Proteomic Analysis of NCK1/2 Adaptors Uncovers Paralog-specific Interactions That Reveal a New Role for NCK2 in Cell Abscission During Cytokinesis

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 , but not 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.

Selective role of Nck1 in atherogenic inflammation and plaque formation

“…To explore the non-compensating role of the Nck1 SH2 and SH3.1 domains in disturbed flow-induced endothelial activation, we sought to identify potential binding partners that could mediate endothelial activation by disturbed flow. A previous report utilizing the BioID proximity biotinylation system to identify Nck1 and Nck2 binding proteins found IRAK-1 as a specific interacting partner for Nck1 (24). While this interaction was proposed to be SH2-dependent using computer modeling software, the specific domain mediating this interaction was not determined.…”

“…Even though they are expressed by different genes, Nck1 and Nck2 proteins share a high sequence identity (68% overall) (10) and their functions are generally regarded as overlapping (21). However, emerging evidence has suggested the independent contribution of the two isoforms in a variety of responses, including T cell activation, cytokinesis, and podocyte cytoskeletal dynamics (22)(23)(24). To investigate the selective roles of Nck1 and Nck2, we utilized Nck1 and Nck2 selective siRNAs that result in a 75% and 85% knockdown, respectively without affecting the expression of the other isoform ( Figure 3A).…”

“…While these data suggest a critical role for Nck1 in atherogenic endothelial activation, the Nck1 binding partner involved remains unknown. A proteomics study by Jacquet et al identified IRAK-1 as a Nck1-specific SH2 interacting partner (24). IRAK-1, a serine/threonine kinase extensively investigated in the interleukin-1 signaling pathway, is required for interleukin-1-induced NF-B activation (31).…”

Uncovering the In Vivo Proxisome Using Proximity‐Tagging Methods

“…Up to now, BioID has been successfully used in diverse contexts. In mammalian cells, BioID was applied to probe the architecture of different subcellular locations such as the nuclear pore complex, focal adhesion complexes, or the centrosome–cilium interface, and also to get insights about molecular mechanisms such as mitochondrial proteostasis, autophagy, mitosis, or viral infection . BioID was also elegantly adapted to investigate RNA–protein interactions that play important roles in cellular functions and diseases.…”

“…With slow tagging kinetics, the BioID and PUP‐IT assays require 16–18 h or 24–36 h of labeling time for efficient biotinylation/pupylation, respectively . These approaches are hence not suited for capturing transient protein interactions but rather provide a history of protein associations over long periods of time, as used in cell cycle or in viral infection . TurboID and miniTurbo were designed to improve BioID assay by reducing the labeling time from 16–18 h to 10 min .…”