Identification and Characterisation of proteins interacting with the melanoma associated adaptor protein RaLP/ShcD
thesisposted on 07.06.2013, 13:33 by Dal-Hee Chung
Cell signalling by Receptor Tyrosine Kinases (RTKs) is widely known to regulate several cellular processes. Activated RTKs can associate with many cellular transducer proteins containing PTB (phosphotyrosine-binding) domains and/or SH2 (Src homology 2) domains. The Shc (Src Homology and Collagen) family of adaptor proteins contain both of these critical domains and bind to many growth factor receptors. Four Shc family members have been identified including Shc/ShcA, Sli/ShcB, Rai/ShcC, and the latest member, RaLP/ShcD. Although all the family members share a similar domain modularity, their tissue expression and biological roles are different. As the most recently identified Shc protein, RaLP/ShcD remains poorly characterised. RaLP/ShcD has been shown to have a migratory role in melanoma cells in humans, to interact with Muscle-Specific Kinase (MuSK) receptor in mice, and to regulate cell differentiation during stem cell development. In order to further understand its role in these or other processes, this study aimed to identify novel interacting partners of RaLP/ShcD. The SH2 domain of RaLP/ShcD was shown to interact with another signalling scaffold protein, Gab1, through phosphorylated tyrosine 183, as revealed by GST pull-down and co-immunoprecipitation experiments. Notably, a Gab1Y183F mutant was unable to recruit RaLP/ShcD to the cell membrane in ruffles upon growth factor stimulation. By screening a yeast two-hybrid library,Peg3/Pw1 and HP1α were isolated as novel binding partners for the collagen homology domain 1 (CH1 domain) of RaLP/ShcD. The ability of RaLP/ShcD to interact with both Peg3/Pw1 and HP1α was confirmed by GST pull-down and co-immunoprecipitation assays using transfected human cell lines. Interestingly, a small portion of RaLP/ShcD co-localised with Peg3/Pw1 in the nucleus. Finally, using an affinity column comprising purified CH1 domain coupled to sepharose beads, vimentin was purified from melanoma cell extracts.