Tumor metastasis suppressor nm23H1 regulates Rac1 GTPase by interaction with Tiam1

Abstract

The putative tumor metastasis suppressor nm23H1 was originally identified in murine melanomas by subtraction cloning. It displays nucleoside diphosphate kinase activity and regulates cellular events, including growth and development. Recently nm23H1 has been reported to also act as a GTPase-activating protein of the Ras-related GTPase Rad. We attempted to determine whether nm23H1 also regulates Rho-family GTPases. Although we were unable to detect a direct association between nm23H1 and Rho-family GTPases, nm23H1 was shown to be associated with a Rac1-specific nucleotide exchange factor, Tiam1, by interaction with its amino-terminal region in extracts from the cells expressing exogenous Tiam1 and from native tissue. Overexpression of nm23H1 inhibited the Tiam1-induced production of GTP-bound Rac1 and activation of c-Jun kinase. On the other hand, forced overexpression of the wild type, but not the kinase-inactivated mutant of nm23H1, converted the GDP-bound forms of Rac1, Cdc42, and RhoA to their GTP-bound forms in vitro by its nucleoside diphosphate kinase activity, but nm23H1 alone apparently did not produce the GTP-bound form of these GTPases in vivo. These results suggest that nm23H1 negatively regulates Tiam1 and inhibits Rac1 activation in vivo. Moreover, adhesion-stimulated membrane ruffles of Rat1 fibroblasts were reduced by overexpression of nm23H1. Based on these observations, we concluded that we had identified a function of nm23H1 as a regulator of Rac1 and that it may be related to the effect of nm23H1 as a tumor metastasis suppressor.

Figure 1

Schematic representation of wild-type and truncated Tiam1 cDNA constructs used in this study. Proteins are depicted to scale. M, myristoylation signal; P, region rich in proline, glutamate, serine, and threonine; PHn and PHc, NH₂-terminal and COOH-terminal pleckstrin homology domains; DHR, Discus-large homology region; DH, Dbl homology domain; WT, wild type.

Figure 2

Nm23H1 specifically associates with the guanine nucleotide exchanging factor Tiam1. (a) 293T cells were transiently transfected with plasmid encoding myc or HA-tagged nm23H1 together with the Tiam1 constructs indicated above the lanes. Cells were lysed and immunoprecipitated (IP) with anti-myc (lanes 1–4) or anti-Tiam1-c (lanes 5–7), which reacts with epitopes located at the carboxyl terminus of Tiam1. Bound proteins were immunoblotted (IB) with the antibodies indicated. Expression of Tiam1 and nm23H1 in individual cell lysates (total lysate) was confirmed by immunoblotting (Bottom). (b) 293T cells were transiently cotransfected with flag-tagged wild type or ΔKpn nm23H1 together with Tiam1 constructs as indicated. Cell lysates were immunoprecipitated with anti-flag and immunoblotted with the antibodies indicated (lanes 1–3). HA-tagged wild-type or ΔKpn nm23H1 constructs were transiently transfected with flag-tagged wild-type or ΔKpn nm23H1 constructs (lanes 4–6). Immunoprecipitation and immunoblotting were performed as indicated. Expression of nm23H1 and Tiam1 is shown at the bottom. (c) 293T cells were transiently transfected with plasmid encoding myc-tagged nm23H1 together with Tiam1, HA-tagged βPIX, or Vav1 as indicated. Cell lysates were immunoprecipitated with anti-myc and immunoblotted sequentially with anti-Tiam1-c and anti-HA antibodies. (d) Whole brains of adult Institute for Cancer Research (ICR) mice were lysed and immunoprecipitated with anti-Tiam1-c antibody, normal rabbit Ig fraction, or peptide-neutralized anti-Tiam1-c antibody. Precipitants were subjected to immunoblot with the indicated antibodies. These experiments were performed at least three times.

