Visit our Expo - Redox and Inflammation signaling 2012

Session X : Cell death in cancer Poster X, 91
Functional analysis of the tumorsuppressor Ras Association Family Domain 1
(RASSF1A) reveals an important role of the SARAH-domain
Claudia Seidel, Antje Klagge, Reinhard Dammann
AWG Tumor Genetics of the Medical Faculty, Martin-Luther-University Halle-
Wittenberg, 06097 Halle/Saale, Germany
INTRODUCTION: RASSF1A is a tumor suppressor gene, which is often epigenetically
inactivated in several cancer types. In lung cancer, a rate of between 30 to 45% of
hypermethylation of the RASSF1A promoter has been found. In several cancer types a
correlation between hypermethylation of RASSF1A and an impaired prognosis for cancer
patients was reported. The promoter of the RASSF1C isoform is never hypermethylated. The
two isoforms RASSF1A and RASSF1C only differ in the first domain, which is a
diacylglycerol binding site and lacks in RASSF1C. Moreover RASSF1A encodes a Ras
association domain and a SARAH domain, which is a protein-protein interaction domain.
METHODS: In order to investigate the function of RASSF1A, we created mutations and
deletions of these different domains and performed functional experiments in the lung cancer
cell line A549. Proliferation analyses as well as soft agar experiments were performed to
investigate the influence of the aberrant RASSF1A forms. The ability of the different forms of
RASSF1A to induce apoptosis was investigated. To analyse the localisation of RASSF1A and
its altered forms the cells were transient transfected using Yellow Fluorescent Protein (YFP)
constructs. Additionally, the interaction of RASSF1A with the proapoptotic kinase MST1 was
determined in precipitation experiments with protein extract from human cells.
RESULTS: The reduced proliferation of A549 cells that express RASSF1A could be
confirmed. Analysis of cells stable transfected with RASSF1A with a deletion of the SARAH
domain (RASSF1ADelSARAH) showed a even lower proliferation rate in comparison to
RASSF1A. A higher rate of apoptosis in cells with RASSF1ADelSARAH could be observed
using transient transfected cells. RASSF1A is when overexpressed as a YFP-fusion
colocalised to the cytoskeleton and during mitosis to the spindles and spindle poles. Using
RASSF1ADelSARAH the mitoses show strong spindles no spindle poles and the
chromosomes are not divided equally. In precipitation experiments MST1 interacts with
RASSF1A but not with RASSF1ADelSARAH.
CONCLUTIONS: The SARAH domain is necessary for the function of the tumor suppressor
RASSF1A. It is responsible for the interaction with MST1 and plays an important role in
regulation of mitotic processes as well as in induction of apoptosis.
This study was supported by BMBF grant 01ZZ0104.
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