SHARPIN-mediated regulation of protein arginine methyltransferase 5 controls melanoma growth
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
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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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 7

MTAP expression determines sensitivity to SHAPIN-mediated growth inhibition of melanoma.

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MTAP expression determines sensitivity to SHAPIN-mediated growth inhibit...
(A) Immunoblot analysis and CFE assay of WM35 cells stably expressing empty vector or MTAP expression plasmids plus scrambled or SHARPIN-specific shRNA. Cells were seeded at 103/well and colonies were visualized and quantified after 14 days in culture. (B) Growth of WM35 cells (2.0 × 106 cells) injected subcutaneously into female nude mice. Stable WM35 transfectants expressing empty vector or MTAP were subjected to KD with scrambled or SHARPIN-specific shRNA. Tumor volume (upper panel) and weight (lower panel) were measured at the indicated time. Immunoblot analysis (right panel) was performed using representative tumor lysates from each group. Data are presented as mean ± SD (at the end point of experiment). n = 8 mice/group. Two-way ANOVA test (tumor volume) or Welch’s test (tumor weight). (C) Cell growth/viability (ATPlite, left panel) of A375 cells expressing scrambled or SHARPIN shRNA, and then treated with indicated amount of MTA for 5 days. KD efficiency of SHARPIN was analyzed with qPCR (right panels). (D) As in C, qPCR analysis (left) and growth/viability assay (ATPlite, right) of A375 cells expressing shRNAs, scrambled, shSHARPIN, and/or shMTAP. (E) CFE assay of A375 cells expressing scrambled, SHARPIN, or MTAP (nos. 1 and 5) shRNAs. Cells (2.5 × 103/well) were seeded and cultured for 14 days. Colonies were visualized and quantified (ImageJ). All quantitation data and qPCR data are presented as mean ± SD (n = 3). Statistical significance was calculated using 1-way (D, left) or 2-way (A and B, D, right, E) Tukey’s test. *P < 0.05; **P < 0.05; ***P < 0.0005. (A, C–E) Data represent results from 2 to 3 independent experiments.