Figure 3

Nm23H1 down-regulates Rac1 by association with Tiam1. (a–c) 293T cells transfected with plasmids indicated at the top were labeled with ³²P_i for 2 h. Lanes 4 and 5 (a): transfected with 0.5 μg and 0.2 μg, respectively, of plasmid encoding nm23H1 tagged with myc. The total amount of transfected plasmid DNA was equalized by adding empty vector pCS2+. The labeled guanine nucleotides that bound GST-tagged Rac1 were separated by TLC and visualized and quantitated with imaging analyzer (BAS 1000; Fuji). Comparable expression of Tiam1, GST-Rac1, and nm23H1 was confirmed by immunoblotting (Bottom). ori, origin of the TLC plate.

Figure 4

Nm23H1 down-regulates Rac1 by association with Tiam1. (a) 293T cells were transfected with the plasmids indicated at the top. A total of 0.25 μg (lanes 3 and 5) and 0.5 μg (lanes 4 and 6) of HA-tagged plasmids encoding wild-type or H118C mutant nm23H1 (HC), respectively, was transfected. Cells were lysed for affinity precipitation (AP) with immobilized GST-PBD as described in Materials and Methods. Precipitated endogenous GTP-bound Rac1 was detected by immunoblotting with anti-Rac1 antibody. Expression of Tiam1 and nm23H1 was detected by immunoblotting (Bottom). (b and c) 293T cells were transfected with the plasmids at the Top. GST-tagged JNK1 was purified from cell lysates and subjected to in vitro kinase assay with GST-c-jun as substrate. Comparable expression of Tiam1, GST-JNK1, and nm23H1 in individual lysates was confirmed by immunoblotting (Bottom). (c) The relative activity of JNK1 against the reference (Lane 2) was calculated by adjusting the densitometric value and standardization. Similar results were obtained in three independent experiments.

Figure 5

Nm23H1 transphosphorylates to Rac1 in vitro. (a) GDP-loaded GST-Rac1 on glutathione Sepharose (1 μg) was UV-irradiated as described in Materials and Methods, followed by incubation with 10 μCi [γ-³²P]ATP in the presence of 0.1 μg GST-nm23H1, GST-nm23H1 (H118C), or control GST, as indicated above the lanes. The reactants were washed and resolved by 12% SDS/PAGE. Precipitated GST-tagged Rac1 was detected by Coomassie stain (Bottom). (b) 293T cells were transfected with pEBG-Rac1 in combination with either mock vector, wild type (WT), H118C mutant of nm23H1 or wild-type Tiam1, as indicated above. Cells were labeled with ³²P_i for 2 h. The labeled guanine nucleotides bound to GST-tagged GTPases were separated by TLC. Expression of GST-tagged Rac1 in individual lysates was confirmed by immunoblotting (Bottom). Similar results were obtained in three independent experiments.

Figure 6

Overexpression of nm23H1 reduces the adhesion-stimulated membrane ruffles of Rat1 cells. (a–d) Rat1 cells were stained for Tiam1 (a and c) and for F-actin (b and d) on uncoated slides (a and b) or replated on fibronectin-coated slides for 15 min (c and d). (e–j and l–o) Rat1 cells stably expressing GFP (e, g, i, l, and n) or GFP-tagged nm23H1 (f, h, j, m, and o) were either untransfected (e–j) or were transfected with Tiam1 C1199 (myc tagged at the C terminus) (l–o), then replated on fibronectin-coated slides for 15 min and stained for F-actin (e, f, l, and m), endogenous Tiam1 (g and h), or Tiam1 C1199 (anti-myc) (n and o). The same cells are shown in a–d, g, i, and h, j. (i and j) Stable expression of GFP or GFP-nm23H1. Expression of endogenous or transfected Tiam1 and nm23H1 in Rat1 cells is shown by immunoblot (k and p). Three independent clones expressing GFP-tagged nm23H1 or GFP were examined, and similar results were obtained.