Protein scaffolds play an important role in signal transduction, functioning to facilitate protein interactions and localize key pathway components to specific signaling sites. Connector enhancer of KSR-2 (CNK2) is a neuronally expressed scaffold recently implicated in nonsyndromic, X-linked intellectual disability (NS-XLID) [1-3]. NS-XLID patients have deficits in cognitive function and their neurons often exhibit dendritic spine abnormalities , suggesting a role for CNK2 in synaptic signaling and/or spine formation. To gain insight regarding how CNK2 might contribute to these processes, we used mass spectrometry to identify proteins that interact with the endogenous CNK2 scaffold. Here, we report that the major binding partner of CNK2 is Vilse/ARHGAP39 and that CNK2 complexes are enriched for proteins involved in Rac/Cdc42 signaling, including Rac1 itself, α-PIX and β-PIX, GIT1 and GIT2, PAK3 and PAK4, and members of the cytohesin family. Binding between CNK2 and Vilse was found to be constitutive, mediated by the WW domains of Vilse and a proline motif in CNK2. Through mutant analysis, protein depletion and rescue experiments, we identify CNK2 as a spatial modulator of Rac cycling during spine morphogenesis and find that the interaction with Vilse is critical for maintaining RacGDP/GTP levels at a balance required for spine formation.
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