Structural insights into the small GTPase specificity of the DOCK guanine nucleotide exchange factors

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Highlights

  • The DOCK family of GEFs regulates cytoskeletal dynamics.

  • DOCK proteins activate Rac1 and/or Cdc42 GTPases.

  • Human DOCK proteins function in developmental processes and the immune system.

  • This review focuses on structural insights into the substrate specificity of DOCK.

  • Specific recognition of GTPases by DHR-2 domains is discussed.

Abstract

The dedicator of cytokinesis (DOCK) family of guanine nucleotide exchange factors (GEFs) regulates cytoskeletal dynamics by activating the GTPases Rac and/or Cdc42. Eleven human DOCK proteins play various important roles in developmental processes and the immune system. Of these, DOCK1–5 proteins bind to engulfment and cell motility (ELMO) proteins to perform their physiological functions. Recent structural studies have greatly enhanced our understanding of the complex and diverse mechanisms of DOCK GEF activity and GTPase recognition and its regulation by ELMO. This review is focused on gaining structural insights into the substrate specificity of the DOCK GEFs, and discuss how Rac and Cdc42 are specifically recognized by the catalytic DHR-2 and surrounding domains of DOCK or binding partners.

Introduction

The Rho family of small GTPases plays a central role in regulating cytoskeletal dynamics and is associated with many cellular processes, including cell division, morphogenesis, polarity, and migration [1, 2, 3, 4]. Similar to other GTPases, Rho GTPases act as molecular switches by cycling between an inactive GDP-bound state and an active GTP-bound state. The signaling activity of Rho GTPases is tightly regulated by guanine nucleotide exchange factors (GEFs), GTP hydrolysis activity promoting factors (GAPs), and guanine nucleotide dissociation inhibitors (GDIs). GEFs directly activate Rho GTPase by facilitating GDP/GTP exchanges. Rho GTPase activation is associated with multiple cancers [5,6]. Thus, Rho GEFs are considered molecular targets in the process of remedying cancer metastasis [7••].

Over 80 proteins are categorized as mammalian Rho GEF proteins. These are divided into two families: (a) the Dbl family, comprising 70 members with a Dbl homology (DH) domain [8,9], and (b) the dedicator of cytokinesis (DOCK) family, comprising 11 members with two DOCK-homology regions (DHRs) [10,11]; (Figure 1a). DHR-1 mediates the binding of specific phospholipids [12, 13, 14] while DHR-2 is responsible for GEF activity [15]. Based on sequence similarity, the 11 DOCK proteins are divided into four subfamilies [16,17]: two DOCK-A/B subfamilies (DOCK1–5) containing an SH3 domain; the DOCK-D subfamily (DOCK9–11) containing a PH domain at the N-terminus; and the DOCK-C subfamily (DOCK6–8) with no known domains other than the two DHRs. Substrate specificity of DOCK proteins is essentially divided between DOCK-A/B and DOCK-C/D (Figure 1b): DOCK-A/B exclusively activates Rac, whereas DOCK-C/D predominantly activates Cdc42 while its subsets (DOCK6, 7, and 10) activate both Rac and Cdc42 [18, 19, 20]. DOCK proteins play several important roles in developmental processes, such as cell migration, phagocytosis, myogenesis, myoblast fusion, cardiovascular development, neuronal axon guidance, and the immune system [21, 22, 23, 24••].

Dbl family proteins show specificity for a wide range of Rho GTPases, such as RhoA–C, RhoG, Rac, and Cdc42 [8,9]. The structural basis for the specific GTPase recognition by the Dbl GEFs, which is based on several representative DH domain-GTPase complex structures [8,25,26], is better understood by comparing the structures of the 13 known complexes [27]. Regarding the DOCK family, recent structural studies have greatly advanced our understanding of the complex and diverse regulatory mechanisms involved. Because a recent review has provided a biological and structural overview of the DOCK family [28••], this review focuses on gaining structural insights into small GTPase specificity of DOCK GEFs.

Section snippets

Structural overview of the DHR-2 domain

The DHR-2 domain in DOCK9, a Cdc42-specific GEF, was the first DHR-2 structure to be reported [29]. The crystal structure of the DOCK9-Cdc42 complex revealed DHR-2 to be a three-lobed structure (Figure 1a, c). Lobe A consists of helical repeats and mediates homodimerization while lobes B and C serve as binding sites for Cdc42. Subsequent biochemical studies on DOCK1 and DOCK8 revealed that the lobe B/C fragment is the minimal region required for GEF activity and selective GTPase recognition [30,

Substrate recognition by DOCK

DOCK recognizes its substrates, Rac1 as well as Cdc42, via lobes B and C of DHR-2 (Figure 2, Figure 3a). Primary substrate selection by DOCK takes place at lobe C, which is the active center of GEF reaction. The 56th amino acid of Cdc42 is phenylalanine, as opposed to Rac1 and other Rho GTPases, the 56th amino acid of which is mostly tryptophan (Figure 2a). The W56F mutation in Rac1 greatly suppressed Rac1 activation by DOCK1 and DOCK2 and gained activation by DOCK9 to some extent

How is dual substrate recognition achieved by DOCK?

