ERK1/2 is a negative regulator of homeodomain protein Arix/Phox2a.

The homeodomain protein Arix/Phox2a plays a role in the development and maintenance of the noradrenergic cell type by regulating the transcription of genes involved in the biosynthesis and metabolism of noradrenaline. Previous work has shown that Arix/Phox2a is a phosphoprotein, and the phosphorylated form of Arix/Phox2a exhibits poorer DNA-binding activity than does the dephosphorylated form. Here, we demonstrate that Arix/Phox2a is phosphorylated by extracellular signal-related kinase (ERK)1/2 at two sites within the N-terminal transactivation domain. The phosphorylation level of Arix in cultured SH-SY5Y neuroblastoma cells is reduced when cells are treated with the mitogen activated protein kinase kinase 1 (MEK1) inhibitor UO126. Treatment of sympathetic neurons with the MEK1 inhibitor, PD98059, results in an elevation of mRNAs encoding noradrenergic proteins, dopamine beta-hydroxylase (DBH) and norepinephrine transporter (NET), but not tyrosine hydroyxlase (TH). Treatment of neuroblastoma cultures with PD98059 increases the interaction of Arix with DBH and NET genes, but not the TH gene. Together, these results suggest that phosphorylation of Arix by ERK1/2 inhibits its ability to interact with target genes, and that both specificity of expression and modulation by external stimuli are monitored through the same transcription factor.

Autocrine growth factor signaling by insulin-like growth factor-II mediates MyoD-stimulated myocyte maturation.

Skeletal muscle differentiation, maturation, and regeneration are regulated by interactions between intrinsic genetic programs controlled by myogenic transcription factors, including members of the MyoD and MEF2 families, and environmental cues mediated by hormones and growth factors. Insulin-like growth factors (IGFs) also play key roles in muscle development, and in the maintenance and repair of mature muscle, but their mechanisms of interaction with other muscle regulatory networks remain undefined. To evaluate the potential interplay between MyoD and IGF signaling pathways, we have studied muscle differentiation in C3H 10T1/2 fibroblasts acutely converted to myoblasts by quantitative infection with a recombinant adenovirus encoding mouse MyoD. In these cells, IGF-II gene and protein expression are induced as early events in differentiation, and the IGF-I receptor and downstream signaling molecules, including Akt, are rapidly activated. Interference with IGF-II production by a tetracycline-inhibited adenovirus expressing an IGF-II cDNA in the antisense orientation reversibly inhibited both production of muscle-specific structural proteins and myocyte fusion to form multinucleated myotubes. Similar results were achieved with a tetracycline-inhibited adenovirus expressing dominant-negative Akt. Our observations identify a robust autocrine amplification network in which MyoD enhances the later steps in muscle differentiation by induction of a locally acting growth factor.