Extracellular signal-regulated kinase 1/2-mediated phosphorylation of p300 enhances myosin heavy chain I/  gene expression via acetylation of nuclear factor of activated T cells c1

Abstract

The nuclear factor of activated T-cells (NFAT) c1 has been shown to be essential for Ca2+-dependent upregulation of myosin heavy chain (MyHC) I/β expression during skeletal muscle fiber type transformation. Here, we report activation of extracellular signal-regulated kinase (ERK) 1/2 in Ca2+-ionophore-treated C2C12 myotubes and electrostimulated soleus muscle. Activated ERK1/2 enhanced NFATc1-dependent upregulation of a −2.4 kb MyHCI/β promoter construct without affecting subcellular localization of endogenous NFATc1. Instead, ERK1/2-augmented phosphorylation of transcriptional coactivator p300, promoted its recruitment to NFATc1 and increased NFATc1–DNA binding to a NFAT site of the MyHCI/β promoter. In line, inhibition of ERK1/2 signaling abolished the effects of p300. Comparison between wild-type p300 and an acetyltransferase-deficient mutant (p300DY) indicated increased NFATc1–DNA binding as a consequence of p300-mediated acetylation of NFATc1. Activation of the MyHCI/β promoter by p300 depends on two conserved acetylation sites in NFATc1, which affect DNA binding and transcriptional stimulation. NFATc1 acetylation occurred in Ca2+-ionophore treated C2C12 myotubes or electrostimulated soleus. Finally, endogenous MyHCI/β gene expression in C2C12 myotubes was strongly inhibited by p300DY and a mutant deficient in ERK phosphorylation sites. In conclusion, ERK1/2-mediated phosphorylation of p300 is crucial for enhancing NFATc1 transactivation function by acetylation, which is essential for Ca2+-induced MyHCI/β expression.

Positive Regulation of Interleukin-2 Expression by a Pseudokinase, Tribbles 1, in Activated T Cells

“…46,47) Calcineurin and NFAT2 are essential for upregulation of myosin heavy chain I/β isoform (MyHCI/β) promoter activity and mRNA expression, and ERK1/2-mediated phosphorylation of p300 is crucial for enhancing the transactivation function of NFAT2 by acetylation, which in turn is essential for Ca 2+ -induced MyHCI/β expression. 48) Our results indicate that TRB1 enhances transactivation by NFAT2 and promotes dissociation of HDAC1 from NFAT2. This displacing activity of TRB1 may contribute to the enhancement of IL-2 promoter activation by dissociating HDAC1, one of the components of co-suppressor complex from NFAT on the IL-2 promoter.…”

Gα13 ablation reprograms myofibers to oxidative phenotype and enhances whole-body metabolism

“…Thus, we propose that Gα13 may serve as a switch that controls the overall sensitivity of multiple receptors associated with energy metabolism. NFATc1 is the most studied regulator of myofiber type-associated gene expression (20)(21)(22). Our results reveal the physiological link between Gα13 and NFAT signaling both in vitro and in vivo.…”

“…Gα13 inhibits NFATc1 activity and regulates muscle oxidative capacity. Nuclear factor of activated T cells 1 (NFATc1) signaling is necessary and sufficient to transform fibers into oxidative type fibers (20)(21)(22). Therefore, we narrowed our focus to NFATc1 as a potential primary regulator of the phenotype transition resulting from Gα13 ablation.…”

Mitogen-Activated Protein Kinase-Activated Protein Kinases 2 and 3 Regulate SERCA2a Expression and Fiber Type Composition To Modulate Skeletal Muscle and Cardiomyocyte Function

“…MEFs were transiently transfected at 50 to 80% confluence. Nonmodified branched polyethylenimine (Sigma-Aldrich, Taufkirchen, Germany) (4.5 l of a 1-mg/ml solution) was added to a mixture of 1.5 g DNA and 90 l serum-and antibiotic-free Dulbecco modified Eagle medium (DMEM), with the subsequent addition of 900 l serum-and antibiotic-free DMEM (27). Cells were transfected using 1,000 ng of one of the following promoter firefly luciferase reporter gene constructs: a human SERCA2 promoter fragment extending from bp Ϫ2577 to ϩ321 (Ϫ2577 fragment; ϩ1 indicates the transcription start site), one of several SERCA2 promoter deletion mutants, a porcine MyHCIId/x promoter fragment extending from bp Ϫ2742 to ϩ9 (Ϫ2.8 fragment) (26), a rabbit MyHCI/␤ promoter fragment extending from bp Ϫ2345 to ϩ99 (Ϫ2.4 fragment) (26), or a murine PGC-1␣ promoter fragment extending from bp Ϫ3036 to ϩ119 (Ϫ3.0 fragment).…”

“…In a dual-luciferase reporter gene assay, firefly luciferase activities were normalized for transfection efficiency using Renilla activity. Firefly luciferase reporter gene assays were performed as described previously (27). Renilla activity was measured by using a Glomax microplate reader (Promega, Mannheim, Germany) and 20 l of lysate, injecting 100 l of 1ϫ Renilla substrate buffer (100 mM phosphate buffer [pH 7.8], 1 mM EDTA, 0.5 M NaCl, 4 M coelenterazine [Promega]) into the microplate reader, and reading for 10 s after a 2-s delay.…”