Characterization of a Rac1- and RhoGDI-associated lipid kinase signaling complex

Abstract

Rho family GTPases regulate a number of cellular processes, including actin cytoskeletal organization, cellular proliferation, and NADPH oxidase activation. The mechanisms by which these G proteins mediate their effects are unclear, although a number of downstream targets have been identified. The interaction of most of these target proteins with Rho GTPases is GTP dependent and requires the effector domain. The activation of the NADPH oxidase also depends on the C terminus of Rac, but no effector molecules that bind to this region have yet been identified. We previously showed that Rac interacts with a type I phosphatidylinositol-4-phosphate (PtdInsP) 5-kinase, independent of GTP. Here we report the identification of a diacylglycerol kinase (DGK) which also associates with both GTP- and GDP-bound Rac1. In vitro binding analysis using chimeric proteins, peptides, and a truncation mutant demonstrated that the C terminus of Rac is necessary and sufficient for binding to both lipid kinases. The Rac-associated PtdInsP 5-kinase and DGK copurify by liquid chromatography, suggesting that they bind as a complex to Rac. RhoGDI also associates with this lipid kinase complex both in vivo and in vitro, primarily via its interaction with Rac. The interaction between Rac and the lipid kinases was enhanced by specific phospholipids, indicating a possible mechanism of regulation in vivo. Given that the products of the PtdInsP 5-kinase and the DGK have been implicated in several Rac-regulated processes, and they bind to the Rac C terminus, these lipid kinases may play important roles in Rac activation of the NADPH oxidase, actin polymerization, and other signaling pathways.

FIG. 1

Association of Rac1 with a DGK. (A) GST and GST fusion proteins of Rac1, RhoA, and Cdc42 bound to GSH beads were loaded with GTPγS or GDPβS and then incubated with rat brain homogenate. The beads were washed and assayed simultaneously for DGK and PtdInsP 5-kinase activity. GST-Rac which had not been exposed to lysate was also assayed as a negative control (lanes 8 and 9). (B) Rac1 was immunoprecipitated from rat brain homogenate, washed, and assayed for DGK and PtdInsP 5-kinase activities. An immunoprecipitation using nonimmune (NI) serum was done as a negative control. The migration positions of the lipid standards in both experiments are indicated. The data are representative of five experiments.

FIG. 2

The C terminus of Rac is necessary for binding to the DGK and the PtdInsP 5-kinase. GST and GST fusion proteins of Rho family members, Rac point mutants, and Rac-Rho chimeras bound to GSH beads were incubated with rat brain homogenate, washed, and assayed for lipid kinase activities. The data are representative of 12 experiments.

FIG. 3

A peptide corresponding to the C terminus of Rac competes with GST-Rac for binding to the lipid kinases. Increasing concentrations of a peptide corresponding to the C terminus of Rac (residues 166 to 188) were preincubated with rat brain homogenate for 30 min. GST-Rac beads were added to the homogenate and incubated for an hour. The beads were then washed and assayed for DGK activity (A) and PtdInsP 5-kinase activity (B). Control peptides corresponding to the C termini of RhoA and Cdc42 (splice variants a and b) were also used in the experiment at 1 mM. The activity of the lipid kinases associated with GST-Rac in the absence of peptide was taken as 100%. Data shown are the mean ± standard error of the mean of 11 experiments.

FIG. 4

The C terminus of Rac is sufficient for binding to PtdInsP 5-kinase and DGK. GST, GST-Rac, and a deletion mutant (RacCT) which contained only the C-terminal 27 amino acid residues of Rac fused to GST (residues 165 to 192) were incubated with rat brain homogenate, washed, and assayed for lipid kinase activities. The migration positions of the lipid standards are indicated. The results shown are representative of three experiments.

FIG. 5

The PtdInsP 5-kinase and DGK interact with Rac as a complex. GST-Rac bound to GSH-agarose beads was incubated with rat brain homogenate. The beads were washed and eluted with a NaCl gradient. (A) The fractions containing PtdInsP 5-kinase and DGK activities were pooled, diluted, and loaded onto a heparin-Sepharose column. The column was eluted with a 0.25 to 0.8 M NaCl gradient, and the fractions were assayed for PtdInsP 5-kinase (circles) and DGK (squares) activities. (B) The active fractions were pooled, desalted on a Sephadex G-25 column, and applied to a Mono-Q column. The Mono-Q column was eluted with a 0 to 1 M linear NaCl gradient, and the fractions were assayed for PtdInsP 5-kinase (squares) and DGK (circles) activities. The results are representative of four experiments. The salt concentrations were monitored with a conductivity meter, and the protein concentration was determined by monitoring absorbance at 280 nm.

FIG. 6

RhoGDI associates with both PtdInsP 5-kinase and DGK. (A) GST and GST-RhoGDI bound to GSH-agarose beads were incubated with rat brain homogenate. The beads were washed and assayed simultaneously for DGK and PtdInsP 5-kinase activities. (B) RhoGDI was immunoprecipitated from rat brain homogenate, washed, and assayed for DGK and PtdInsP 5-kinase activities. An immunoprecipitation using nonimmune (NI) serum was done as a negative control. The migration positions of the lipid standards in both experiments are indicated. The data are representative of five experiments.

FIG. 7

Rac mediates the association between RhoGDI and the lipid kinases. Increasing concentrations of the Rac C-terminal peptide (Rac 166-188) were preincubated with rat brain homogenate for 30 min. GST-RhoGDI bound to GSH-agarose beads was added to the homogenate and incubated for an additional hour. The beads were then washed and assayed for DGK (A) and PtdInsP 5-kinase (B) activities. A control peptide corresponding to residues 130 to 148 of Rac (Rac 130-148) was also used in the experiment at 1 mM. The activity of the lipid kinases associated with GST-RhoGDI in the absence of peptide was taken as 100%. Data shown are the mean ± standard error of the mean of three experiments. (C) GST-RhoGDI bound to GSH-agarose was incubated with SF9 cell lysate expressing Rac1, washed, and then treated with or without 1 mM Rac peptides. After incubation with peptides, the supernatant was collected from each sample (S). The RhoGDI beads were washed, and then the beads (B) and supernatant from each sample were analyzed by Western blotting using Rac1 antiserum. The results shown are representative of three experiments.

FIG. 8

Effects of lipids on the interaction between Rac and the PtdInsP 5-kinase and DGK. GST-RacV12 expressed in COS 7 cells was purified with GSH-agarose beads, washed, preincubated with 25 μM lipid, and then incubated with rat brain homogenate. Following the incubation, the beads were washed and assayed for DGK and PtdInsP 5-kinase (PIPK) activities. The data shown are the mean ± standard error of the mean of three experiments. PC, phosphatidylcholine.

FIG. 9

Model for the function of the Rac-associated lipid kinase complex. PtdInsP (PIP) 5-kinase, DGK, and Rac may exist as a preformed complex bound to RhoGDI. Upon stimulation, the complex may be shuttled to the membrane, where the DGK could synthesize PA, which would stimulate PI-4,5-P₂ production. These phospholipid products may mediate dissociation of the Rac-RhoGDI complex and/or stimulate nucleotide exchange. Newly synthesized lipids could also bind to actin regulatory proteins and induce actin uncapping and new actin polymerization as well as target other Rac signaling pathways such as the NADPH oxidase. GEFs, guanine nucleotide exchange factors.