Abstract
Muc4, a heterodimeric transmembrane mucin containing EGF-like domains, has been described as an ErbB2-binding protein which modulates signaling via the ErbB2-ErbB3 pathway. In Muc4-transfected MCF-7 cells, Muc4 expression resulted in alteration of both the time course and phosphorylation levels of NRG beta 1 induced phosphorylation and activation of both ErbB2 and ErbB3. Muc4 significantly enhanced the autophosphorylation of ErbB2 over the early (defined 0-30 min) and intermediate (30-120 min) NRG beta 1 treatment times at three sites, Y1248, Y1221 and Y1139. The sites displayed differential maximal phosphorylation times. At Y1248 and Y1139, maximal phosphorylation occurred entirely during the early treatment phase. However, Y1221/2 showed maximal phosphorylation during the intermediate phase with a smaller peak during the early phase. The ratio of phosphorylated ErbB3 and total receptor level was significantly enhanced (in cells that expressed Muc4 compared without Muc4) over both the early and intermediate NRG beta 1 treatment time at the Y1289 site. This motif is one of several similar ErbB3 motifs whose phosphorylation mediates the binding of PI3-kinase. This phospholipid kinase is a key modulator of numerous cellular pathways leading to proliferation, motility and survival. Aberrancies in the ErbB2-ErbB3 signaling pathway have been implicated in the aggressive behavior of tumor cells, and the identification and characterization of modulators of this pathway are being sought as targets of potential therapeutic interventions. Muc4 significantly enhanced activated ERK in the absence of NRG beta 1 treatment while a NRG beta 1 mediated activation of AKT was observed. At early NRG beta 1 treatment time phases, Muc4 co-localized with phosphorylated ErbB2 (pY1248) independent of NRG beta 1 treatment; co-localization of Muc4 and ErbB2 receptor (activated/receptor forms) was observed at the apical surface or around the cell surface membrane. These data provide evidence in the Muc4-transfected MCF-7 cells for the biological NRG beta 1 mediated ErbB2 and ErbB3 activation. Our data suggests that Muc4 affects steady state phosphorylation levels and duration of the phosphorylation signal of both ErbB receptors, and that NRG beta 1 might affect ErbB2 and ErbB3 signaling differently. Additionally, the results of the timing of phosphorylation studies suggest the possibility that temporal aspects of phosphorylation at different sites may determine the pathways activated preferentially in the subsequent signaling cascades.