Src Family Kinases Are Required for Prolactin Induction of Cell Proliferation

Prolactin (PRL) is a pleiotropic cytokine promoting cellular proliferation and differentiation. Because PRL activates the Src family of tyrosine kinases (SFK), we have studied the role of these kinases in PRL cell proliferation signaling. PRL induced [(3)H]thymidine incorporation upon transient transfection of BaF-3 cells with the PRL receptor. This effect was inhibited by cotransfection with the dominant negative mutant of c-Src (K>A295/Y>F527, SrcDM). The role of SFK in PRL-induced proliferation was confirmed in the BaF-3 PRL receptor-stable transfectant, W53 cells, where PRL induced Fyn and Lyn activation. The SFK-selective inhibitors PP1/PP2 and herbimycin A blocked PRL-dependent cell proliferation by arresting the W53 cells in G1, with no evident apoptosis. In parallel, PP1/PP2 inhibited PRL induction of cell growth-related genes c-fos, c-jun, c-myc, and odc. These inhibitors have no effect on PRL-mediated activation of Ras/Mapk and Jak/Start pathways. In contrast, they inhibited the PRL-dependent stimulation of the SFKs substrate Sam68, the phosphorylation of the tyrosine phosphatase Shp2, and the PI3K-dependent Akt and p70S6k serine kinases. Consistently, transient expression of SrcDM in W53 cells also blocked PRL activation of Akt. These results demonstrate that activation of SFKs is required for cell proliferation induced by PRL.

催乳素（Prolactin，PRL）是一种多效性细胞因子，可促进细胞增殖与分化。鉴于PRL可激活酪氨酸激酶Src家族（Src family of tyrosine kinases，SFK），本研究探讨了此类激酶在PRL介导的细胞增殖信号通路中的作用。当通过PRL受体对BaF-3细胞进行瞬时转染后，PRL可诱导[³H]胸腺嘧啶核苷掺入；该效应可通过共转染c-Src显性负突变体（K>A295/Y>F527，简称SrcDM）得到抑制。在稳定表达PRL受体的BaF-3转染细胞W53中，研究团队进一步验证了SFK在PRL诱导的细胞增殖中的作用：PRL可激活Fyn与Lyn激酶。选择性SFK抑制剂PP1/PP2及除莠霉素A（herbimycin A）可通过将W53细胞阻滞于G1期而阻断PRL依赖的细胞增殖，且未引发明显细胞凋亡。与此同时，PP1/PP2可抑制PRL诱导的细胞生长相关基因c-fos、c-jun、c-myc及odc的表达。此类抑制剂对PRL介导的Ras/Mapk及Jak/Start通路的激活无影响；与之相反，它们可抑制SFK底物Sam68的PRL依赖性激活、酪氨酸磷酸酶Shp2的磷酸化，以及磷脂酰肌醇3-激酶（PI3K）依赖的Akt与p70S6k丝氨酸激酶的活化。与此一致的是，在W53细胞中瞬时表达SrcDM同样可阻断PRL对Akt的激活。上述结果表明，SFK的激活是PRL诱导细胞增殖所必需的。