Mechanisms of plexin-mediated signalling
Final Report Abstract
In summary, we were able to fulfil the majority of the aims of the project. First, we successfully further characterized Plexin-B1-Met-mediated signalling and identified Grb2 as an important mediator thereof. Furthermore, we could show that a Grb2-dependent signalling mechanism is required for Sema4D-induced, Plexin-B1-mediated regulation of cell migration. These data identify an important new component of plexin-B-mediated signalling and fill the gap between Met-mediated phosphorylation of Plexin-B1 and its ability to deactivate RhoA via p190 RhoGAP. Moreover, on the cellular level, we were able to show that Grb2 is crucial for Plexin-B1-mediated inhibition of cellular motility. In the second part of the project we concentrated on characterization of the Plexin-B1-ErbB-2-mediated signalling pathway. We performed siRNA-based screens and identified the members of the IKK-complex as crucial regulators of Plexin-B1 signalling. On the cellular level, we observed that the Sema4D-dependent, Plexin-B1-mediated migration and dedifferentiation of osteoblasts requires IKK-complex activity, thereby indicating that the significance of the identified IKK signalling mechanism is not only restricted to cancer cells but also extends to other non-malignant cell types. These both findings not only represent a significant advance in our understanding of Plexin-B signalling, but may provide the basis for a future translational effort. Especially the IKK-complex might represent a novel therapeutic target for the treatment of B-plexin-dependent tumours as well as in osteoporosis and other bone diseases. Additionally, we identify over 20,000 phosphorylation sites, of which approximately 1 % is regulated upon TNF-α stimulation. We identify various potential novel IKKβ substrates, including kinases and regulators of cellular trafficking. Moreover, we show that one of the candidates, AEG-1/MTDH/LYRIC, is directly phosphorylated by IKKβ on serine 298. We provide strong evidence that IKKβ-mediated AEG-1 phosphorylation is essential for IκBα degradation as well as NF-κB-dependent gene expression and cell proliferation, which correlate with cancer patient survival in vivo. These findings provide a valuable dataset that can further be used by other scientists exploring IKKβ-mediated signalling. Moreover, we succeeded in describing the way in which AEG-1 regulates IκBα degradation, and we could also show that the levels of AEG-1 phosphorylation correlate with patient survival, thus establishing the possibility of future clinical applications for these findings.
Publications
- 2012. Grb2 mediates semaphorin-4D-dependent RhoA inactivation. J Cell Sci 125:3557-67
T. Sun, R. Krishnan, J. M. Swiercz
(See online at https://doi.org/10.1242/jcs.101063) - 2014 The IκB kinase complex is required for plexin-B-mediated activation of RhoA. PLoS One. 9:e105661
M. Zielonka, R. K. Krishnan, J. M. Swiercz, S. Offermanns
(See online at https://doi.org/10.1371/journal.pone.0105661) - Quantitative analysis of the TNF-α-induced phosphoproteome reveals AEG-1/MTDH/LYRIC as an IKKβ substrate. Nature Communications volume 6, Article number: 6658 (2015), 15 S.
R. K. Krishnan, H. Nolte, H. Kaur, K. Sreenivasan, M. Looso, T. Sun, S. Offermanns, M. Krüger, J. M. Swiercz
(See online at https://doi.org/10.1038/ncomms7658)
