer therapy.Nat. Rev. Cancer. 2003; 3: 11-22Crossref PubMed Scopus (2520) Google Scholar, Lavoie and Therrien, 2015Lavoie H. Therrien M. Regulation of RAF protein kinases in ERK signalling.Nat. Rev. Mol. Cell Biol. 2015; 16: 281-298Crossref PubMed Scopus (385) Google Scholar, Mendoza et al., 2011Mendoza M.C. Er E.E. Blenis J. The Ras-ERK and PI3K-mTOR pathways: cross-talk and compensation.Trends Biochem. Sci. 2011; 36: 320-328Abstract Full Text Full Text PDF PubMed Scopus (1188) Google Scholar, Samatar and Poulikakos, 2014Samatar A.A. Poulikakos P.I. Targeting RAS-ERK signalling in cancer: promises and challenges.Nat. Rev. Drug Discov. 2014; 13: 928-942Crossref PubMed Scopus (745) Google Scholar). The RAS family has three major isoforms, KRAS, HRAS, and NRAS (Barbacid, 1987Barbacid M. ras genes.Annu. Rev. Biochem. 1987; 56: 779-827Crossref PubMed Scopus (3777) Google Scholar, Malumbres and Barbacid, 2003Malumbres M. Barbacid M. RAS oncogenes: the first 30 years.Nat. Rev. Cancer. 2003; 3: 459-465Crossref PubMed Scopus (1427) Google Scholar) that share 92%–98% sequence identity in their amino-terminal region, and oncogenic mutations of RAS family members are commonly found in 20%–30% of all human tumors (Prior et al., 2012Prior I.A. Lewis P.D. Mattos C. A comprehensive survey of Ras mutations in cancer.Cancer Res. 2012; 72: 2457-2467Crossref PubMed Scopus (1254) Google Scholar, Stephen et al., 2014Stephen A.G. Esposito D. Bagni R.K. McCormick F. Dragging ras back in the ring.Cancer Cell. 2014; 25: 272-281Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). The prevailing NRAS mutation in melanoma occurs at position 61, where glutamine is substituted by arginine, lysine, or leucine (Q61R/K/L) (Bos, 1989Bos J.L. ras oncogenes in human cancer: a review.Cancer Res. 1989; 49: 4682-4689PubMed Google Scholar, Hayward et al., 2017Hayward N.K. Wilmott J.S. Waddell N. Johansson P.A. Field M.A. Nones K. Patch A.M. Kakavand H. Alexandrov L.B. Burke H. et al.Whole-genome landscapes of major melanoma subtypes.Nature. 2017; 545: 175-180Crossref PubMed Scopus (739) Google Scholar, Jakob et al., 2012Jakob J.A. Bassett Jr., R.L. Ng C.S. Curry J.L. Joseph R.W. Alvarado G.C. Rohlfs M.L. Richard J. Gershenwald J.E. Kim K.B. et al.NRAS mutation status is an independent prognostic factor in metastatic melanoma.Cancer. 2012; 118: 4014-4023Crossref PubMed Scopus (502) Google Scholar). This mutation impairs the intrinsic GTP hydrolysis activity and traps NRAS in a constitutive GTP-bound active conformation, which recruits RAF to the inner membrane for dimerization and activation (Marais et al., 1995Marais R. Light Y. Paterson H.F. Marshall C.J. Ras recruits Raf-1 to the plasma membrane for activation by tyrosine phosphorylation.EMBO J. 1995; 14: 3136-3145Crossref PubMed Scopus (526) Google Scholar, Smith et al., 2013Smith M.J. Neel B.G. Ikura M. NMR-based functional profiling of RASopathies and oncogenic RAS mutations.Proc. Natl. Acad. Sci. USA. 2013; 110: 4574-4579Crossref PubMed Scopus (159) Google Scholar). Oncogenic activation of NRAS leads to growth factor-independent proliferation of melanocytes and ultimately transformation to melanoma (Ji et al., 2012Ji Z. Flaherty K.T. Tsao H. Targeting the RAS pathway in melanoma.Trends Mol. Med. 2012; 18: 27-35Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Thus, NRAS Q61 mutations are critical drivers of melanomagenesis and important therapeutic targets. However, unlike the well-defined inhibitors targeting the oncogenic BRAFV600E mutation (Lavoie and Therrien, 2015Lavoie H. Therrien M. Regulation of RAF protein kinases in ERK signalling.Nat. Rev. Mol. Cell Biol. 2015; 16: 281-298Crossref PubMed Scopus (385) Google Scholar), the development of NRAS-selective inhibitors has been unsuccessful in the past decades (Cox et al., 2014Cox A.D. Fesik S.W. Kimmelman A.C. Luo J. Der C.J. Drugging the undruggable RAS: Mission possible?.Nat. Rev. Drug Discov. 