Domain–domain interaction of P-Rex1 is essential for the activation and inhibition by G protein βγ subunits and PKA
Introduction
Signaling cascades from the G protein-coupled receptor (GPCR) to Rho-family GTPases (Rho-GTPases) mediate various cellular responses, such as cell migration and adhesion, through the reorganization of the actin cytoskeleton, cell-cycle progression, and the production of reactive oxygen species [1], [2], [3]. Rho-GTPases, which include Rho, Rac, and Cdc42, are active in the GTP-bound form and hydrolyze GTP to the inactive GDP-bound form. The activities of Rho-GTPases are controlled by the following regulatory molecules: GEFs, which catalyze the transition from the GDP- to the GTP-state on Rho-GTPases [4], [5], and GTPase-activating proteins (GAPs), which enhance the intrinsic GTPase activity. To date, about 70 Rho-family GEFs possessing the tandem Dbl-homology (DH) and pleckstrin-homology (PH) domains have been identified. Most GEFs exhibit tissue-specific expressions and substrate specificities for Rho, Rac, or Cdc42. Generally, the catalytic activities of Rho-family GEFs are suppressed at the steady state and activated by regulatory molecules, such as heterotrimeric G proteins. For instance, the catalytic activities of p115-RhoGEF and the related GEF members LARG and PDZ-RhoGEF are regulated by Gα12 and Gα13 [6], [7], [8]. P-Rex1 and P-Rex2 are synergistically activated by binding with Gβγ and phosphatidylinositol-3,4,5-trisphosphate (PIP3) [9], [10]. In other examples, Dbl, Ost, and Kalirin were bound with Gβγ through their SEK14-like domain [11], and p114-RhoGEF was activated by Gβγ, possibly through the amino-terminal DH/PH domain and the carboxyl-terminal region [12].
P-Rex1 was originally purified as a PIP3-dependent Rac-activator from pig neutrophil lysates and identified as a Dbl-like GEF mainly for Rac [10]. It is mainly expressed in the peripheral blood leukocytes and brain [10]. Neutrophils derived from P-Rex1-deficient mice showed a reduction of Rac2 activation and ROS production in response to fMLP or C5a [13], [14]. Furthermore, dominant negative P-Rex1 perturbed radial neuronal migration in mouse fetal cerebral cortex [15], although P-Rex1-deficient mice did not present severe abnormalities in brain development [13], [14]. In vitro, P-Rex1 exhibits guanine nucleotide exchange activity for Rac and Cdc42 and is synergistically activated by Gβγ and PIP3 [10]. Furthermore, PKA phosphorylates P-Rex1 in vitro and inhibits Gβγ-induced activation [16]. P-Rex1 has a multi-domain structure containing an amino-terminal DH domain, a PH domain, two tandem DEP domains, two tandem PDZ domains, and a carboxyl-terminal IP4P domain. The DH/PH domain shows nucleotide exchange activity [17]. PDZ domain and DEP domain are known as protein-binding domain. The IP4P domain was designated according to its primary structural similarity to inositol polyphosphate-4-phosphatase, but it has not been demonstrated the phosphatase activity [9], [10]. Previously, Gβγ-induced P-Rex1 activation was investigated using GDP/GTP exchange assays on Rac [17]. Gβγ activated both the isolated DH/PH domains and the P-Rex1 mutant lacking the PH domain. Thus, the DH domain is thought to be necessary for Gβγ-dependent activation. However, P-Rex1 mutants lacking two DEP, two PDZ, or an IP4P domain decreased the maximal activation by Gβγ relative to the full-length P-Rex1. Hence, these domains were expected to be responsible for activation by Gβγ.
In this study, we investigated the regulatory mechanisms of Gβγ-dependent P-Rex1 activation and PKA-dependent inhibition. We constructed various P-Rex1 mutants and examined the binding with Gβγ, the Gβγ-induced Rac-GEF activities in vitro and in cells. In conclusion, we found that P-Rex1 possesses a domain–domain interaction between its second DEP/first PDZ domains and IP4P domain and the interaction is required for the Gβγ-induced activation. Moreover, we showed that PKA phosphorylation of P-Rex1 inhibits the Gβγ-dependent activation by preventing the domain–domain interaction.
Section snippets
Plasmids and antibodies
Mouse P-Rex1 cDNA was provided by Kazusa DNA Research Institute as mKIAA1415 (GenBank accession number AK173168). Full-length P-Rex1 (residues 1–1650), ΔDH (residues 1–41, 248–1650), DH/PH (residues 1–392), ΔIP4P (residues 1–783), IP4P (residues 763–1650), DEP2/PDZ1 (residues 504–713), PDZ1 (residues 580–713), DEP2 (residues 504–658), PDZ1AAAA (residues 1–1650, DYGF629–632AAAA), PDZ2AAAA (residues 1–1650, ALSF713–716AAAA), ΔIP4P-PDZ1AAAA (residues 1–783, DYGF629–632AAAA), and ΔIP4P-PDZ2AAAA
Interaction of P-Rex1 and Gβγ
P-Rex1 was purified from neutrophil cytosol as a PtdIns(3,4,5)P3- and Gβγ-sensitive activator of Rac [10]. Although the stimulation of GEF activity of P-Rex1 by Gβγ was demonstrated in vitro [10], [17], [25], the direct binding of P-Rex1 to Gβγ has not yet been analyzed. First, we assessed the equilibrium-binding constant between Gβγ and P-Rex1 by surface plasmon resonance analysis (Fig. 1A). Recombinant Gβ1γ2 was covalently coupled to a CM5-sensor chip, and five different concentrations (25,
Discussion
In this study, we focused on P-Rex1 regulation by Gβγ and demonstrated that the domain–domain interaction between the second DEP/first PDZ domains and the IP4P domain of P-Rex1 was required for Gβγ-induced activation and PKA-induced inhibition. To date, the P-Rex family is the only Rho-family GEF defined as that directly activated by Gβγ. The DH domain of P-Rex1 has been reported to show Gβγ-sensitive activation [17]. However, as shown in Fig. 1C, the DH domain of P-Rex1 was not required for
Acknowledgements
We thank Junji Yamauchi and Yuki Miyamoto for participating in valuable discussions and providing helpful advice for guanine nucleotide exchange assays. This work was supported by Grants-in-Aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (17079006, and 19370055).
References (37)
- et al.
Trends Pharmacol. Sci.
(2001) - et al.
J. Biol. Chem.
(1999) - et al.
FEBS Lett.
(2004) - et al.
Cell
(2002) - et al.
FEBS Lett.
(1999) - et al.
Curr. Biol.
(2005) - et al.
Curr. Biol.
(2005) - et al.
J. Biol. Chem.
(2006) - et al.
J. Biol. Chem.
(2005) - et al.
Biochem. Biophys. Res. Commun.
(1995)
J. Biol. Chem.
J. Biol. Chem.
Methods Enzymol.
J. Biol. Chem.
Cell
J. Biol. Chem.
FEBS Lett.
Cell
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