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. 2013 Jun 25;52(25):4354-63.
doi: 10.1021/bi301688g. Epub 2013 Jun 14.

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

Yeyun Zhou  1 , Jared L Johnson, Richard A Cerione, Jon W Erickson
Affiliations

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

Yeyun Zhou et al. Biochemistry. .

Abstract

The unconventional guanine nucleotide exchange factor (GEF) family comprising 11 DOCK180 related proteins is classified into four subfamilies, A through D, based on their relative GEF activity toward the closely related Rac and Cdc42 GTPases. DOCK proteins participate in the remodeling of the actin cytoskeleton and are key regulators of cell motility, phagocytosis, and adhesion. Here we show that the guanine nucleotide exchange domain of DOCK7, DHR2 (for DOCK homology region 2), is a potent GEF for prenylated Cdc42 and Rac1 in a model liposome system, demonstrating that the prenylation and membrane localization of Cdc42 or Rac1 are necessary for their activation by DOCK7. Additionally, we identify DOCK7 residues that confer GTPase GEF specificity. Finally, using our liposome reconstitution assay, we show that a more narrowly defined GEF domain of DHR2 (designated DHR2s) harbors an N-terminal site distinct from the GEF active site that binds preferentially to the active, GTP-bound forms of Cdc42 and Rac1 and thereby recruits free DHR2s from solution to the membrane surface. This recruitment results in a progressive increase in the effective concentration of DHR2s at the membrane surface that in turn provides for an accelerated rate of guanine nucleotide exchange on Cdc42. The positive cooperativity observed in our reconstituted system suggests that the action of DOCK7 in vivo may involve the coordinated integration of Cdc42/Rac signaling in the context of the membrane recruitment of a DOCK7 GEF complex.

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Figures

Figure 1
Figure 1
Protein expression (A) Schematic representation of the DOCK7 and DHR2 constructs that were examined in this study. (B) SDS-PAGE and Colloidal Blue-staining of wild-type proteins used in this study.
Figure 2
Figure 2
DHR2s and DHR2c of DOCK7 do not catalyze the nucleotide exchange activity of non-isoprenylated Cdc42 or Rac1. (A) DOCK7-DHR2s (160 nM) was added at t=0 with Mant-GDP with or without bacterially expressed GST-Cdc42 (200 nM). The same concentration of Cdc42 without DHR2s was EDTA treated (10 mM) to demonstrate maximum Mant-GDP binding. (B) As in (A), DOCK7-DHR2s (160 nM) was added at t=0 with Mant-GDP (1 µM) with or without E. coli-expressed GST-Rac (200 nM). To test the exchange reactivity of Rac, 200 nM (final concentration) of E. coli-expressed GST-Rac was mixed with 200 nM (final concentration) of the DOCK1 exchange domain, DHR2c. (C) 20 µM liposomes (bulk concentration) were mixed with or without DOCK7-DHR2s (160 nM) and 1 mM Mant-GDP, with 200 nM E. coli-expressed 6His-Cdc42. (D) Insect cells expressing 6His-Cdc42 were separated into soluble and particulate fractions and Cdc42 purified from the soluble fraction (200 nM Cdc42) was tested for reactivity with E. coli-expressed DHR2s (200 nM).
Figure 3
Figure 3
DOCK7-DHR2s and DOCK7-DHR2c activate isoprenylated Cdc42 and Rac1 in the presence of liposomes. 20 µM liposomes (bulk lipid concentration) pre-incubated with either added DOCK7-DHR2s (50 nM) (red) or DOCK7-DHR2c (50 nM) (blue) at the zero-time point to initiate the GDP to Mant-GDP exchange reaction with insect cell-expressed, isoprenylated Cdc42 (200 nM) (A) or Rac1 (200 nM) (B). No GEF was added in the control curves. (C) The activation of Cdc42 (200 nM) by DHR2s of DOCK7 was dose-dependent with the DHR2s concentrations indicated. Similar Ymax values for each condition were attained after sufficient reaction time. The exchange data were fit to a single exponential (Ft=Fmax[1−e(−k’t)] where Ft is the observed fluorescence at time t, Fmax is the fluorescence observed at the completion of exchange, and k’ is apparent first order rate constant). This yielded values for {F(max), k’(s−1)} of {100.8+/−2.5, 0.028+/−0.001)}, {89.4+/−0.3, 0.083+/−0.001} and {102.9+/−0.3, 0.115+/−0.001} for the 5 nM, 20 nM and 50 nM curves, respectively.
Figure 4
Figure 4
Residues specific for Cdc42 and Rac nucleotide exchange activity. (A) Sequence comparison in the specificity encoding region for DOCK subfamilies A, C, and D members: DOCK1, DOCK7, and DOCK9, respectively. Numbered residues shown refer to the human DOCK7 sequence (Acc # AAZ38451). (B) SDS-PAGE and Colloidal Blue-staining of wild-type DOCK7-DHR2c and DOCK7-DHR2c mutants tested in this figure. (C) Substitution of methionine 1875 of DHR2c to leucine increases its GEF activity toward Cdc42; while mutation of glutamine 1878 of DHR2c to asparagine decreases its GEF activity toward Cdc42. The DHR2c protein concentration for each mutant was added to a final concentration of 40 nM at t=0. The concentration of insect cell-expressed isoprenylated Cdc42 was 200 nM for each trace with Mant-GDP present at 1 µM. The exchange-deficient DOCK7 mutant V1885A serves as a negative control as does liposome-bound Cdc42 alone. All traces are representative of 4 replicates of each condition and were repeated with at least two different protein purifications.
Figure 5
Figure 5
DOCK7-DHR2s, but not DOCK7-DHR2c, preferentially binds to the active (GTP-bound) forms of Cdc42 and Rac1. (A) DOCK7-DHR2s was incubated with isoprenylated Cdc42 (left panel) or Rac1 (right panel) pre-bound to liposomes. EDTA-treated (nucleotide-free), and GDP- and GTPγS-bound forms of the GTPases are indicated. (B) DOCK7-DHR2s or DOCK7-DHR2c at 300 nM was incubated with either E. coli-expressed GST-Cdc42 or GST-Rac1 (0.4 nmol=20 µg) and affinity precipitated with glutathione-conjugated beads after loading the respective GTPases with GDP, GTPγS, or leaving them nucleotide-free.
Figure 6
Figure 6
DOCK7-DHR2s activates Cdc42 with a nucleotide-dependent exchange rate that is accelerated by GTPγS. Cdc42 (200 nM final concentration) pre-loaded with Mant-GDP, and then separated from excess Mant-GDP, was mixed with 50 µM unlabeled GDP or GTPγS prior to the addition of DHR2s (160 nM final concentration; at t=0 indicated by arrow).
Figure 7
Figure 7
Model for Cdc42/Rac1 activation by DOCK7 via a recruitment feed-back loop. In solution, DHR2s of DOCK7 could not stimulate the activation of Cdc42 or Rac1, whereas in liposomes, DHR2s initiated nucleotide exchange on Cdc42 or Rac1. Addition of Cdc42-GTPγS accelerated the nucleotide exchange reaction.
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