2014; 13: 828-851Crossref PubMed Scopus (1191) Google Scholar). The serine/threonine-protein kinase 19 (STK19) was originally reported to phosphorylate α-casein at serine/threonine residues and histones at serine residues (Gomez-Escobar et al., 1998Gomez-Escobar N. Chou C.F. Lin W.W. Hsieh S.L. Campbell R.D. The G11 gene located in the major histocompatibility complex encodes a novel nuclear serine/threonine protein kinase.J. Biol. Chem. 1998; 273: 30954-30960Crossref PubMed Scopus (23) Google Scholar). Recently, it has been implicated in a transcription-related DNA damage response (Boeing et al., 2016Boeing S. Williamson L. Encheva V. Gori I. Saunders R.E. Instrell R. Aygün O. Rodriguez-Martinez M. Weems J.C. Kelly G.P. et al.Multiomic analysis of the UV-induced DNA damage response.Cell Rep. 2016; 15: 1597-1610Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). However, the role of STK19 in cancer initiation and development is poorly appreciated. Importantly, STK19 harbors significant somatic hotspot mutations in 5% of melanoma (Hodis et al., 2012Hodis E. Watson I.R. Kryukov G.V. Arold S.T. Imielinski M. Theurillat J.P. Nickerson E. Auclair D. Li L. Place C. et al.A landscape of driver mutations in melanoma.Cell. 2012; 150: 251-263Abstract Full Text Full Text PDF PubMed Scopus (1852) Google Scholar) and 10% of skin basal cell carcinoma (Bonilla et al., 2016Bonilla X. Parmentier L. King B. Bezrukov F. Kaya G. Zoete V. Seplyarskiy V.B. Sharpe H.J. McKee T. Letourneau A. et al.Genomic analysis identifies new drivers and progression pathways in skin basal cell carcinoma.Nat. Genet. 2016; 48: 398-406Crossref PubMed Scopus (285) Google Scholar) respectively, and is listed among the top melanoma driver genes (Lawrence et al., 2014Lawrence M.S. Stojanov P. Mermel C.H. Robinson J.T. Garraway L.A. Golub T.R. Meyerson M. Gabriel S.B. Lander E.S. Getz G. Discovery and saturation analysis of cancer genes across 21 tumour types.Nature. 2014; 505: 495-501Crossref PubMed Scopus (2100) Google Scholar). This strong genetic evidence implies an important, but uncharacterized role of STK19 in melanocyte malignant transformation and melanoma progression. In this study, we set out to use melanoma as a model to identify novel strategies for targeting oncogenic RAS signaling by identifying kinases that regulate NRAS activity. We discovered STK19 as an NRAS-activating kinase with frequent gain-of-function mutations and provide evidence that blockade of STK19 represents an effective therapeutic strategy for NRAS mutant melanomas. Activation of NRAS signaling depends on its association with effector proteins, such as RAF and PI3K, that contain a common RAS-binding domain (RBD) (Pylayeva-Gupta et al., 2011Pylayeva-Gupta Y. Grabocka E. Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web.Nat. Rev. Cancer. 2011; 11: 761-774Crossref PubMed Scopus (1221) Google Scholar). Disrupting the NRAS-RBD protein interaction would represent an effective therapy in NRAS mutant melanoma. To achieve this objective, we screened for kinases that could regulate the activity of NRASQ61R, the most prevalent NRAS mutation found in melanoma (Jakob et al., 2012Jakob J.A. Bassett Jr., R.L. Ng C.S. Curry J.L. Joseph R.W. Alvarado G.C. Rohlfs M.L. Richard J. Gershenwald J.E. Kim K.B. et al.NRAS mutation status is an independent prognostic factor in metastatic melanoma.Cancer. 