Motor neurons modulate acetylcholine receptor (AChR) gene expression in skeletal muscle by two signalling pathways: the transmitter‐evoked depolarization of muscle membrane inhibits AChR gene transcription throughout the myofibre presumably via activation of a serine/threonine kinase, while the transcription rates of AChR genes in the synaptic region are increased by nerve‐derived trophic factors including AChR‐inducing activity (ARIA). To gain further insight into these interactions we characterized the receptor for heregulin (HRG)/ARIA in muscle. We showed that HRG increases AChR alpha‐subunit mRNA levels via tyrosine phosphorylation of ErbB3 and ErbB2/neu in myotubes. The protein tyrosine phosphatase inhibitor, pervanadate, potentiated the responses to HRG that were in turn blocked by the tyrosine kinase inhibitor erstatin, indicating the relevance of tyrosine phosphorylation to these events. The effects of HRG were inhibited by enhanced cellular serine/threonine phosphorylation which has been implicated in the repression of AChR genes by electrical activity. Immunocytochemical analysis of adult rat muscle revealed that while ErbB2/neu is present throughout the entire surface of the myofibre membrane, ErbB3 expression is exclusively restricted to the endplate suggesting its involvement in synapse‐specific transcription of AChR genes by HRG/ARIA.

Binding of heregulin (HRG) to its receptor, ErbB3, results in a dimerization with ErbB2/neu and activation of their intrinsic tyrosine kinases, initiating a cascade of events resulting in the stimulation of acetylcholine receptor (AChR) genes in muscle. Here we have examined the signalling downstream of the HRG receptor. We show that phosphatidylinositol 3′‐kinase (PI3K) and SHC bind to the HRG‐activated ErbB3 in myotubes. Subsequently, p70S6 kinase (p70S6k), and MAP kinase ERK2 and thereby p90rsk are activated. However, inhibition of PI3K and p70S6k by wortmannin and rapamycin, respectively, failed to antagonize AChR α‐subunit gene expression stimulated by HRG, despite the fact that the activities of the kinases were inhibited. In contrast, these inhibitors elevated AChR α‐subunit mRNA levels, by themselves, independently of muscle electrical activity. On the other hand, the 17mer antisense oligonucleotide, EAS1, caused a specific depletion of ERK2 and eliminated the ability of HRG to stimulate AChR α‐subunit gene expression. These results indicate that HRG stimulates expression of AChR genes via ERK2 activation, and provide a physiological example of neurotrophic factor‐associated repression of AChR genes by stimulation of p70S6k activity which may contribute to the expression of adult type AChR genes at the neuromuscular junction.

The breast cancer susceptibility gene BRCA1 encodes a nuclear phosphoprotein that acts as a tumor suppressor. Phosphorylation of BRCA1 has been implicated in altering its function, however, the pathway(s) that leads to the phosphorylation of BRCA1 has not been described. Here, a signaling pathway by which heregulin induces cell cycle-independent phosphorylation of BRCA1 was delineated. We showed that heregulin stimulation induced the phosphorylation of BRCA1 and concomitant activation of the serine/threonine kinase AKT in T47D human breast cancer cells. Heregulin-induced phosphorylation of BRCA1 was abrogated by phosphatidylinositol 3-kinase (PI3K) inhibitors and by a dominant-negative AKT. In the absence of heregulin, the ectopic expression of the constitutively active p110 subunit of PI3K was sufficient to induce BRCA1 phosphorylation. Furthermore, the purified glutathione Stransferase/AKT kinase phosphorylated BRCA1 in vitro. We have also shown that the phosphorylation of BRCA1 by AKT occurs on the residue Thr-509, which is located in the nuclear localization signal. These results reveal a novel signaling pathway that links extracellular signals to the phosphorylation of BRCA1 in breast cancer cells.Heregulins (NDF/neuregulin) are a group of growth factors that regulate growth, differentiation, and survival of various breast cancer cell lines (1). Heregulins activate the ErbB-2 receptor through direct binding to ErbB-3 and ErbB-4 receptors and initiate a cascade of events resulting in the stimulation of Ras/Erk and phosphatidylinositol 3-kinase (PI3K) 1 pathways (1, 2). PI3K appears to regulate the phosphorylation and consequently the activity of the p70S6 kinase, protein kinase C isoforms, and the serine/threonine kinase AKT (3, 4). AKT activity is regulated both by binding of PI3K lipid products to its pleckstrin homology (PH) domain and by phosphorylation of Thr-308 and Ser-473 residues located within its activation loop and the C terminus, respectively (4). Activated AKT provides a survival signal that protects cells from apoptosis and mediates growth factor-induced cell proliferation (3-5). The aberrant expression of AKT has also been implicated in cell transformation (6). However, the regulation of AKT activity by heregulin and its potential importance in breast cancer cells are not known.The hereditary breast cancer susceptibility gene product BRCA1 (7-10) has been shown to have tumor suppressive activity (11, 12) and to play a role in the differentiation of mammary epithelial cells (13, 14), apoptosis (15), and DNA recombination (16,17). Despite several lines of evidence suggesting that serine phosphorylation of BRCA1 during cell cycle progression and in response to DNA-damaging agents may affect its function (16 -19), the signaling pathway(s) involved in BRCA1 phosphorylation is unclear.Here, we have studied the regulation of AKT activity by heregulin and its impact on the phosphorylation of BRCA1 in T47D cells. We show that heregulin stimulates phosphorylation of BRCA1 via PI3K/AKT in breast cancer ...