C77
The proapoptotic kinases MST1 and MST2 interact with RASSF2 translocating it from the nucleus to the cytoplasm
Wendy Cooper1, Luke Hesson1, Ashraf Dallol1, Eamonn Maher1, Walter Kolch2, Farida Latif1
1Department of Medical and Molecular Genetics, Institute of Biomedical Research, University of Birmingham, Birmingham, UK, 2The Beatson Institute for Cancer Research, Glasgow, UK
RASSF2 is a recently described tumour suppressor that in common with the rest of the RASSF family contains both Ras association and SARAH domains. Proteomic approaches (yeast two hybrid and FLAG immunoprecipitation / mass spectrometry) identified the proapoptotic kinases MST1 and MST2 as the most significant binding partners of RASSF2. Like RASSF2, MST1 and MST2 contain the SARAH protein interaction motif.
Here we demonstrate that endogenous RASSF2 interacts with both endogenous MST1 and endogenous MST2 and that the majority of MST1 and MST2 present in A549 cells is associated with RASSF2. To confirm that RASSF2 is not sequestering MST1 or MST2 in an inactive form we demonstrated that immunoprecipitation of FLAG tagged RASSF2 precipitates MST that is fully active in a kinase assay. Deletion and mutation analysis of RASSF2 demonstrates that this interaction is indeed mediated via the RASSF2 SARAH domain. Furthermore, we demonstrated that RASSF2 and MST2 do indeed colocalise, but whilst RASSF2 alone is nuclear the presence of MST1 or MST2 results in colocalisation in the cytoplasm, and deletion of the SARAH domain from RASSF2 results in resistance to the MST1 or MST2 induced translocation of RASSF2. Interestingly, non-phosphorylatable and kinase inactive mutants of MST2 were unable to induce RASSF2 translocation.
Therefore we propose that phosphorylation of MST1/MST2 is required for nuclear/cytoplasmic shuttling of the RASSF2 tumour suppressor gene.
