Prolactin modulates phosphorylation, signaling and trafficking of epidermal growth factor receptor in human T47D breast cancer cells

Abstract

Prolactin (PRL) is a polypeptide hormone produced by the anterior pituitary gland and other sites that acts both systemically and locally to cause lactation and other biological effects by interacting with the PRL receptor, a Janus kinase (JAK)2-coupled cytokine receptor family member, and activating downstream signal pathways. Recent evidence suggests PRL is a player in the pathogenesis and progression of breast cancer. Epidermal growth factor (EGF) also has effects on breast tissue, working through its receptors, epidermal growth factor receptor (EGFR) and ErbB-2 (c-neu, HER2), both intrinsic tyrosine kinase growth factor receptors. EGFR promotes pubertal breast ductal morphogenesis in mice, and both EGFR and ErbB-2 are relevant in pathogenesis and behavior of breast and other human cancers. Previous studies showed that PRL and EGF synergize to enhance motility in the human breast cancer cell line, T47D. In this study, we explored crosstalk between the PRL and EGF signaling pathways in T47D cells, with an ultimate aim of understanding how these two important factors might work together in vivo to affect breast cancer behavior. Both PRL and EGF caused robust signaling in T47D cells; PRL acutely activated JAK2, signal transducer and activator of transcription-5 (STAT5), and extracellular signal-regulated kinase-1 and -2 (ERK1 and ERK2), whereas EGF caused EGFR activation and consequent src homology collagen (SHC) activation and ERK activation. Notably, PRL also caused phosphorylation of the EGFR and ErbB-2 at sites detected by PTP101, an antibody that recognizes threonine phosphorylation at consensus motifs for ERK-induced phosphorylation. PRL-induced PTP101-reactive phosphorylation was prevented by pretreatment with PD98059, an ERK pathway inhibitor. Furthermore, PRL synergized with EGF in activating SHC and ERK and transactivating a luciferase reporter driven by c-fos gene enhancer elements, suggesting that PRL allowed markedly enhanced EGF signaling. This was accompanied by substantial inhibition of EGF-induced EGFR downregulation when PRL and EGF cotreatment was compared to EGF treatment alone. This effect of PRL was abrogated by ERK pathway inhibitor pretreatment. Our data suggest that PRL synergistically augments EGF signaling in T47D breast cancer cells at least in part by lessening EGF-induced EGFR downregulation and that this effect requires PRL-induced ERK activity and threonine phosphorylation of EGFR.

Differential roles of ERK and Akt pathways in regulation of EGFR-mediated signaling and motility in prostate cancer cells

“…Thus, the degree and kinetics of EGFR activity and downregulation are believed to be critical modulators of EGF actions. We previously uncovered that, in addition to tyrosine phosphorylation, EGFR also undergoes threonine phosphorylation in response to EGF, which is ERK activation dependent and can slow the pace of ligand-induced receptor downregulation (Huang et al, 2003(Huang et al, , 2006Li et al, 2008). In this study, we found that blockade of the ERK pathway, but not of the Akt pathway, eliminates EGF-induced EGFR threonine phosphorylation, which in turn potentiates receptor tyrosine phosphorylation (activation) and subsequently enhances EGFR endocytic downregulation in prostate cancer cells.…”

“…Cell starvation, inhibitor treatment, stimulation, protein extraction, immunoprecipitation, immunoblotting and densitometry These procedures were performed as previously described (Huang et al, 2003(Huang et al, , 2006Ma et al, 2009). For experiments in which ubiquitination was assayed, N-ethylmaleimide (5 mM) was added to the lysis buffer to prevent post-lysis deubiquitination of proteins (Deng et al, 2007).…”

“…We also evaluated the degree of Cbl tyrosine phosphorylation and the level of Cbl proteins associated with EGFR under these conditions (Figures 3c-e) by In a reconstitution cell system, we have recently shown that elimination of EGFR phosphorylation at 669 T by a point mutation (threonine to alanine) resulted in accelerated ligand-induced receptor loss (Li et al, 2008). To determine whether the effect of PD98059 on EGF-induced activation, ubiquitination (by Cbl) and downregulation of EGFR was related to receptor threonine phosphorylation in prostate cancer cells that endogenously express EGFR, we used a monoclonal antibody (PTP101) that specifically recognizes phosphorylated threonine residues residing within the ERK substrate consensus sites (Huang et al, 2003(Huang et al, , 2004(Huang et al, , 2006Li et al, 2008). As shown in Figure To confirm that these observations were due to inhibition of ERK and not to an off-target effect of the PD98059 and UO126 compounds, we introduced a constitutively active MEK1 (c.a.…”

“…MEK1 in DU145 cells enhanced EGFR mass and promoted EGFR threonine phosphorylation, when compared with the cells expressing lacZ (control). Our previous work using other cell systems (Huang et al, 2003(Huang et al, , 2006Li et al, 2008) suggests that EGF-induced ERK-mediated threonine phosphorylation of EGFR could serve as a brake on EGFR downregulation. Taken together, the current data indicate that inhibition of ERK signaling eliminates EGF-induced EGFR threonine phosphorylation and leads to the release of the 'brake', which in turn enhances receptor activation, ubiquitination (by Cbl) and downregulation.…”

“…Further data suggest that signaling can also emanate from the EGFR in the process of postendocytic trafficking (Wiley, 2003;Sebastian et al, 2006). Indeed, we have previously shown that phosphorylation of EGFR at threonine-669 by ERK can influence receptor signaling and trafficking (Huang et al, 2003(Huang et al, , 2004(Huang et al, , 2006Li et al, 2008). However, the effects that ERKdependent EGFR phosphorylation has on the quantitative and qualitative output from EGFR and cancerous behaviors, such as invasive migration, remain poorly understood.…”