Characterisation of the novel melanoma associated Shc adaptor ShcD/RaLP

RaLP is a recently identified signalling adaptor protein found to be up-regulated in invasive melanoma. To characterise RaLP and investigate its cellular role, a polyclonal antibody was raised against RaLP and purified successfully; it was considered to be a good tool for RaLP characterisation. Moreover, stable cell lines that express either GFPRaLP or FLAG-RaLP were generated. These cells showed a slightly higher increase in the percentage of G1 cells when compared to the parental counterparts using FACS analysis, and increased levels of p21/cip, a marker of G1 arrest. The underlying mechanism for the increased level of p21/cip remains unclear, but this may contribute to enhanced melanoma migration or survival induced by RaLP. The subcellular localisation of RaLP, as revealed by fluorescence microscopy and subcellular fractionation, was not restricted to the cytoplasm and cell membrane as has been reported previously, but a proportion of the protein was found to be present in the nucleus and mitochondria. Herein we show for the first time that RaLP shuttles between the nucleus and cytoplasm, and that the RaLP-CH2 domain has a critical role in RaLP export from the nucleus. Analysis of deletion and point mutants revealed that the sequence [superscript 83]LCTLIPRM[superscript 90] represents a functional nuclear export signal within the CH2 domain. Moreover, we have demonstrated that RaLP accumulates in the nucleus upon oxidative stress. Preliminary experiments showed that RaLP associates with the DNA and might play a role in gene transcription. Moreover, using mass spectrometry we were able to identify a phosphorylation site at Thr156/159 within the CH2 domain that is induced upon oxidative stress, while Ser132 was phosphorylated in unstressed conditions as well. These phosphorylation events may play a role in nuclear localisation or function of RaLP. Further support of a role for RaLP in the nucleus and mitochondria was provided by analysis of protein complexes containing GFP-RaLP by mass spectrometry. Proteins identified included a nuclear pore complex protein, importin-β transcriptional regulators, and mitochondrial proteins, in addition to the receptor tyrosine kinases Ret and TrkC. In a previous study, the invasion suppressor Nischarin was identified as an interacting partner with the CH2 domain of RaLP using a yeast two-hybrid library screen. Here we confirm that RaLP co-immunoprecipitates with Nischarin when overexpressed in cells or when present at endogenous levels. Mapping the RaLP-CH2 domain for the sequence required for Nischarin binding revealed that the first 93 amino acids are required for RaLP/Nischarin interaction, while interaction is greatly reduced upon deletion of the first 24 amino acids. Interestingly, Nischarin was also identified by mass spectrometry as part of a complex precipitated by GFP-RaLP from N1E-115 cells. Confocal microscopy provided additional evidence of co-localisation within melanoma cells. While we were unable to observe any effect of the RaLP/Nischarin association on the LIMK/cofilin pathway, very preliminary evidence suggested that RaLP might relieve suppression of Erk activation induced by Nischarin. Taken together, our results point to diverse roles for RaLP in cellular signalling and migration.