| Summary: | Semaphorin/Plexin signalling pathways modulate many biological processes such as neuronal axon pathfinding, angiogenesis, organogenesis, immune response, and cell migration. Given the fact that Semaphorins/Plexins have a strong influence on cytoskeleton structure through their interactions with receptor tyrosine kinases (RTKs) and Rho-like small GTPases, it has been suggested that they may also have a role in tumourigenesis. Recently we have identified a number of mutations in the gene PLXNB1 which encodes the Sema4D receptor protein Plexin-Bl that are associated with prostate cancer metastasis. This study aims at elucidating the effects of three of the identified mutations in PLXNB1, A5359G (amino acid change: T1697A), A5653G (T1795A) and T5714C (L1815P). None of the mutations affected the Sema4D and PDZ-RhoGEF binding function of Plexin-Bl in coimmunoprecipitation assays. In addition, no influence on the Rho regulation function of Plexin-Bl from the mutations could be detected. However by GST-pull down assays we found that Plexin-B1LT5714C was defective in binding RacL61, a constitutively active Rac mutant. This Rac binding defect renders Plexin-B1T5714C unresponsive to the cell surface trafficking effect of RacL61. Moreover, Plexin-B1T5714C failed to sequester and inhibit Rac-GTP as evident from the fact that it could neither inhibit RacL61 mediated Pak phosphorylation nor inhibit the cell spreading process mediated by RacL61. Our results suggest that the prostate cancer related PLXNB1 mutation T5714C has important effects on the functioning of Plexin-Bl by disrupting the interaction of Plexin-B 1 with Rac.
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