SHARPIN-mediated regulation of protein arginine methyltransferase 5 controls melanoma growth
Hironari Tamiya, … , Kazuhiro Iwai, Ze’ev A. Ronai
Hironari Tamiya, … , Kazuhiro Iwai, Ze’ev A. Ronai
Published December 11, 2017
Citation Information: J Clin Invest. 2018;128(1):517-530. https://doi.org/10.1172/JCI95410.
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Research Article Cell biology Oncology

SHARPIN-mediated regulation of protein arginine methyltransferase 5 controls melanoma growth

Abstract

SHARPIN, an adaptor for the linear ubiquitin chain assembly complex (LUBAC), plays important roles in NF-κB signaling and inflammation. Here, we have demonstrated a LUBAC-independent role for SHARPIN in regulating melanoma growth. We observed that SHARPIN interacted with PRMT5, a type II protein arginine methyltransferase, and increased its multiprotein complex and methyltransferase activity. Activated PRMT5 controlled the expression of the transcription factors SOX10 and MITF by SHARPIN-dependent arginine dimethylation and inhibition of the transcriptional corepressor SKI. Activation of PRMT5 by SHARPIN counteracted PRMT5 inhibition by methylthioadenosine, a substrate of methylthioadenosine phosphorylase, which is codeleted with cyclin-dependent kinase inhibitor 2A (CDKN2A) in approximately 15% of human cancers. Collectively, we identified a LUBAC-independent role for SHARPIN in enhancing PRMT5 activity that contributes to melanomagenesis through the SKI/SOX10 regulatory axis.

Authors

Hironari Tamiya, Hyungsoo Kim, Oleksiy Klymenko, Heejung Kim, Yongmei Feng, Tongwu Zhang, Jee Yun Han, Ayako Murao, Scott J. Snipas, Lucia Jilaveanu, Kevin Brown, Harriet Kluger, Hao Zhang, Kazuhiro Iwai, Ze’ev A. Ronai

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Figure 5

PRMT5 increases SOX10 and PAX3 expression by arginine methylation–dependent inhibition of SKI.

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PRMT5 increases SOX10 and PAX3 expression by arginine methylation–depend...
(A) Luciferase reporter assay of TGF-β signaling in WM793 cells expressing scrambled shRNA or HOIP-, HOIL-1L–, or SHARPIN-specific shRNAs. Cells transfected with TGF-β reporter (CAGAx9-luc) and pCMV-Cypridina-luc (internal control) reporter plasmids were treated with TGF-β (20 ng/ml) for 8 or 24 hours. (B) qPCR analysis of SOX10, PAX3, and MITF expression in WM115 cells expressing empty vector (control), SHARPIN, or PRMT5 expression plasmids and treated with TGF-β (20 ng/ml) for 0, 24, or 48 hours. (C) Immunoblot and qPCR analysis of WM115 cells transfected with scrambled (control) siRNA or SKI-specific SMARTpool siRNA. (D) ChIP analysis of WM115 cells expressing empty vector, SHARPIN, or PRMT5 and treated with vehicle (Veh) or TGF-β (20 ng/ml) for 18 hours. ChIP analysis was performed with anti-SKI antibody, and coimmunoprecipitated SOX10 or PAX3 promoter sequences were quantified by qPCR. (E) Immunoblot analysis of SKI and p53 in WM115 cell lysates immunoprecipitated with normal rabbit serum (NRS) or SYM10 antibody. (F) Analysis as in E except WM115 cells expressed scrambled or PRMT5-specific shRNA. (G) Immunoblot analysis of anti-Flag immunoprecipitates of A375 cells coexpressing WT, Arg8 mutated (R8K), or Arg658/660 mutated (RR/KK) SKI with either PRMT5 or SHARPIN. Asterisks and arrowhead indicate SHARPIN and tubulin, respectively. (H) Immunoblot analysis of anti-Myc immunoprecipitates of HEK293T cells expressing Myc-tagged SHARPIN and Flag-tagged WT, R8K mutant, or RR/KK mutant SKI proteins. (I) Immunoblot analysis of IgG or anti-SKI immunoprecipitates of SK-Mel-28 cells. (J) ChIP analysis as in D of WM115 cells expressing WT or R8K mutant SKI proteins. Lysates were immunoprecipitated with indicated antibodies. For immunoprecipitation analyses, input indicates 5% of lysates. Statistical significance was calculated using 2-way ANOVA (Dunnett’s test, A and B) or 2-tailed Student’s t test (C, D, J). Data are presented as mean ± SD (n = 3). *P < 0.05; **P < 0.005; ***P < 0.0005.