Interaction Analyses of 14-3-3ζ, Dok1, and Phosphorylated Integrin β Cytoplasmic Tails Reveal a Bi-molecular Switch in Integrin Regulation

Integrins are hetero-dimeric (alpha and beta subunits) type I transmembrane proteins that facilitate cell adhesion and migration. The cytoplasmic tails (CTs) of integrins interact with a plethora of intra-cellular proteins that are required for integrin bidirectional signaling. In particular, the beta CTs of integrins are known to recruit a variety of cytosolic proteins that often have overlapping recognition sites. However, the chronological sequence of beta CTs/cytosolic proteins interactions remains to be fully characterized. Previous studies have shown that the scaffold protein 14-3-3 zeta binds to phosphorylated beta CTs in activated integrins, whereas interactions of Dok-1 with phosphorylated beta CTs maintained integrins in the resting state. In this study, we examined the binding interactions between 14-3-3 zeta, Dok1, and phosphorylated integrin beta 2 and beta 3 CTs. We show that the scaffold protein 14-3-3 zeta interacts with the phosphotyrosine binding (PTB) domain of Dok1 even in the absence of the phosphorylated integrin beta CTs. The interactions were mapped onto the beta-sheet region of the PTB domain of Dok1. Furthermore, we provide evidence that the 14-3-3 zeta/Dok1 binary complex is able to bind to their cognate phosphorylated sequence motifs in the integrin beta CTs. We demonstrate that Thr phosphorylated pTTT beta 2 CT or pTST beta 3 CT can bind to 14-3-3 zeta that is in complex with the Dok1 PTB domain, whereas Ser phosphorylated beta 2 CT or Tyr phosphorylated beta 3 CT interacted with Dok1 in 14-3-3 zeta/Dok1 complex. Based on these data, we propose that 14-3-3 zeta/Dok1 complex could serve as a molecular switch providing novel molecular insights into the regulating integrin activation. (C) 2018 Elsevier Ltd. All rights reserved.