NLK is required for Ras/ERK/SRF/ELK signaling to tune skeletal muscle development by phosphorylating SRF and antagonizing the SRF/MKL pathway

Shang-Ze Li,Qi-Peng Shu,Jie Gao,Chao Li,Xue-Hua Du,Junli Wang,Zhi-Hao Ding,Ming-You Dong,Ze-Yan Zhang,Xiao-Dong Zhang,Jie Chen,Xin-Yi Liang,Run-Lei Du

doi:10.1038/s41420-021-00774-9

Abstract

Serum response factor (SRF) regulates differentiation and proliferation by binding to RhoA-actin-activated MKL or Ras-MAPK-activated ELK transcriptional coactivators, but the molecular mechanisms responsible for SRF regulation remain unclear. Here, we show that Nemo-like kinase (NLK) is required for the promotion of SRF/ELK signaling in human and mouse cells. NLK was found to interact with and phosphorylate SRF at serine residues 101/103, which in turn enhanced the association between SRF and ELK. The enhanced affinity of SRF/ELK antagonized the SRF/MKL pathway and inhibited mouse myoblast differentiation in vitro. In a skeletal muscle-specific Nlk conditional knockout mouse model, forming muscle myofibers underwent hypertrophic growth, resulting in an increased muscle and body mass phenotype. We propose that both phosphorylation of SRF by NLK and phosphorylation of ELKs by MAPK are required for RAS/ELK signaling, confirming the importance of this ancient pathway and identifying an important role for NLK in modulating muscle development in vivo.

Highlights

Serum response factor (SRF) is a member of the myocyte enhancer factor-2 (MEF2) family of MADS-box transcription factors, including MCM1, Agamous, and Deficiens, that precisely orchestrates the growth and development of skeletal muscle [1]
GAPDH was used as a loading control
To determine the roles of kinase activity in ETS-like proteins (ELKs)/SRF signaling, NLKKM, a kinase-dead Nemo-like kinase (NLK) mutant with replacement of lysine 167 with methionine, was employed [15, 16]. Both an ELK/SRF reporter and an EGR1 reporter were significantly activated by NLK but not NLKKM, which means that NLK kinase activity is required for ELK/SRF signaling (Fig. 1C, D)

Summary

Introduction

SRF is a member of the myocyte enhancer factor-2 (MEF2) family of MADS-box transcription factors, including MCM1, Agamous, and Deficiens, that precisely orchestrates the growth and development of skeletal muscle [1]. SRF modulates cell proliferation and morphology by binding different coactivator ETS-like proteins (ELKs) and myocardin-like factors (MKLs) to induce the expression of specific types of genes [2]. The immediate early genes (IEGs) induced by serum or growth factors, such as EGR1, C-FOS, and TCF7L2, are largely dependent on SRF through binding to members of the SRF coactivator ternary complex factor (TCF) family, including ELK1, ELK4 (SAP1) and NET (ELK3, SAP2, or ERP), which are phosphorylated and activated by mitogen-activated protein kinase (MAPK) in the RAS-ERK axis [3,4,5]. SRF regulates muscle differentiation genes by binding members of another coactivator family, MKL1 and MKL2 ( called myocardin-related transcription factors (MRTFs)), in response to the Rho-actin pathway [6]. The balance between these two pathways is precisely controlled and affects cell proliferation and adhesive properties [9, 10]

Methods

Results

Conclusion