2012; 118: 4014-4023Crossref PubMed Scopus (502) Google Scholar). We therefore established HEK293FT cells expressing HA-tagged NRASQ61R and screened a primary human kinome small interfering RNA (siRNA) library using a modified active NRAS chemiluminescence assay as a readout. Specifically, HA-NRASQ61R from siRNA-transfected cells was captured using a GST-CRAF-RBD fusion protein on glutathione-coated plates and detected with an anti-HA tag antibody conjugated with horseradish peroxidase for luminescence quantification (Figure 1A). We initially identified 12 kinases whose knockdown led to more than 50% inhibition of NRASQ61R activity and six kinases whose knockdown led to at least 2-fold upregulation of NRASQ61R activity (Figure 1B). Among these genes, STK19 was one of the candidates whose knockdown caused the highest inhibition on NRAS activity (35.1% of control group) (Figure 1B). To identify the status of STK19 in human melanomas, we investigated STK19 alteration in the TCGA melanoma cohort (PanCancer Atlas) and found STK19 to be altered in 91 of 363 (25.07%) sequenced skin cutaneous melanoma cases (Figure 1C). This is consistent with the analysis of large-scale melanoma exome data that discovered STK19 as one of six novel melanoma genes (PPP6C, RAC1, SNX31, TACC1, STK19, and ARID2) with a statistically significant functional mutation burden (Hodis et al., 2012Hodis E. Watson I.R. Kryukov G.V. Arold S.T. Imielinski M. Theurillat J.P. Nickerson E. Auclair D. Li L. Place C. et al.A landscape of driver mutations in melanoma.Cell. 2012; 150: 251-263Abstract Full Text Full Text PDF PubMed Scopus (1852) Google Scholar). As such, STK19 has been listed as an oncogenic candidate among the Broad Institute melanoma driver genes (Lawrence et al., 2014Lawrence M.S. Stojanov P. Mermel C.H. Robinson J.T. Garraway L.A. Golub T.R. Meyerson M. Gabriel S.B. Lander E.S. Getz G. Discovery and saturation analysis of cancer genes across 21 tumour types.Nature. 2014; 505: 495-501Crossref PubMed Scopus (2100) Google Scholar). Analysis of STK19 alteration in melanomas collected in different cBioPortal for Cancer Genomics databases further confirmed this discovery (Barretina et al., 2012Barretina J. Caponigro G. Stransky N. Venkatesan K. Margolin A.A. Kim S. Wilson C.J. Lehár J. Kryukov G.V. Sonkin D. et al.The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity.Nature. 2012; 483: 603-607Crossref PubMed Scopus (4859) Google Scholar, Hodis et al., 2012Hodis E. Watson I.R. Kryukov G.V. Arold S.T. Imielinski M. Theurillat J.P. Nickerson E. Auclair D. Li L. Place C. et al.A landscape of driver mutations in melanoma.Cell. 2012; 150: 251-263Abstract Full Text Full Text PDF PubMed Scopus (1852) Google Scholar, Hugo et al., 2016Hugo W. Zaretsky J.M. Sun L. Song C. Moreno B.H. Hu-Lieskovan S. Berent-Maoz B. Pang J. Chmielowski B. Cherry G. et al.Genomic and transcriptomic features of response to anti-PD-1 therapy in metastatic melanoma.Cell. 2016; 165: 35-44Abstract Full Text Full Text PDF PubMed Scopus (1752) Google Scholar). However, the function of STK19 is largely uncharacterized, particularly in melanoma initiation and progression. We found that STK19 alteration was significantly mutually exclusive with BRAF in human melanomas (p = 0.002) (Figure S1A), consistent with BRAF lying downstream of NRAS activation (Lavoie and Therrien, 2015Lavoie H. Therrien M. Regulation of RAF protein kinases in ERK signalling.Nat. Rev. Mol. Cell Biol. 2015; 16: 281-298Crossref PubMed Scopus (385) Google Scholar). Given these observations, we further investigated the potential role of STK19 in regulating NRAS functions in melanomas.Figure S1STK19 Is a Critical Regulator of NRAS Function, Related to Figure 1Show full captionA. Mutual exclusivity between STK19 and BRAF alterations in TCGA PanCancer Atlas.B. STK19 was