Phosphorylation of Rac1 T108 by extracellular signal-regulated kinase in response to epidermal growth factor: a novel mechanism to regulate Rac1 function

Abstract

Accumulating evidence has implicated Rho GTPases, including Rac1, in many aspects of cancer development. Recent findings suggest that phosphorylation might further contribute to the tight regulation of Rho GTPases. Interestingly, sequence analysis of Rac1 shows that Rac1 T108 within the (106)PNTP(109) motif is likely an extracellular signal-regulated kinase (ERK) phosphorylation site and that Rac1 also has an ERK docking site, (183)KKRKRKCLLL(192) (D site), at the C terminus. Indeed, we show here that both transfected and endogenous Rac1 interacts with ERK and that this interaction is mediated by its D site. Green fluorescent protein (GFP)-Rac1 is threonine (T) phosphorylated in response to epidermal growth factor (EGF), and EGF-induced Rac1 threonine phosphorylation is dependent on the activation of ERK. Moreover, mutant Rac1 with the mutation of T108 to alanine (A) is not threonine phosphorylated in response to EGF. In vitro ERK kinase assay further shows that pure active ERK phosphorylates purified Rac1 but not mutant Rac1 T108A. We also show that Rac1 T108 phosphorylation decreases Rac1 activity, partially due to inhibiting its interaction with phospholipase C-γ1 (PLC-γ1). T108 phosphorylation targets Rac1 to the nucleus, which isolates Rac1 from other guanine nucleotide exchange factors (GEFs) and hinders Rac1's role in cell migration. We conclude that Rac1 T108 is phosphorylated by ERK in response to EGF, which plays an important role in regulating Rac1.

Fig 1

Rac1 sequences/motifs and the interaction between Rac1 and ERK. (A) Human Rac1 sequences and various motifs related to this research. (B) Co-IP of ERK and p-ERK with Rac1. COS-7 cells were transfected with GFP-Rac1. Following EGF stimulation for the indicated times, GFP-Rac1 was immunoprecipitated with antibody to GFP; the coimmunoprecipitated ERK and p-ERK were revealed by immunoblotting with antibodies to ERK and p-ERK. Con, immunoprecipitation with normal IgG was used as a control. (C) EGF-stimulated phosphorylation of ERK. COS-7 cells were stimulated with EGF for the indicated times, and the phosphorylation of ERK was determined by immunoblotting with antibody to p-ERK. (D) Interaction between endogenous Rac1 and ERK in COS-7, MCF-7, and MDA-MB-231 cells. Endogenous Rac1 was immunoprecipitated by anti-Rac1 antibody, and the co-IP of endogenous ERK was determined by immunoblotting with antibodies to ERK. (E) Interaction between ERK and GST-fused Rac1. Lysates of the COS-7 cells with or without EGF stimulation were incubated with GST-Rac1 attached to glutathione-agarose beads. The agarose beads were then separated and subjected to immunoblotting analysis with an antibody against ERK. GST fusion protein loading was verified by amido black staining of the membrane. (F) The effects of GTP loading on the interaction between Rac1 and ERK. GTP-γS was added to COS-7 cell lysate during incubation with GST-Rac1, and the interaction between ERK and GST-Rac1 was then examined as described in panel E.

Fig 2

The effects of Rac1 D site on the interaction between Rac1 and ERK. (A) For GST pulldown assays, glutathione-agarose bead-conjugated GST-Rac1 or mutant GST-Rac1ΔD was incubated with lysate of COS-7 cells with or without EGF stimulation. The agarose beads were then separated and subjected to immunoblotting analysis with an antibody against ERK. GST fusion protein loading was verified by amido black stain of the membrane. (B) For IP experiments, COS-7 cells were transfected with either GFP-Rac1 or GFP-Rac1ΔD. With or without EGF stimulation, GFP-Rac1 and GFP-Rac1ΔD were immunoprecipitated by rabbit anti-GFP antibody. The coimmunoprecipitated ERK was examined by rabbit anti-ERK antibody.

Fig 3

EGF-induced phosphorylation of Rac1 at T108. (A) EGF-induced threonine phosphorylation of Rac1 by ERK. COS-7 cells were transfected with GFP-Rac1 and stimulated with EGF (50 ng/ml) for the indicated times with or without preincubation with U0126 (5 μM) for 30 min. GFP-Rac1 was immunoprecipitated with rabbit anti-GFP antibody, and the threonine phosphorylation of GFP-Rac1 was examined by antibody to phosphothreonine (pT). The input GFP, p-ERK, and ERK were determined by immunoblotting using whole-cell lysate. (B) The effects of T108 mutation of EGF-induced threonine phosphorylation of Rac1. Mutant GFP-Rac1 T108A was expressed in COS-7 cells. Following EGF stimulation, proteins containing pPXTP motif were immunoprecipitated with antibody to pPXTP; the immunoprecipitates were then subjected to immunoblotting with antibody to GFP to reveal the threonine phosphorylation of GFP-Rac1 and GFP-Rac1 T108A.

