Phosphorylation of the rod-tail hinge region of cingulin regulates its interaction with nonmuscle myosin-2B

The tight junction (TJ) protein cingulin binds directly to nonmuscle myosin 2B (NM2B) through sequences in its C-terminal rod-tail region and recruits it to tight junctions (TJ) to control membrane cortex mechanics, epithelial morphogenesis and cingulin conformation. However, the minimal sequence required for cingulin-NM2B interaction and how this interaction is regulated is not known. Here we identify a 19-aminoacid sequence at the hinge between the cingulin rod and tail that is required for cingulin-NM2B interaction, and we investigate the role of phosphorylation of Ser residues within this region in regulating this interaction. Immunofluorescence microscopy localization of NM2B in cingulin-KO cells rescued with mutant cingulin constructs shows that phospho-mimetic but not dephospho-mimetic cingulin mutants inhibit NM2B recruitment to junctions and downstream regulation of cingulin conformation and TJ tortuosity, correlating with cingulin-NM2B interaction, as determined by GST pulldown analysis. In contrast, either phospo-or dephospho-mimetic mutants of Ser residues within the cingulin head domain do not affect either NM2B recruitment to TJ, or cingulin conformation and localization in cells, or TJ membrane tortuosity. Finally, Ser residues within the hinge display the consensus sequence for protein kinases CK1 and CK2, and, through in vitro phosphorylation, site mutation analysis and use of inhibitors, we identify a complex interplay between CGN phospho-sites, with a prominent negative role of Ser1162 phosphorylation in the regulation of cingulin-NM2B interaction. In summary, we show that cingulin-NM2B interaction is regulated by cingulin phosphorylation within the hinge and identify a potential role for CK1 and CK2 kinases in cingulin phosphorylation.