depleted in A375, UACC62, SK-MEL-2 and WM2032 cells. Active NRAS proteins were pulled down by GST-CRAF RBD fusion protein. Active NRAS levels and activation of NRAS downstream signaling were detected by immunoblots.C. STK19 was depleted in HPM, A375, UACC62, SK-MEL-2 and WM2032 cells, and the cell proliferation rates were measured. Data are means ± SD relative to individual control group (n = 6). ∗∗∗p < 0.001, Student’s t test.D. qRT-PCR analysis of STK19 mRNA levels in SK-MEL-2 and WM2032 cells infected with STK19 shRNAs. Error bars indicate 95% confidence interval of triplicates.E. STK19 was depleted in SK-MEL-2 and WM2032 cells. Active NRAS proteins were pulled down by GST-CRAF RBD fusion protein. Active NRAS levels and activation of NRAS downstream signaling were detected by immunoblots.F. SK-MEL-2 and WM2032 cells with depletion of STK19 were infected with retroviruses encoding empty Flag or Flag-tagged STK19WT. Active NRAS proteins were pulled down by GST-CRAF RBD fusion protein. Active NRAS levels and activation of NRAS downstream signaling were detected by immunoblots.G. Immunoblots to show depletion of STK19 and overexpression of HA-NRAS proteins in hTERT/p53DD/CDK4(R24C) melanocytes.H. hTERT/p53DD/CDK4(R24C) melanocytes expressing empty HA, HA-NRASWT or HA-NRASQ61R were introduced with control shRNA or STK19 shRNA, and then seeded for cell proliferation assay. Data are means ± SD relative to control group (n = 6). ∗∗∗p < 0.001, Student’s t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A. Mutual exclusivity between STK19 and BRAF alterations in TCGA PanCancer Atlas. B. STK19 was depleted in A375, UACC62, SK-MEL-2 and WM2032 cells. Active NRAS proteins were pulled down by GST-CRAF RBD fusion protein. Active NRAS levels and activation of NRAS downstream signaling were detected by immunoblots. C. STK19 was depleted in HPM, A375, UACC62, SK-MEL-2 and WM2032 cells, and the cell proliferation rates were measured. Data are means ± SD relative to individual control group (n = 6). ∗∗∗p < 0.001, Student’s t test. D. qRT-PCR analysis of STK19 mRNA levels in SK-MEL-2 and WM2032 cells infected with STK19 shRNAs. Error bars indicate 95% confidence interval of triplicates. E. STK19 was depleted in SK-MEL-2 and WM2032 cells. Active NRAS proteins were pulled down by GST-CRAF RBD fusion protein. Active NRAS levels and activation of NRAS downstream signaling were detected by immunoblots. F. SK-MEL-2 and WM2032 cells with depletion of STK19 were infected with retroviruses encoding empty Flag or Flag-tagged STK19WT. Active NRAS proteins were pulled down by GST-CRAF RBD fusion protein. Active NRAS levels and activation of NRAS downstream signaling were detected by immunoblots. G. Immunoblots to show depletion of STK19 and overexpression of HA-NRAS proteins in hTERT/p53DD/CDK4(R24C) melanocytes. H. hTERT/p53DD/CDK4(R24C) melanocytes expressing empty HA, HA-NRASWT or HA-NRASQ61R were introduced with control shRNA or STK19 shRNA, and then seeded for cell proliferation assay. Data are means ± SD relative to control group (n = 6). ∗∗∗p < 0.001, Student’s t test. To confirm whether STK19 knockdown inhibits NRAS activity, we investigated whether the downstream signaling of NRAS was inhibited after STK19 silencing. Retroviruses encoding empty HA, HA-tagged wild-type NRAS, or NRASQ61R were introduced into human primary melanocytes (HPMs) depleted of STK19. The active-RAS pull-down assay indicated that depletion of STK19 markedly decreased the active fraction of both wild-type and oncogenic NRAS, and consequently, signaling downstream of NRAS via the RAF-MEK-ERK and PI3K-AKT pathways was diminished (Figure 1D). We also used melanoma cells with different NRAS mutation status to identify the role of STK19 in regulating endogenous NRAS. To this end, STK19 was silenced in A375 and UACC62 cells, both with BRAFV600E and wild-type NRAS, or SK-MEL-2 and WM2032 cells that both express NRASQ61R and wild-type BRAF (Barretina et al., 2012Barretina J. Caponigro G. Stransky N. Venkatesan K. Margolin A.A. Kim S. Wilson C.J. Lehár J. Kryukov G.V. Sonkin D. et al.The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity.Nature. 