Regulation of Rac and Cdc42 Pathways by Gi during Lysophosphatidic Acid-induced Cell Spreading

Hiroshi Ueda,Rika Morishita,Junji Yamauchi,Hiroshi Itoh,Kanefusa Kato,Tomiko Asano

doi:10.1074/jbc.m007541200

Hiroshi Ueda, Rika Morishita + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m007541200

Copy DOI

Abstract

The pertussis toxin-sensitive G protein, G(i), has been implicated in lysophosphatidic acid-induced cell mitogenesis and migration, but the mechanisms remain to be detailed. In the present study, we found that pertussis toxin blocks lysophosphatidic acid-induced cell spreading of NIH 3T3 fibroblasts on fibronectin. This prevention of cell spreading was eliminated by the expression of constitutively active mutants of Rho family small GTP-binding proteins, Rac and Cdc42, but not by Rho. In addition, activation of the endogenous forms was suppressed by pertussis toxin, indicating that G(i)-induced cell spreading is mediated through the Rac and Cdc42 pathway. Transfection of constitutively active mutants of G alpha(i) and G alpha(11) and G beta gamma subunits enhanced spreading of pertussis toxin-treated cells. G beta(1) with G gamma(12), a major G gamma form in fibroblasts, was more effective for increasing cell spreading than G beta(1)gamma(2) or G beta(1) plus G gamma(12)S2A, a mutant in which Ser-2, a phosphorylation site for protein kinase C, is replaced with alanine. In addition, a protein kinase C inhibitor diminished G beta(1)gamma(12)-induced cell spreading, suggesting a role for phosphorylation of the protein. These findings indicate that both G alpha(i) and G beta gamma stimulate Rac and Cdc42 pathways with lysophosphatidic acid-induced cell spreading on fibronectin.

Highlights

The pertussis toxin-sensitive G protein, Gi, has been implicated in lysophosphatidic acid-induced cell mitogenesis and migration, but the mechanisms remain to be detailed
We found that pertussis toxin blocks lysophosphatidic acid-induced cell spreading of NIH 3T3 fibroblasts on fibronectin
To investigate the role of Gi signaling during cell spreading, serum-starved NIH 3T3 fibroblasts were incubated for 16 h in the absence or presence of pertussis toxin (PTX), replated on fibronectin-coated plastic dishes, exposed to 10 ␮M lysophosphatidic acid (LPA) for various times, and stained with rhodamine-phalloidin to label F-actin (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Materials—pCMV5-G␣i2Q205L, pCMV5-G␣11Q209L, pCMV5G␣sQ227L, pCMV5-G␣12Q229L, pCMV5-G␥2, pCMV5-G␥12, pCMV5G␥12S2A, pCMV5-G␤1, the pCMV5 carboxyl terminus of ␤-adrenergic receptor kinase 1 (␤ARKct), pCMV5-FLAG-RhoAT19N, pCMV5-FLAGRac1T17N, pCMV5-FLAG-Cdc42HsT17N, pCMV5-FLAG-RhoAG14V, pCMV5-FLAG-Rac1G12V, and pCMV5-FLAG-Cdc42HsG12V were constructed as detailed previously (14 –20). GST-Myc-CRIB fusion protein-bound beads were suspended in phosphate-buffered saline, and glycerol was added to a final concentration of 50% and stored at Ϫ20 °C. After replating on fibronectin-coated dishes, cells were washed with phosphate-buffered saline, incubated for 5 min at 0 °C in buffer B (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 2 mM MgCl2, 10% glycerol, 0.2 mM phenylmethylsulfonyl fluoride, and 2 ␮g/ml trypsin inhibitor) containing 1% Nonidet P-40, and centrifuged at 21,000 ϫ g for 5 min at 4 °C. Aliquots were taken from the supernatant to compare protein amounts For this purpose, incubation with bacterially produced GST-Myc-CRIB fusion proteins bound to glutathione-coupled Sepharose 4B beads at 4 °C for 60 min was followed by washing three times in an excess of buffer B containing 0.5% Nonidet P-40. Detection was with a chemiluminescence reagent (PerkinElmer Life Sciences) and densitometry analysis was performed using the LAS1000 system (Fujifilm)

RESULTS

Regulation of Cell Spreading by Gi

DISCUSSION