16K human prolactin inhibits vascular endothelial growth factor-induced activation of Ras in capillary endothelial cells

Signaling pathways mediating the antiangiogenic action of 16K human (h)PRL include inhibition of vascular endothelial growth factor (VEGF)-induced act ivation of the mitogen-activated protein kinases (MAPK). To determine at wh ich step 16K hPRL acts to inhibit VEGF-induced MAPK activation, we assessed more proximal events in the signaling cascade. 16K hPRL treatment blocked VEGF-induced Raf-l activation as well as its translocation to the plasma me mbrane. 16K hPRL indirectly increased cAMP levels; however, the blockade of Raf-l activation was not dependent on the stimulation of cAMP-dependent pr otein kinase (PKA), but rather on the inhibition of the GTP-bound Ras. The VEGF-induced tyrosine phosphorylation of the VEGF receptor, Flk-1, and its association with the Shc/Grb2/Ras-GAP (guanosine triphosphatase-activating protein) complex were unaffected by 16K hPRL treatment. In contrast, 16K hP RL prevented the VEGF-induced phosphorylation and dissociation of Sos from Grb2 at 5 min, consistent with inhibition by 16K hPRL of the MEK/MAPK feedb ack on Sos. The inhibition of Ras activation was paralleled by the increase d phosphorylation of 120 kDa proteins comigrating with Ras-GAP. Taken toget her, these findings show that 16K hPRL inhibits the VEGF-induced Ras activa tion; this antagonism represents a novel and potentially important mechanis m for the control of angiogenesis.