2012; 483: 603-607Crossref PubMed Scopus (4859) Google Scholar, Herlyn et al., 1985Herlyn M. Thurin J. Balaban G. Bennicelli J.L. Herlyn D. Elder D.E. Bondi E. Guerry D. Nowell P. Clark W.H. et al.Characteristics of cultured human melanocytes isolated from different stages of tumor progression.Cancer Res. 1985; 45: 5670-5676PubMed Google Scholar). Interestingly, inhibition of active NRAS and its downstream effectors (p-MEK, p-ERK1/2, p-AKT) was highly efficient in SK-MEL-2 and WM2032 (NRASQ61R, BRAFWT) cells, but the inhibition was much weaker in A375 and UACC62 (BRAFV600E, NRASWT) cells (Figure S1B). Consistently, depletion of STK19 significantly decreased the growth rate of SK-MEL-2 and WM2032 (NRASQ61R, BRAFWT) cells, but had a much smaller effect on the BRAFV600E, NRASWT cells, or primary melanocytes (Figure S1C). To confirm that inhibition of NRAS signaling was mediated by STK19 knockdown, two new sets of STK19-targeted small hairpin RNA (shRNA) were exploited and the resulting depletion of STK19 notably inhibited NRAS activity and its downstream signaling in SK-MEL-2 and WM2032 cells (Figures S1D and S1E). Furthermore, we performed rescue experiments and infected the STK19-depleted melanoma cells with retroviruses encoding empty Flag or Flag-tagged STK19. The ectopic expression of STK19-Flag restored the activation of NRAS signaling (Figure S1F), confirming that the inhibition of NRAS signaling using STK19-specific shRNAs was mediated by knockdown of STK19 rather than the off-target effects. Collectively, these results suggest that STK19 has a crucial role in activating oncogenic NRASQ61R -driven signaling. To explore the potential role of STK19 in NRASQ61R-driven tumorigenesis, the role of STK19 in melanocyte proliferation and malignant transformation was evaluated using genetically engineered human immortalized melanocytes (hTERT/p53DD/CDK4(R24C)) (Lissanu Deribe et al., 2016Lissanu Deribe Y. Shi Y. Rai K. Nezi L. Amin S.B. Wu C.C. Akdemir K.C. Mahdavi M. Peng Q. Chang Q.E. et al.Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma.Proc. Natl. Acad. Sci. USA. 2016; 113: E1296-E1305Crossref PubMed Scopus (32) Google Scholar) expressing NRASQ61R together with STK19 silencing. The results indicated that silencing STK19 (Figure S1G) substantially inhibited the colony formation capacity of NRASQ61R-transformed melanocytes (Figure 1E), as well as their proliferation (Figure S1H) and tumor-forming ability in xenografts (Figures 1F–1H). These results suggest that STK19 is critical for NRASQ61R-driven melanomagenesis. To identify the direct substrates of STK19, cell lysates collected from SK-MEL-2 cells expressing human recombinant Flag-tagged STK19 protein were purified using anti-Flag beads and associated factors analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify STK19-interacting proteins (Figure S2A). NRAS was identified as one of the most abundant STK19-interacting proteins on the basis of total number of unique peptides (13) (Figure 2A). Reciprocal co-immunoprecipitation confirmed that endogenous STK19 strongly interacted with NRAS in SK-MEL-2 and WM2032 cells (NRASQ61R), but bound less well in A375 and UACC62 cells (expressing wild-type NRAS) (Figures S2B and S2C). The NRASQ61R-STK19 interaction was confirmed by reciprocal coimmunoprecipitation from HPMs exogenously expressing STK19-Flag and HA-NRAS wild-type (WT) or Q61R mutant (Figures 2B and 2C). These results indicate that STK19 directly interacts with NRAS, and especially with NRASQ61R.Figure 2STK19 Phosphorylates NRAS Protein at Serine 89Show full caption(A) Mass spectral peptide count of STK19-interacting proteins.