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 3

SHARPIN affects melanoma growth and survival through regulation of SOX10, PAX3, and MITF expression.

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SHARPIN affects melanoma growth and survival through regulation of SOX10...
(A) Pigmentation (upper panels), immunoblot (lower panels), and qPCR (right panel) analysis of MITF and upstream/downstream regulatory proteins in WM115 or WM35 melanoma cells overexpressing SHARPIN or a control empty vector (EV). qPCR data are presented as relative mRNA levels compared with control cells. (B) Immunoblot (left panel) and qPCR (right panels) analysis of WM115 cells expressing scrambled or SHARPIN-specific siRNAs or shRNAs. Statistical significance was calculated using Student’s t test (unpaired, 2 tailed, siRNA) or 1-way ANOVA and Dunnett’s test (shRNA). (C) Immunoblot analysis of WM35, WM793 and WM115 cells expressing scrambled or SHARPIN-specific shRNAs. Arrow and NS indicate SHARPIN and nonspecific bands, respectively. (D) Immunoblot analysis and CFE assay of WM115 melanoma cells expressing scrambled or SHARPIN-specific shRNA and ectopically expressing control or SOX10 (rescue). Middle and right panels show representative images of colonies and quantification of CFE 14 days after cells were seeded at 103/well. CFE (ratio to scrambled/control) was presented as mean ± SD, and statistical significance was calculated using 2-way ANOVA and Tukey’s test (n = 6 from 2 independent experiments). All qPCR data are presented as mean ± SD (n = 3).*P < 0.05; **P < 0.005; ***P < 0.0005 (2-tailed, Student’s t test). (A–D) Data are representative of 2 experiments.