RSK2 Mediates Muscle Cell Differentiation through Regulation of NFAT3

Abstract

RSK2, an ERK downstream kinase, is a novel mediator of skeletal muscle cell differentiation through its regulation of NFAT3 activity. We found that the N-terminal (amino acids (aa) 1-68) and C-terminal (aa 416 -674) kinase domains of RSK2 directly interacted with nuclear localization signal 1, the Ser/Pro repeat, and the polyproline domains (aa 261-365) of NFAT3. Upon A23187 stimulation, RSK2 induced nuclear localization of NFAT3. RSK2 phosphorylated NFAT3 in vitro (K m ‫؍‬ 3.559 M), and activation of NFAT3 by RSK2 enhanced the promoter activity of NFAT3 downstream target genes in vivo. Furthermore, nuclear accumulation of NFAT3 was attenuated markedly in RSK2 ؊/؊ cells compared with wild-type RSK2 ؉/؉ cells. Notably, RSK2 and NFAT3 induced a significant differentiation of C2C12 myoblasts to multinucleated myotubes. Multinucleated myotube differentiation was inhibited by small interfering RNA against RSK2, ERK1/2, or NFAT3. These results demonstrate that RSK2 is an important kinase for NFAT3 in mediating myotube differentiation.Myoblast cell differentiation to muscle fibers depends on myogenic transcription factors, particularly myogenin (1). Many studies addressing the involvement of the mitogen-activated protein kinase (MAPK) 2 pathways are controversial because results showed both positive and negative effects of this signaling pathway on myogenesis. The MAPK cascades are implicated in the regulation of cell proliferation, survival, growth, and motility (2, 3) as well as tumorigenesis (4). Extracellular signal-regulated kinase (ERK)-1 and ERK2 (5, 6) mediate the 90-kDa ribosomal S6 kinases (RSKs), which are a family of broadly expressed serine/threonine kinases that respond to many growth factors, peptide hormones, and neurotransmitters (7,8). When activated, RSK2 translocates to the nucleus, where it can phosphorylate various nuclear proteins, including c-Fos, Elk-1, histones, and cAMP-responsive element-binding protein (CREB) (9 -13); activating transcription factor-4 (14); and p53 (15). Moreover, because RSKs have broad substrate specificity, RSK2 may be able to bind and/or phosphorylate a number of diverse substrates that regulate cell proliferation or differentiation or the cell cycle, depending on the specific situation.The nuclear factor of activated T cell (NFAT) family of transcription factors has been characterized primarily in immune cells (16). However, accumulating evidence has demonstrated that NFAT transcription factors are present in a wide range of cell types and tissues (17-22). NFAT3 and NFAT4 have an ϳ48% amino acid homology in the whole protein and exhibit an especially high degree of similarity (i.e. ϳ80%) in the Rel homology domain, which contains the DNA-binding motif. Moreover, the N-terminal domains (amino acids (aa) 1-360) of NFAT3 and NFAT4 have only a 30% amino acid similarity. Therefore, we proposed that the N-terminal domains of NFAT3 and NFAT4 might bind to different proteins, which might also phosphorylate key residues. c-Jun N-terminal kinase (JNK)-2 phosphorylates NFAT4, but...

Chromatin-bound mitogen-activated protein kinases transmit dynamic signals in transcription complexes in β-cells

“…ERK1/2 may also have indirect effects on proteins present in promoter complexes, for example, through downstream protein kinases such as Rsk, which may phosphorylate NFAT (29). A second indirect effect may arise from synergistic interactions of transcription factors on the insulin gene promoter.…”

Neogenin Regulates Skeletal Myofiber Size and Focal Adhesion Kinase and Extracellular Signal-regulated Kinase Activities In Vivo and In Vitro

“…ERK activity has both positive and negative roles in C2C12 cell differentiation (Wu et al, 2000;Li and Johnson, 2006;Cho et al, 2007;Yokoyama et al, 2007); it is also implicated in fiber type identity in vivo (Murgia et al, 2000;Shi et al, 2008). The ERK substrate p90RSK has also been implicated in C2C12 cell differentiation (Cho et al, 2007). Given the lower than normal levels of the phosphorylated forms of these kinases in Neo1 Gt/Gt embryos and myoblasts, it is logical to suggest that they are involved in neogenin's promyogenic function.…”

“…FAK activity is required for efficient myotube formation during muscle regeneration in response to injury, in fusion and costamere development of primary mouse myoblasts, and in survival of proliferating C2C12 myoblasts and their differentiation into myotubes (Clemente et al, 2005;Quach and Rando, 2006;Quach et al, 2009). ERK activity has both positive and negative roles in C2C12 cell differentiation (Wu et al, 2000;Li and Johnson, 2006;Cho et al, 2007;Yokoyama et al, 2007); it is also implicated in fiber type identity in vivo (Murgia et al, 2000;Shi et al, 2008). The ERK substrate p90RSK has also been implicated in C2C12 cell differentiation (Cho et al, 2007).…”

Synthesis, activity evaluation, and docking analysis of barbituric acid aryl hydrazone derivatives as RSK2 inhibitors

“…The 90 kDa ribosomal S6 kinases (RSKs) are serine/ threonine kinases and lie downstream of Ras/mitogenactivated protein kinase (MAPK) pathway, which are responsible for activating some important cellular components including transcription factors and histones [1][2][3][4][5][6][7][8] . This family consists of four human isoforms (RSK1-4) with each being a product of a separate gene 9 , among which RSK2 has attracted more attention, for its over expression and aberrant activation have been linked to many human diseases, including breast cancer, prostate cancer 10 .…”