Received: 14 May 2018 | Accepted: 2 July 2018 DOI: 10.1002/jcp.27113 ORIGINAL RESEARCH ARTICLE PDZRN3 regulates differentiation of myoblasts into myotubes through transcriptional and posttranslational control of Id2 Takeshi Honda | Makoto Inui Department of Pharmacology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan Correspondence Makoto Inui, Department of Pharmacology, Yamaguchi University Graduate School of Medicine, 1‐1‐1 Minami‐Kogushi, Ube, Yamaguchi 755‐8505, Japan. Email: minui@yamaguchi-u.ac.jp Funding information Japan Society for the Promotion of Science, Grant/Award Number: Scientific Research (C) Number 15K08235 PDZRN3 (also known as LNX3) is a member of the PDZ domain‐containing RING finger protein family. We previously showed that PDZRN3 is essential for differentiation of myoblasts into myotubes and that depletion of PDZRN3 inhibits such differentiation downstream of the upregulation of myogenin, a basic helix‐loop‐helix (bHLH) transcription factor required for completion of the differentiation process. However, the mechanism by which PDZRN3 controls this process has remained unclear. Myogenin is rendered active during the late stage of myogenic differentiation by the downregulation of Id2, a negative regulator of bHLH transcription factors. We now show that depletion of PDZRN3 inhibits the differentiation of C2C12 cells by inducing the upregulation of Id2 and thereby delaying its downregulation. Knockdown of Id2 by RNA interference restores the differentiation of PDZRN3‐depleted cells. Luciferase reporter assays revealed that a putative binding site for STAT5b in the Id2 gene promoter is required for the upregulation of Id2 expression by PDZRN3 depletion. In addition, the amount of phosphorylated Id2 was reduced and that of the nonpho- sphorylated protein concomitantly increased in PDZRN3‐depleted cells, with the inhibitory effect of Id2 on bHLH transcription factors having previously been shown to be attenuated by phosphorylation of Id2 catalyzed by the complex of Cdk2 with cyclin A2 or E1. Indeed, the expression of cyclin A2, but not that of cyclin E1, was reduced in PDZRN3‐depleted cells. Our results thus indicate that PDZRN3 plays a key role in the differentiation of myoblasts into myotubes by regulating Id2 at both transcriptional and posttranslational levels. KEYWORDS basic helix‐loop‐helix (bHLH) transcription factor, cell differentiation, Id2, mesenchymal stem cell, myoblast, myogenesis, PDZRN3, skeletal muscle 1 | INTRODUCTION PDZ domain‐containing RING finger family protein 3 (PDZRN3, also known as LNX3) was identified in silico as a homolog of LNX1 (PDZRN2), which is thought to contribute to cell fate determination through inhibition of Notch signaling as a consequence of its function as an E3 ubiquitin ligase for NUMB (Katoh, 2004). Whereas LNX1 consists of an NH 2 ‐terminal RING domain, four PDZ domains in the central region, and a COOH‐terminal consensus motif for binding of the PDZ domain, PDZRN3 has only two PDZ domains in the central region in addition to the RING domain and the consensus motif for binding of the PDZ domain. We previously cloned the complemen- tary DNA (cDNA) of PDZRN3 from a human heart library with the use of a yeast two‐hybrid screen for proteins that bind to the PDZ domains of PSD‐95 (Ko et al., 2006). PDZRN3 is expressed in a variety of human tissues including heart, skeletal muscle, brain, and J Cell Physiol. 2018;1–10. wileyonlinelibrary.com/journal/jcp © 2018 Wiley Periodicals, Inc. | 1