The semaphorins are a family of proteins originally identified as axon-guiding molecules in the developing nervous system that have been recently shown to regulate many cellular functions, including motility, in a variety of cell types. We have previously shown that in endothelial cells Semaphorin 4D acts through its receptor, Plexin-B1, to elicit a pro-angiogenic phenotype that involves the activation of the phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway. Here we show through the

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... e of a receptor chimeric approach, Plexin-B1 mutants, and dominant negative and pharmacological inhibitors that this response is dependent upon the activation of RhoA and its downstream target, Rho kinase (ROK). Indeed, we demonstrate that in endothelial cells, Semaphorin 4D promotes the formation of focal adhesion complexes, stress fibers, and the phosphorylation of myosin light chain, a response that was abolished by the use of ROK inhibitors and absent from cells expressing Plexin-B1 mutant constructs incapable of signaling to RhoA. Stress fiber polymerization and contraction are in turn necessary for RhoA-dependent pro-angiogenic signaling through Plexin-B1. Furthermore, we observed that in endothelial cells Plexin-B1 promotes the integrin-mediated activation of Pyk2, resulting in the stimulation of PI3K, Akt, and ERK. These findings provide evidence that Plexin-B1 promotes endothelial cell motility through RhoA and ROK by regulating the integrin-dependent signaling networks that result in the activation of PI3K and Akt.