Heregulin-beta is especially potent in activating phosphatidylinositol 3-kinase in nontransformed human mammary epithelial cells

The neu differentiation factors/heregulins (HRGs) comprise a family of poly peptide growth factors that activate p185(erbB-2) through direct binding to either erbB-3 or erbB-4 receptor tyrosine kinases. We have previously show n that HRG-beta is mitogenic for various human mammary epithelial cell line s that coexpress c-erbB-2 and c-erbB-3. Phosphatidylinositol 3-kinase (PI3K ) is activated by p185(erbB-2)/erbB-3 heterodimers in cells stimulated by H RG, and PI3K is constitutively activated by p185(erbB-2)/erbB-3 in breast c arcinoma cells that overexpress c-erbB-2. To better understand the relative abilities of HRGs, epidermal growth factor (EGF), or insulin to activate P I3K under normal physiological conditions, we compared the levels of recrui tment of the 85-kDa regulatory subunit of PI3K when activated by the type I (erbB) or type II [insulin-like growth factor (IGF)] receptor tyrosine kin ases in two different nontransformed human mammary epithelial cell lines. T he nontransformed H16N-2 cells isolated from normal tissue express EGFR, p1 85(erbB-2), and erbB-3, and are highly responsive to the mitogenic effects of HRG-beta as well as to the combination of EGF and insulin in serum-free culture. We measured the stoichiometry of p85 recruited by tyrosine-phospho rylated proteins induced in H16N-2 cells by either the alpha or the beta is oform of HRG. HRG-beta was greater than 10-fold more potent in inducing p85 recruitment than was the less biologically active HRG-alpha isoform. HRG-b eta was also a more potent inducer of p85 recruited by tyrosine-phosphoryla ted proteins than was either EGF, insulin, or EGF and insulin combined. Fur thermore, erbB-3 principally mediated the direct recruitment of p85 in cell s stimulated by HRG or EGF, indicating that, in addition to the high-level activation of PI3K by p185(erbB-2)/erbB-3, EGFR/erbB-3 heterodimer interact ion is essential for the weak but significant level of PI3K activated by EG F in cells that express normal EGFR levels. Studies using the PI3K inhibito r wortmannin also indicated that PI3K activation was required for the proli feration of H16N-2;cells induced by either HRG-beta or EGF and insulin in s erum-free culture. Finally, HRG-beta was also an especially potent inducer of PI3K in the nontransformed MCF-10A cells, which were derived spontaneous ly from normal reduction mammoplasty tissue. These data show, for the first time, a side-by-side quantitative comparison of the relative degree of PI3 K activated by different growth factors in nontransformed growth factor-dep endent cells under precisely defined conditions in culture. J. Cell. Physio l. 183:301-313, 2000. (C) 2000 Wiley-Liss. Inc.