The HER4 cytoplasmic domain, but not its c terminus, inhibits mammary cell proliferation

Unlike the proliferative action of other epidermal growth factor (EGF) receptor family members, HER4/ ErbB4 is often associated with growth- inhibitory and differentiation signaling. These actions may involve HER4 two- step proteolytic processing by intramembraneous gamma- secretase, releasing the soluble, intracellular 80- kDa HER4 cytoplasmic domain, s80(HER4). We demonstrate that pharmacological inhibition of either gamma- secretase activity or HER4 tyrosine kinase activity blocked heregulin- dependent growth inhibition of SUM44 breast cancer cells. We next generated breast cell lines stably expressing GFP-s80(HER4) [green fluorescent protein (GFP) fused to the N terminus of the HER4 cytoplasmic domain, residues 676- 1308], GFP-CTHER4 (GFP fused to N terminus of the HER4 C-terminus distal to the tyrosine kinase domain, residues 989-1308), or GFP alone. Both GFP-s80(HER4) and GFP-CTHER4 were found in the nucleus, but GFP- s80(HER4) accumulated to a greater extent and sustained its nuclear localization. s80HER4 was constitutively tyrosine phosphorylated, and treatment of cells with a specific HER family tyrosine kinase inhibitor 1) blocked tyrosine phosphorylation; 2) markedly diminished GFP- s80(HER4) nuclear localization; and 3) reduced signal transducer and activator of transcription (STAT) 5A tyrosine phosphorylation and nuclear localization as well as GFP-s80(HER4): STAT5A interaction. Multiple normal mammary and breast cancer cell lines, stably expressing GFP-s80(HER4) (SUM44, MDA- MB- 453, MCF10A, SUM102, and HC11) were growth inhibited compared with the same cell line expressing GFP- CTHER4 or GFP alone. The s80(HER4)- induced cell number reduction was due to slower growth because rates of apoptosis were equivalent in GFP-, GFP- CTHER4-, and GFP- s80(HER4)- expressing cells. Lastly, GFP- s80(HER4) enhanced differentiation signaling as indicated by increased basal and pro-lactin-dependent beta- casein expression. These results indicate that surface HER4 tyrosine phosphorylation and ligand- dependent release of s80(HER4) are necessary, and s80(HER4) signaling is sufficient for HER4- dependent growth inhibition.