Fig 4

Phosphorylation of purified Rac1 by purified active ERK in vitro. (A) The phosphorylation of Rac1 by purified active ERK was performed with an in vitro ERK kinase assay kit in the presence of [γ-³²P]ATP. His-Rac1 (5 μg) or His-Elk1 (5 μg) was incubated with 0.5 μg of purified active ERK1. The kinase reaction mixture was resolved by SDS-PAGE and transferred to polyvinylidene difluoride membrane. The autoradiography of ³²P was detected on an X-ray film. Elk1 was used as a positive-control substrate for ERK. (B) The phosphorylation of GST-Rac1 and mutant GST-Rac1 T108A by purified ERK. The phosphorylation of GST-Rac1 (2 μg) and GST-Rac1 T108A (2 μg) by purified active ERK was performed as described in panel A. GST was used as a negative control. (C) Quantification of the data from three independent experiments as described in panel B. The intensities of the bands of ³²P were normalized against the intensity of the GST loading. The error bar is standard error. **, P < 0.01; *, P < 0.05.

Fig 5

The effects of Rac1 and ERK phosphorylation on the interaction between ERK and Rac1. (A) COS-7 cells were transfected with GFP-Rac1 and GFP-Rac1 T108A and stimulated with EGF for the indicated times. GFP-Rac1 and GFP-Rac1 T108A were immunoprecipitated with mouse anti-GFP antibody. The coimmunoprecipitated ERK and p-ERK were determined by immunoblotting with antibody to ERK and p-ERK. (B) COS-7 cells were transfected with GFP-Rac1 and treated with EGF (50 ng/ml) for 15 min with or without preincubation with U0126 (5 μM) for 30 min. The cell lysates were incubated with GST-Rac1 or GST-Rac1 T108A bound to glutathione-agarose beads. Bound ERK and p-ERK were analyzed by immunoblotting with antibodies to ERK and p-ERK. GST fusion protein loading was verified by amido black staining of the membrane.

Fig 6

The effects of T108 phosphorylation on the activity of Rac1. (A) COS-7 cells were transfected with GFP-Rac1, GFP-Rac1 T108A, GFP-Rac1 T108E, GFP-Rac1ΔD, GFP-Rac1 Q61L, or GFP-Rac1 T17N. Lysates were incubated with GST fusion Rac-binding domain of PAK (GST-PAK). The active Rac1 that binds to GST-PAK was determined by immunoblotting with antibody to GFP.

Fig 7

The effects of Rac1 T108 phosphorylation on Rac1 interaction with PLC-γ1. (A) COS-7 cells were transfected with GFP, GFP-Rac1, GFP-Rac1 T108A, or GFP-Rac1 T108E. With or without EGF stimulation, cell lysates were incubated with a GST fusion PLC-γ1 SH3 domain attached to glutathione-agarose beads. The binding of wild-type and mutant Rac1 to GST-PLC-γ1 SH3 was examined by immunoblotting of the isolated beads with antibody to GFP. GST protein loading was verified by amido black staining of the membrane. (B) COS-7 cells lysates were incubated with GST-Rac1, GST-Rac1 T108A, or GST-Rac1 T108E attached to glutathione-agarose beads. The agarose beads were then separated and subjected to immunoblotting analysis with an antibody against PLC-γ1. GST protein loading was verified by amido black staining of the membrane.

Fig 8

The effects of Rac1 T108 phosphorylation on Rac1 nuclear localization. COS-7 cells were transfected with GFP-Rac1, GFP-Rac1 T108A, and GFP-Rac1 T108E. Following EGF (50 ng/ml) stimulation, the subcellular localization of wild-type and mutant Rac1 was examined by fluorescence microscopy (A and B) and subcellular fractionation (C and D). (A) Fluorescence images show the subcellular localization of Rac1 and the mutants with or without EGF stimulation. Scale bar, 10 μm. (B) Quantitation of the data from panel A. Each value is the mean of at least three experiments with at least 20 transfected cells counted for each experiment. The error bar is standard error. *, P < 0.05; **, P < 0.01; ***, P < 0.001. WT, wild type. (C) Subcellular fractionation. The transfected COS-7 cells were homogenized, and the cell homogenates were separated into nuclear and nonnuclear fractions as described in Materials and Methods. Equal cell equivalents of each fraction were analyzed by Western blotting. Nu, nuclear fraction; Non, nonnuclear fraction. (D) Quantitation of the data from panel C. Each value is the mean of at least three experiments. The error bar is standard error.

Fig 9

Subcellular distribution of endogenous Rac1 after EGF treatment. (A) COS-7 cells were separated into nuclear, total membrane, and cytosolic fractions as described in Materials and Methods. One-third of the nuclear fraction, one-half of the membrane fraction, and 3% of the cytosolic fraction were analyzed by immunoblotting using Rac1-specific antibody with no cross-reactivity with Rac2, Rac3, and Cdc42. (B) Percentage of Rac1 in each fraction of the cells. The error bar is standard error of the mean. *, P < 0.05. Each value is the mean of at least three experiments.

Fig 10

Effect of Rac1 T108 phosphorylation on cell migration as measured by wound-healing assay. (A) After transfection with GFP vector, GFP-Rac1, GFP-Rac1 T108A, and GFP-Rac1 T108E, confluent monolayers of serum-starved COS-7 cells were scratched and either not treated or treated with EGF (50 ng/ml) for the indicated times. Cell migration was examined by wound-healing assay as described in Materials and Methods. (B) Quantitation of the data from panel A. *, P < 0.05 compared with the cells transfected with GFP.