Subcellular distribution and funciton of the neuronal JNK signalling pathway scaffold protein JIP3

The JNKs have been shown to regulate cellular processes ranging from apoptosis to differentiation and survival, depending on cell-tissue type and cell context. JIP3 was identified as a putative scaffold protein functioning in the JNK signalling pathway. JIP3 is predominantly expressed in neurons, and is thought to have a role in neuronal vesicular transport. The function of JIP3 in the context of JNK signalling has, however, not been established.;A polyclonal JIP3 antiserum was generated in the course of this study, which was suitable for western blotting, immunprecipitation and indirect immunofluorescence microscopy experiments. This antiserum showed greater efficacy than any other available antisera, and was essential in subsequent experiments.;Biochemical fractionation and immunofluorescence microscopy experiments identified the association of endogenous JIP3 with an unconventional vesicular species. The majority of JIP3 was found to localise predominantly in growth cone regions of differentiated N1E-115 and PC-12 cells. In these growth cone regions, significant co-localisation was identified between JIP3 and JNK pathway components, but was not apparent in other subcellular compartments. Immunoprecipitation experiments showed increased association of JIP3 with JNK pathway components upon differentiation of PC-12 cells.;The proposed function of the JNK pathway in neurite outgrowth led to the investigation of the potential for JIP3 regulation of this neuronal differentiation process. Overexpression of JIP3, which acts as a partial inhibitor of JNK pathway signalling, was found to significantly suppress neurite outgrowth in N1E-115 and PC-12 cells. Moreover, re-constitution of a JIP3 mediated JNK signalling module was sufficient to induce neurite outgrowth in PC-12 cells.;Finally, JIP3 co-localised with the cytoskeletal regulator paxillin specifically within growth cone regions of differentiated PC-12 cells. This suggests that JIP3 may direct JNK pathway signalling towards a growth cone associated substrate, paxillin, and thus regulate neurite outgrowth.