Kohler, Reto. Functional characterization of human NDR kinases : novel regulatory mechanisms and a path towards direct substrates. 2012, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_9858
We investigated the role of human MOB proteins in NDR/LATS kinase regulation. We found that three hMOB proteins did not bind to or activate all human NDR kinases and that hMOB2 was an NDR-specific binder. Furthermore, we describe competitive binding of hMOB1A/B and hMOB2 towards the NTR of human NDR1/2. Interestingly, in contrast to hMOB1A/B, hMOB2 is bound to unphosphorylated NDR1/2. Moreover, RNAi-mediated depletion of hMOB2 protein resulted in increased NDR activity. Consistent with these findings, hMOB2 overexpression impaired not only okadaic acid-induced activation of NDR but also the functional roles of NDR in death receptor-induced apoptosis and centrosome duplication. In summary, our data indicate that hMOB2 is a negative regulator of human NDR1/2 kinases.
Additionally, we established a basis for the discovery of additional human NDR kinase substrates. We employed the chemical genetic method developed by Shokat and colleagues to create analog-sensitive variants of NDR1/2 kinases. Subsequently, we have tried to identify direct targets of analog-sensitive NDR1(M166G) by performing in vitro kinase assays on cell lysates and immunocomplexes in the presence of a radiolabeled ATP analog and observed a specific and reproducible pattern of labeled bands in reactions containing NDR1(M166G) immunocomplexes. Our data together with the recent identification of the first in vivo substrate of human NDR1/2 kinases, p21, should stimulate further efforts to dissect the downstream signaling of mammalian NDR kinases.
|Advisors:||Hemmings, Brian A.|
|Committee Members:||Wymann, Matthias Paul and Affolter, Markus|
|Faculties and Departments:||03 Faculty of Medicine > Departement Biomedizin > Division of Biochemistry and Genetics > Cancer- and Immunobiology (Wymann)|
|Bibsysno:||Link to catalogue|
|Number of Pages:||135 S.|
|Last Modified:||30 Jun 2016 10:48|
|Deposited On:||08 May 2012 08:50|
Repository Staff Only: item control page