(B and C) Exogenous interactions between HA-NRAS and STK19-Flag were detected by immunoprecipitation in HPMs.(D) HPMs were introduced with retroviruses encoding empty HA, HA-NRASWT or HA-NRASQ61R and/or STK19-Flag. The serine-, threonine- and tyrosine-phosphorylation of HA-NRAS isoforms were detected by immunoblots with specific antibodies.(E) Mass spectrometry analysis to identify serine 89 (S89) as the phosphorylation residue by STK19.(F) Schematic diagram showing the evolutionarily conserved serine residue (S89) in NRAS.(G) An in vitro kinase assay was performed using purified recombinant human STK19 protein and indicated purified recombinant human NRAS isoform proteins, followed by detection of phosphorylation in NRAS.(H) HPMs were introduced with empty HA, HA-NRASWT, HA-NRASS89A, HA-NRASQ61R, or HA-NRASQ61R/S89A. The NRAS-effector protein:protein interaction (including BRAF, CRAF, and PI3Kα), active HA-NRAS levels and activation of NRAS downstream signaling were detected by immunoblots after infection with retroviruses encoding empty Flag or Flag-tagged STK19.(I) hTERT/p53DD/CDK4(R24C) melanocytes expressing empty HA, HA-NRASQ61R or HA-NRASQ61R/S89A were introduced with retroviruses encoding empty Flag or Flag-tagged STK19, and then seeded for colony formation assay. Data are means ± SD relative to control group with empty HA vector and empty Flag vector (n = 6). ∗∗∗p < 0.001, n.s., not significant, Student’s t test.(J–L) Growth curve (J), tumor weight (K), and dissected tumors (L) for the xenograft experiments with indicated cells inoculated subcutaneously into flanks of nude mice. Visible tumors were measured every 3 days. Data are means ± SEM relative to control group (n = 7). ∗∗∗p < 0.001, Student’s t test.See also Figure S2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Mass spectral peptide count of STK19-interacting proteins. (B and C) Exogenous interactions between HA-NRAS and STK19-Flag were detected by immunoprecipitation in HPMs. (D) HPMs were introduced with retroviruses encoding empty HA, HA-NRASWT or HA-NRASQ61R and/or STK19-Flag. The serine-, threonine- and tyrosine-phosphorylation of HA-NRAS isoforms were detected by immunoblots with specific antibodies. (E) Mass spectrometry analysis to identify serine 89 (S89) as the phosphorylation residue by STK19. (F) Schematic diagram showing the evolutionarily conserved serine residue (S89) in NRAS. (G) An in vitro kinase assay was performed using purified recombinant human STK19 protein and indicated purified recombinant human NRAS isoform proteins, followed by detection of phosphorylation in NRAS. (H) HPMs were introduced with empty HA, HA-NRASWT, HA-NRASS89A, HA-NRASQ61R, or HA-NRASQ61R/S89A. The NRAS-effector protein:protein interaction (including BRAF, CRAF, and PI3Kα), active HA-NRAS levels and activation of NRAS downstream signaling were detected by immunoblots after infection with retroviruses encoding empty Flag or Flag-tagged STK19. (I) hTERT/p53DD/CDK4(R24C) melanocytes expressing empty HA, HA-NRASQ61R or HA-NRASQ61R/S89A were introduced with retroviruses encoding empty Flag or Flag-tagged STK19, and then seeded for colony formation assay. Data are means ± SD relative to control group with empty HA vector and empty Flag vector (n = 6). ∗∗∗p < 0.001, n.s., not significant, Student’s t test. (J–L) Growth curve (J), tumor weight (K), and dissected tumors (L) for the xenograft experiments with indicated cells inoculated subcutaneously into flanks of nude mice. Visible tumors were measured every 3 days. Data are means ± SEM relative to control group (n = 7). ∗∗∗p < 0.001, Student’s t test. See also Figure S2. To address the significance of the STK19-NRASQ61R interaction, we asked whether NRAS was an STK19 substrate. Phosphorylated-serine, -threonine, and -tyrosine in NRAS were detected in cellular lysates collected from HPMs expressing ectopic STK19 and NRAS (WT or Q61R) after NRAS immunoprecipitation (Figure 2D). Strikingly, serine, but not threonine or tyrosine phosphorylation of NRASQ61R mutant, was substantially increased by STK19 expression, and only a marginal increase in phosphorylation of NRASWT was detected. We also observed robust endogenous NRAS phosphorylation in a panel of melanoma cells expressing NRASQ61R (Figure S2D) whereas phosphorylation of NRAS at serine residues was barely detectable in cells following STK19 silencing (Figures S2E and S2F). The upregulation of phosphorylated serine levels in NRAS was also diminished by overexpression of a kinase-dead STK19 K317P mutant (Gomez-Escobar et al., 1998Gomez-Escobar N. Chou C.F. Lin W.W. Hsieh S.L. Campbell R.D. The G11 gene located in the major histocompatibility complex encodes a novel nuclear serine/threonine protein kinase.J. Biol. Chem. 1998; 273: 30954-30960Crossref PubMed Scopus (23) Google Scholar) (Figures S2G and S2H). These results suggest that STK19 phosphorylates NRAS at serine residues. The preferential phosphorylation of NRASQ61R compared to NRASWT was also observed using an in vitro kinase assay (Figure S2I), suggesting that STK19 has a stronger preference toward GTP-loaded active NRAS. To verify this, we further performed an in vitro kinase assay using purified NRAS recombinant protein preloaded with GDP, GTP, or GTPγS in the presence of recombinant STK19. We found a higher STK19-induced phosphorylation of the GTP- and GTPγS-loaded NRAS than GDP-loaded NRAS (Figure S2J). This was confirmed in experiments that showed STK19-induced phosphorylation of different NRAS mutant isoforms was more efficient than that of NRASWT (Figure S2K). To identify the specific NRAS serine residue(s) phosphorylated by STK19, we performed mass spectrometry after in vitro phosphorylation of recombinant NRASQ61R by purified recombinant STK19 protein. This approach identified phosphorylation of the evolutionarily conserved NRAS serine 89 (S89) (Figures 2E and 2F). In agreement with this observation, mutation of S89 to alanine (S89A) abolished phosphorylation of NRASWT and NRASQ61R (Figure 2G). To confirm that STK19 upregulates NRAS activity through phosphorylating NRAS protein at S89, HA-NRASWT, HA-NRASS89A, HA-NRASQ61R, or HA-NRASQ61R/S89A were expressed in HPMs expressing ectopic STK19-Flag. After immunoprecipitation of NRAS WT or mutants, immunoblots were performed to detect interactions between NRAS and its effectors, including BRAF, CRAF and PI3Kα. We found that STK19 overexpression dramatically enhanced the interaction between NRASQ61R and its effectors, and also stimulated signaling downstream of NRAS as detected by immunoblotting whole cell extracts (Figure 2H). No effects of STK19 were observed on a phosphorylation-defective form of NRAS (NRASS89A) or its signaling. By contrast, the introduction of phosphomimetic NRASQ61R/S89D enhanced NRAS interaction with its effectors and its" @default.
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• W2912706099 title "Pharmacological Targeting of STK19 Inhibits Oncogenic NRAS-Driven Melanomagenesis" @default.
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