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

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.

The inositol 5-phosphatase INPP5B regulates B cell receptor clustering and signaling

“…Considering that PI(4,5)P 2 can regulate a number of actin regulators ( Janmey et al, 2018 ), we would speculate that INPP5B action upon the actin cytoskeleton in this context is likely to be mediated by additional factors. Indeed, we also observed an increase in the level of active Cdc42 in INPP5B-depleted cells, which could be mediated by the PI(4,5)P 2 -binding Rho family GEFs Vav ( Treanor et al, 2011 ; Weber et al, 2008 ) or DOCK8 ( Sakurai et al, 2021 ). Similarly, the actin nucleation promoting factor WASP ( Huang et al, 2020 ; Rey-Suarez et al, 2020 ), which is regulated by PI(4,5)P 2 and Cdc42 ( Rohatgi et al, 2000 ), may also be involved.…”

CRISPR/Cas-Based Gene Editing Strategies for DOCK8 Immunodeficiency Syndrome

“…DOCK proteins consist of two conserved protein domains known as Dock Homology Region 1 and 2, (DHR1 and -2) ( Côté et al, 2005 ). The DHR1 domain is located upstream of the DHR2 domain and mediates the binding to phosphoinositide (PI), which leads to localized GEF activity near the cell membrane ( Sakurai et al, 2021 ; Côté and Vuori, 2002 ). The DHR2 domain interacts with the nucleotide-free form of Rho GTPases such as RhoA, Rac, or Cdc42, depending on the DOCK protein.…”

“…The basis of the various functions of DOCK8 can be divided into either GEF-dependent actin regulation or functions within GEF-independent pathways. When chemokines bind to extracellular receptors, phosphoinositide 3 kinase (PI3K) is activated and initiates the production of phosphatidylinositol (3,4,5)-triphosphate (PIP3), which recruits DOCK8 via the DHR-1 subunit, which consequently leads to membrane-adjacent GEF activity ( Xu et al, 2017 ; Sakurai et al, 2021 ). In addition, when chemokines bind extracellular receptors, PKCα is activated, which phosphorylate DOCK8 for dissociation from Leuchine Rich Repeats And Calponin Homology Domain Containing (LRCH1) (2), thus diminishing its inhibitory impact ( Xu et al, 2017 ).…”

The phosphoinositide code is read by a plethora of protein domains

“…Its crystal structure has been determined, allowing the position of PI(4,5)P 2 and PI(3,4,5)P 3 to be modeled in a basic groove between β hairpin loops [87]. The crystal structure of DOCK8's DHR-1 domain bound to its unresolved PI(4,5)P 2 ligand reveals that the lysine and arginine residues reorient their sidechains accordingly [88]. These calcium-independent PI interactions target guanine nucleotide exchange factor functions to the plasma membrane to help control the organization and dynamics of the actin cytoskeleton and cell migration.…”