Abstract
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 remodeling of the actin cytoskeleton. Evidence from gene inactivation in mouse suggests 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 signaling networks. We used both affinity purification-mass spectrometry and BioID proximity labeling to identify NCK1/2 signaling networks comprised of 98 proteins. Strikingly, we found 30 proteins restricted to NCK1 and 28 proteins specifically associated with NCK2, suggesting differences in their function. We report that Nck2-/-, but not Nck1-/- mouse embryo fibroblasts (MEFs) are multinucleated 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 several proteins specifically associated with either protein. Thus, contrary to what is generally accepted, we demonstrate that NCK1 and NCK2 are not completely redundant, and shed light on a previously uncharacterized function for the NCK2 adaptor protein in cell division.
Highlights
Signals from cell surface receptors are often relayed via adaptor proteins
affinity purification followed by mass spectrometry (AP-MS) and BioID Proximity Labeling Delineate NCK1/2 Adaptor Protein Interactomes and Identify Specific Partners for Each NCK1 and NCK2—To systematically delineate protein interaction networks nucleated by NCK1 and NCK2 adaptor proteins, we generated clonal HEK293T cells stably expressing 3xFLAG-tagged NCK1 or NCK2 at near endogenous levels
We found that the best characterized NCK-associated proteins, such as PAK1/2, CHN1, CBL, WASL and TNIK are common to both NCK1 and NCK2
Summary
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 remodeling of the actin cytoskeleton. We used affinity purification-mass spectrometry (AP-MS) and BioID proximity labeling to delineate NCK1 and NCK2 protein networks, which unexpectedly reveal many interactors specific to each adaptor protein. We find that NCK2, but not NCK1, is localized at the site of furrow ingression and at the midbody during cell division, a process displaying the most extensive reorganization of the actin cytoskeleton.
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