A conserved PI(4,5)P2-binding domain is critical for immune regulatory function of DOCK8.

Tetsuya Sakurai,Takehito Uruno,Mikako Shirouzu,Yoshinori Fukui,Tomoharu Yasuda,Mutsuko Kukimoto-Niino,Shigeyuki Yokoyama,Nana Yamane,Daiji Sakata,Ryosuke Aihara,Kazufumi Kunimura

doi:10.26508/lsa.202000873

Abstract

DOCK8 is a Cdc42-specific guanine-nucleotide exchange factor that is essential for development and functions of various subsets of leukocytes in innate and acquired immune responses. Although DOCK8 plays a critical role in spatial control of Cdc42 activity during interstitial leukocyte migration, the mechanism remains unclear. We show that the DOCK homology region (DHR)-1 domain of DOCK8 binds specifically to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and is required for its recruitment to the plasma membrane. Structural and biochemical analyses reveal that DOCK8 DHR-1 domain consists of a C2 domain-like core with loops creating the upper surface pocket, where three basic residues are located for stereospecific recognition of phosphoinositides. Substitution of the two basic residues, K576 and R581, with alanine abolished PI(4,5)P2 binding in vitro, ablated the ability of DOCK8 to activate Cdc42 and support leukocyte migration in three-dimensional collagen gels. Dendritic cells carrying the mutation exhibited defective interstitial migration in vivo. Thus, our study uncovers a critical role of DOCK8 in coupling PI(4,5)P2 signaling with Cdc42 activation for immune regulation.

Highlights

To elicit innate and adaptive immune responses, inflammatory cells must efficiently migrate through the complex, physiological environments in the body
To test whether the DOCK8 DOCK homology region (DHR)-1 domain is responsible for propidium iodide (PI)(4,5)P2 binding, we obtained BW5147α−β− cells expressing a mutant form of DOCK8 that lacks the DHR-1 domain (DOCK8 ΔDHR-1; Fig 1B)
Unlike wild-type DOCK8, DOCK8 ΔDHR-1 failed to bind to PI(4,5)P2 (Fig 1C), indicating that the DHR-1 domain is solely responsible for the interaction with PI(4,5)P2

Summary

Introduction

To elicit innate and adaptive immune responses, inflammatory cells must efficiently migrate through the complex, physiological environments in the body. Rho family GTPases such as Rho, Rac, and Cdc play a central role in cell migration by controlling directionality, protrusive force generation, adhesion to ECM and actomyosin contraction through the regulation of the membrane-cytoskeletal organization (Hall, 1998; Etienne-Manneville & Hall, 2002; Ridley, 2015). Rho GTPases and their regulators play crucial roles in leukocyte development, activation, differentiation, and migration (Heasman & Ridley, 2008; Tybulewicz & Henderson, 2009). Rho GTPases function as a molecular switch cycling between GDPbound inactive, and GTP-bound active states, conversions of which are catalyzed by two mechanistically distinct classes of regulators: guanine nucleotide exchange factors (GEFs) for activation, and GTPase activating proteins for inactivation (Hall, 1998; EtienneManneville & Hall, 2002; Jaffe & Hall, 2005). Specific localization patterns of GEFs and GTPase-activating proteins, and a molecular network of their regulation underlie the spatiotemporal control of Rho GTPases activities in the cells.

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Results

Conclusion