Investigation of receptor-interacting serine-threonine kinase 4 (RIPK4) andpleimorphic adenoma gene-like 2 (PLAGL2) proteins interaction

Epidermis is a stratified squamous epithelium, composed of a proliferating basal and differentiated suprabasal keratinocyte layers. Epidermal layers are under the control of different signaling pathways, such as TGF-beta, WNT, PI3K and NF-kB, that are important for development. RIPK4, which we have previously associated with Aslan Type Multiple Pterygium syndrome, regulates the epidermal differentiation. However, there is not enough information about the molecular mechanisms of RIPK4 in this process. Within the context of a BAP9749 Project that was conducted by Asst. Prof. Tuba DİNÇER and supported by KTU Scientific Reaserch Coordination Unit, RIPK4 interacting proteins were aimed to be identified to clarify the role of RIPK4 in epidermal differentiation. For this purpose, human keratinocyte cDNA library was screened by large scale yeast two hybrid system using RIPK4 as bait and sixteen proteins were identified. Among them, transcription factor PLAGL2 was chosen for further analyses since it was previously reported to have a role in WNT signaling pathway similar to the RIPK4. Accordingly within the scope of this thesis; we aimed to verify the interaction of RIPK4 and PLAGL2 and identify the domains responsible for his interaction firstly in yeast with small scale yeast two hybrid system and secondly in mammalian cells by coimmunoprecipitation and co-immunoflourescence experiments. In this context, initially the interaction betwwen RIPK4 and PLAGL2 was shownwith small scale yeast two hybrid in yeast. Then interaction of RIPK4 and PLAGL2 was verifien in mammalian cells by co-immunoprecipitation experiment. Furthermore, N-terminal domain of RIPK4 with intact kinase activity and C-terminal domain of PLAGL2 were found to be responsive from this interaction. In addition immunoflourescence experiment showed that when RIPK4 and PLAGL2 co-expressed RIPK4 prevented nuclear trasnsport of PLAGL2 by keeping PLAGL2 in the cytoskeleton filaments and cytoplasmic dot like structures and RIPK4's kinase activity and C-terminal domain was important for PLAGL2-RIPK4 cytoplasmic co-localization.