Neuregulin-1-stimulated phosphorylation of GABP in skeletal muscle cells

Localization of acetylcholine receptors (AChRs) to neuromuscular synapses is mediated, in part, through selective transcription of AChR genes in myofiber synaptic nuclei. Neuregulin-1 (NRG-1) is a good candidate for the extracellular signal that induces synapse-specific gene expression, since NRG-1 is concentrated at synaptic sites and activates AChR synthesis in cultured muscle cells. NRG-1-induced transcription requires activation of Erk and Jnk MAP kinases, but the downstream substrates that mediate this transcriptional response are not known. Previous studies have demonstrated that a consensus binding site for Ets proteins is required both for NRG-1-induced transcription and for synapse-specific transcription in transgenic mice. This regulatory element binds GABP alpha, an Ets protein, and GABP beta, a protein that dimerizes with GABP alpha raising the possibility that phosphorylation of GABP by MAP kinases induces transcription of AChR genes. To determine whether MAP kinases might directly regulate the activity of GABP, we studied MAP kinase-catalyzed and NRG-1-induced phosphorylation of GABP alpha and GABP beta. We show that GABP alpha and GABP beta are phosphorylated in vitro by Erk and by Jnk. Using recombinant proteins containing mutated serine and threonine resides, we show that GABP alpha is phosphorylated predominantly at threonine 280, while serine 170 and threonine 180 are the major phosphorylation sites in GABP beta. We generated antibodies specific to the major phosphorylation site in GABP alpha and show that NRG-1 stimulates phosphorylation of GABP alpha at threonine 280 in vivo. These results suggest that GABP alpha is a target of MAP kinases in NRG-1-stimulated muscle cells and are consistent with the idea that phosphorylation of GABP alpha contributes to transcriptional activation of AChR genes by NRG-1.