PROLACTIN ACTIVATES RAS VIA SIGNALLING PROTEINS SHC, GRB2 AND SOS

Abstract

Identification of the signal transduction pathways used by prolactin (PRL) is essential for understanding the role of PRL receptors in growth and differentiation processes. Early cellular mediators of PRL receptor activation include tyrosine kinases of the JAK and SRC families, with rapid nuclear signalling via tyrosine phosphorylated STAT transcription factors. In the present study we provide the first demonstration of PRL-induced activation of RAS, an oncogenic protein that supports a separate signalling route from the membrane to the nucleus. In rat Nb2-SP lymphoma cells PRL stimulated RAS as detected by a 2.0-fold increase in the GTP-bound state of the molecule (p<0.01). RAS activation was associated with marked tyrosine phosphorylation and increased membrane association of the 52 kDa form of SHC. PRL induced binding of SHC to GRB2 and the guanine-nucleotide exchange factor SOS, 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-GTPase-activating protein was detected, based upon an absence of PRL-inducible tyrosine phosphorylation of these proteins. Collectively, these results provide a molecular bridge between activation of PRL receptor-associated tyrosine kinases and the subsequent stimulation of the serine/threonine kinase RAF-1, an established RAStarget that was recently shown to be activated by PRL in Nb2 cells. In conclusion, PRL is able to activate RAS via recruitment of the signaling proteins SHC, GRB2 and SOS in Nb2 cells. Moreover, prolactin induced tyrosine phosphorylation of SHC in two out of three PRL-responsive human breast cancer cell lines, suggesting that SHC-mediated RAS activation is a commonly used signalling strategy for PRL.