Identification of non-overlapping functions between adaptor proteins NCK1 and NCK2

Signals from cell surface receptors are often relayed via adaptor proteins. NCK1 and NCK2 are Src-Homology (SH) 2 and 3 domain adaptors that regulate processes requiring a remodelling of the actin cytoskeleton. Evidence from gene inactivation in mouse suggest that NCK1 and NCK2 are functionally redundant, although recent reports support the idea of unique functions for NCK1 and NCK2. We sought to examine this question further by delineating NCK1- and NCK2-specific signalling networks. We used both affinity purification-mass spectrometry and BioID proximity labelling to identify NCK1/2 signalling networks comprised of 98 proteins. Strikingly, we found 30 proteins restricted to NCK1 and 28 proteins specifically associated with NCK2, thus suggesting differences in their function. We report that Nck2-/-, but not Nck1-/- mouse embryo fibroblasts (MEFs) are multi-nucleated and display extended protrusions reminiscent of intercellular bridges, which correlate with an extended time spent in cytokinesis as well as a failure of a significant proportion of cells to complete abscission. Our data also show that the midbody of NCK2-deficient cells is not only increased in length, but also altered in composition, as judged by the mislocalization of AURKB, PLK1 and ECT2. Finally, we show that NCK2 function during cytokinesis requires its SH2 domain. Taken together, our data delineate the first high-confidence interactome for NCK1/2 adaptors and highlight a number of proteins specifically associated with either protein. We further demonstrate that contrary to what is generally accepted, NCK1 and NCK2 are not completely redundant, and shed light on a previously uncharacterized function for the NCK2 adaptor protein in cell division.