PROLACTIN ACTIVATES RAS VIA SIGNALING PROTEINS SHC, GROWTH-FACTOR RECEPTOR-BOUND-2, AND SON-OF-SEVENLESS

Identification of the signal transduction pathways used by PRL is esse ntial for understanding the role of PRL receptors in growth and differ entiation processes. Early cellular mediators of PRL receptor activati on include tyrosine kinases of the Janus kinase (JAK) and SRC families , with rapid nuclear signaling via tyrosine phosphorylated signal tran sducers and activators of transcription. In the present study we provi de the first demonstration of PRL-induced activation of Ras, an oncoge nic protein that supports an alternative signaling route from the memb rane to the nucleus. PRL stimulated Ras in rat Nb2-SP lymphoma cells, as detected by a 2.0-fold increase in the GTP-bound state of the molec ule (P < 0.01). This activation was associated with marked tyrosine ph osphorylation and increased membrane association of the 52-kilodalton form of SHC. Moreover, PRL induced binding of SHC to growth factor rec eptor bound 2 and the guanine-nucleotide exchange factor son of sevenl ess, a common method used by growth factor receptors to activate Ras. In contrast, no apparent regulation by PRL of Ras via VAV or p120 Ras- guanosine triphosphatase-activating protein was detected, based upon a n absence of PRL-inducible tyrosine phosphorylation of these proteins. Collectively, these results provide a molecular bridge between activa tion of PRL receptor-associated tyrosine kinases and subsequent stimul ation of the serine/threonine kinase Raf-1, an established Ras target that was recently shown to be activated by PRL in Nb2 cells. We conclu de that PRL is able to activate Ras via recruitment of the signaling p roteins SHC, growth factor receptor bound 2, and son of sevenless in N b2 cells. Moreover, PRL induced tyrosine phosphorylation of SHC in two of three PRL-responsive human breast cancer cell lines, suggesting th at SHC-mediated Ras activation is a commonly used signaling strategy b y PRL.