The role of cytohesins and the ARNO-CNK1 complex in insulin signalling

The tight regulation of signalling cascades is of crucial importance for organisms in order to react to external stimuli and maintain homeostasis. Insulin signalling is a key metabolic pathway in vertebrates and dysregulation as seen in the case of diabetes mellitus poses a major health risk to the ageing society. This work aims at a deeper understanding of the underlying intracellular signalling cascade, especially of the cytohesin-CNK1 complex. Cytohesins are guanine nucleotide exchange factors for GTPases of the ADP-ribosylation factor (Arf) family and were shown to be involved in insulin signalling. Cellular data further suggest that the adaptor protein Connector enhancer of kinase suppressor of ras 1 (CNK1) forms a complex with the cytohesin Arf nucleotide-binding site opener (ARNO). This complex is required for the translocation to the plasma membrane and signal transduction from the insulin receptor to its downstream effector protein kinase B (PKB). To characterise interaction partners of ARNO and elucidate its mode of action within the insulin receptor (IR) signalling network, biochemical and biophysical approaches were undertaken. A direct interaction of ARNO with the IR could not be demonstrated, however this study characterises the interaction between ARNO and CNK1 in depth. Using pull-down, analytical size-exclusion chromatography and isothermal titration calorimetry approaches, a direct physical interaction between ARNO and CNK1 was shown. Furthermore, the interaction site was mapped to the respective coiled-coil domains of both proteins. Structural studies resulted in the generation of protein crystals, which however did not allow for structure determination due to poor X-ray diffraction. To elucidate the effect of complex formation on the catalytic activity of ARNO, an exchange assay using liposomes and myristoylated Arfs was established. The localisation as well as the effect of CNK1 on the exchange rate was tested in vitro. As expected, wildtype CNK1 did not localise to membranes and also did not affect ARNOcatalysed exchange. Since activated CNK1 as well as ARNO are membrane-bound, CNK1 was artificially tethered to functionalised liposomes via a hexahistidine-tag. Although the liposome tethering was successful, no exchange could be monitored under these conditions, and hence the consequences of the interaction on the exchange rate remain to be investigated using a modified approach. Possibly, CNK1 helps localise cytohesins to the plasma membrane which increases the concentration of activated Arf. Sustained Arf activity leads to higher levels in phosphatidylinositol- 4,5-bisphosphate (PIP2) and increased IR signalling. Lysine acetylation within the PH domain of CNK1 might be an important factor to direct CNK1 to the membrane and drive signalling but further studies need to confirm the specific mode of action