%0 Generic
%A Majety, Meher Vinay Krishna Mohan
%D 2006
%F heidok:6929
%K Cell cycle regulation , cilia
%R 10.11588/heidok.00006929
%T Development and application of a high throughput cell based assay to identify novel modulators of ERK1/2 activation and,Functional characterisation of the candidate Radial spokehead like (Rshl1)
%U https://archiv.ub.uni-heidelberg.de/volltextserver/6929/
%X The aim of my project was to identify and functionally characterise novel human proteins that influence cancer relevant cellular processes like cell proliferation, signalling, and apoptosis upon over-expression. The focus of my work was 1) The establishment of a high throughput cell based assay to screen for proteins involved in the modulation of cell signalling pathways, specifically the activation of the ERK1/2 pathway, 2) to apply this assay in a screen of previously uncharacterised proteins, and 3) to characterise one candidate protein from this assay and to validate its association with the ERK1/2 pathway.  The principle of the assay is based on the detection of phosphorylated ERK1/2 in cells over-expressing N- and C-terminal YFP tagged proteins. Data acquisition was done using a flow cytometer with an integrated 96-well plate reader. A total of 200 proteins were screened, out of which eleven novel cancer relevant modulators of ERK1/2 activation were identified.  One of the candidates, the Radial spoke head like -1 (Rshl1), which was identified as an inhibitor of ERK1/2 activation was followed up, and shown to be down regulated in kidney cancer. The protein was identified as an inhibitor of proliferation in another cell based assay. The corresponding gene is located on chromosome 19q13.3 at the primary ciliary dyskinesia locus, and the encoded protein contains a radial spoke domain. However, the biological role of this protein was not described. I found that Rshl1 indeed localizes to primary cilia but also to the cytoplasm and nucleus of human kidney cells. Further, I found that its localisation is cell cycle phase dependent. Rshl1 co-localised with MEK1, ERK1/2 and CDK2 and interacts with MEK1, CDK2 and ERK3. Its role as an inhibitor of proliferation was elucidated by the finding that over-expression of Rshl1 caused a G0/G1 phase arrest in human kidney cells via an up-regulation of p57KIP2 expression and stabilization of ERK3. Rshl1 thus regulates the cell cycle by inhibiting the ERK1/2 kinase. It interacts with critical signalling proteins in the cell and maintains homeostasis by arresting cells in the G0/G1 phase. In conclusion, I screened 200 novel proteins for their influence on ERK1/2 activation and identified eleven novel modulators of ERK1/2 pathway. Detailed functional analysis of Rshl1, which was an inhibitor of ERK1/2 activation, identifies this protein as a novel player in the MAPK pathway, and shed light on its role in homeostasis and tumorigenesis.