The three DOCKs, DOCK6 and DOCK7 (DOCK-C subfamily), and DOCK10 (DOCK-D subfamily), activate both Rac1 and Cdc42 [18, 19, 20]. Of these, only the crystal structure of DOCK7 in the DOCK7-Cdc42 complex has been described [35•]. The structures of lobe B and switch 1 in DOCK7-Cdc42 were almost identical to those found in DOCK9-Cdc42. Although the crystal structure of DOCK7-Rac1 has not been solved yet, MD simulation yielded a structural model of DOCK7-Rac1 with a lobe B conformation similar to that

Role of the PH domain in GEF activity

DOCK-D possesses a PH domain in its N-terminal region. The NMR structure of the PH domain of DOCK9 was solved (PDB code 1WG7). Although previous studies have not indicated whether the PH domain of DOCK-D mediates GEF activity, it has been established that the PH domain of DOCK9 mediates phospholipid binding and membrane targeting [38].

DOCK-A as well as B can form a complex with engulfment and cell motility (ELMO) proteins (ELMO1–3), which have a C-terminal PH domain [39] (Figure 1a). Formation

Conclusions

DOCK and Dbl families are structurally and mechanistically different Rho GEFs. The DOCK family recognizes Rac or Cdc42 via the difference in the 56th amino acid as well as the conformational difference in switch 1 of GTPases, by using lobes C and B of the DHR-2 domain, respectively. Although the Dbl family also recognizes 56th amino acid of GTPases, switch 1 is not involved in this interaction. Thus, the GTPase recognition of DOCK GEFs is more complex than that of Dbl GEFs. At the level of

Funding

This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science by the Japan Science and Technology Agency (grant number 19K06575 to M. K. N).

Conflict of interest statement

Nothing declared.

References (55)

  • M. Kukimoto-Niino et al.

    Structural basis for the dual substrate specificity of DOCK7 guanine nucleotide exchange factor

    Structure

    (2019)
  • Y. Zhou et al.

    Prenylation and membrane localization of Cdc42 are essential for activation by DOCK7

    Biochemistry

    (2013)
  • N. Meller et al.

    Function of the N-terminus of zizimin1: autoinhibition and membrane targeting

    Biochem J

    (2008)
  • M. Patel et al.

    An evolutionarily conserved autoinhibitory molecular switch in ELMO proteins regulates Rac signaling

    Curr Biol

    (2010)
  • L. Chang et al.

    Structure of the DOCK2-ELMO1 complex provides insights into regulation of the auto-inhibited state

    Nat Commun

    (2020)
  • M. Lu et al.

    PH domain of ELMO functions in trans to regulate Rac activation via Dock180

    Nat Struct Mol Biol

    (2004)
  • C.M. Grimsley et al.

    Dock180 and ELMO1 proteins cooperate to promote evolutionarily conserved Rac-dependent cell migration

    J Biol Chem

    (2004)
  • K. Scheffzek et al.

    Pleckstrin homology (PH) like domains – versatile modules in protein–protein interaction platforms

    FEBS Lett

    (2012)
  • A.B. Jaffe et al.

    Rho GTPases: biochemistry and biology

    Annu Rev Cell Dev Biol

    (2005)
  • S.J. Heasman et al.

    Mammalian Rho GTPases: new insights into their functions from in vivo studies

    Nat Rev Mol Cell Biol

    (2008)
  • S. Narumiya et al.

    Rho signaling research: history, current status and future directions

    FEBS Lett

    (2018)
  • R.B. Haga et al.

    Regulation and roles in cancer cell biology

    Small GTPases

    (2016)
  • P. Aspenström

    Activated Rho GTPases in cancer-the beginning of a new paradigm

    Int J Mol Sci

    (2018)
  • M.D.M. Maldonado et al.

    Targeting Rac and Cdc42 GEFs in metastatic cancer

    Front Cell Dev Biol

    (2020)
  • K.L. Rossman et al.

    GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors

    Nat Rev Mol Cell Biol

    (2005)
  • D.R. Cook et al.

    Rho guanine nucleotide exchange factors: regulators of Rho GTPase activity in development and disease

    Oncogene

    (2014)
  • J.F. Côté et al.

    GEF what? Dock180 and related proteins help Rac to polarize cells in new ways

    Trends Cell Biol

    (2007)
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