ARTICLE ABSTRACTSerine/threonine kinase PAK1 is activated by estrogen and plays an important role in breast cancer. However, the integration of PAK1 into the estrogen response is not fully understood. In this study, we investigated the mechanisms underlying the hormone-induced activation of estrogen receptor (ERα, ESR1). We show that estrogen activated PAK1 through both the ERα and GPER1 membrane receptors. Estrogen-dependent activation of PAK1 required the phosphorylation of tyrosine residues by Etk/Bmx and protein kinase A (PKA) within an assembled signaling complex comprising pTyr-PAK1, Etk/Bmx, the heterotrimer G-protein subunits Gβ1, Gγ2, and/or Gγ5, PAK-associated guanine nucleotide exchange factor (βPIX, ARHGEF7), and PKA. Moreover, the PKA RIIβ subunit is a direct target of PAK1, and thus in response to estrogen, the activated pTyr-PAK1 complex reciprocally potentiated PKA activity, suggesting a positive feedback mechanism. We also demonstrate that PKA phosphorylated Ser305-ERα in response to estrogen, but pTyr-PAK1 phosphorylated Ser305-ERα in response to prolactin (PRL), implying that maximal ERα phosphorylation is achieved when cells are exposed to both PRL and estrogen. Furthermore, S305-ERα activation led to enhanced phosphorylation of Ser118-ERα and promoted cell proliferation and tumor growth. Together, these data strongly support a critical interplay between PRL and estrogen via PAK1 and suggest that ligand-independent activation of ERα through PRL/PAK1 may impart resistance to anti-estrogen therapies. Cancer Res; 76(9); 2600–11. ©2016 AACR.