Wrch-1, a novel member of the Rho gene family that is regulated by Wnt-1 - PubMed (original) (raw)

Wrch-1, a novel member of the Rho gene family that is regulated by Wnt-1

W Tao et al. Genes Dev. 2001.

Abstract

We report the isolation and cloning of the Wrch-1 (Wnt-1 responsive Cdc42 homolog) cDNA. Wrch-1 is a novel gene whose mRNA level increases in response to Wnt-1 signaling in Wnt-1 transformed cells, Wnt-1 transgene induced mouse mammary tumors, and Wnt-1 retrovirus infected cells. Wrch-1 encodes a homolog of the Rho family of GTPases. It shares 57% amino acid sequence identity with Cdc42, but possesses a unique N-terminal domain that contains several putative PXXP SH3-binding motifs. Like Cdc42, Wrch-1 can activate PAK-1 and JNK-1, and induce filopodium formation and stress fiber dissolution. Active Wrch-1 stimulates quiescent cells to reenter the cell cycle. Moreover, overexpression of Wrch-1 phenocopies Wnt-1 in morphological transformation of mouse mammary epithelial cells. Taken together, Wrch-1 could mediate the effects of Wnt-1 signaling in the regulation of cell morphology, cytoskeletal organization, and cell proliferation.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Wnt-1 signaling induces Wrch-1, but not Cdc-42. (A) Northern blot analysis of Wrch-1 expression in cells expressing Wnt-1, 4145, or 4145TV was performed using a 393 bp mouse Wrch-1-specific probe (containing codons for amino acids 81–211). Blots were rehybridized with a mouse GAPDH probe for loading control. Fold induction of Wrch-1 over the vector control was represented after normalizing to GAPDH levels. (B) C57MG cells were cocultured with either the parental QT6 cells or the QT6/Wnt-1 cells. Total RNA was extracted from the coculture at indicated time points, and Northern blotting was performed using the mouse Wrch-1 probe described above. Levels of GAPDH as a loading control are shown in C. (C) Cdc42 mRNA levels in the coculture were determined by reprobing the blots described in B with a Cdc42 cDNA probe (containing codons for amino acids 1–103).

Figure 2

Figure 2

Expression of Wrch-1 and Wnt-1 in Wnt-1 transgenic mouse mammary tumors, but not in normal, wild-type mouse mammary glands. Total RNA was extracted from wild-type mouse mammary glands (MG) and two independent MMTV-Wnt-1 transgenic mouse mammary tumors. Levels of the Wrch-1 transcript were assessed by Northern blot analysis. The blots were reprobed with the full-length mouse Wnt-1 cDNA (2.2 kb) to determine levels of the Wnt-1 mRNA.

Figure 3

Figure 3

Amino acid sequences and domain structure of human and mouse Wrch-1. The deduced amino acid sequences of human and mouse Wrch-1 (hWrch-1 and mWrch-1) were aligned with those of human Cdc42, Rac-1, and RhoA. Identity across these proteins is denoted by black boxes. Nucleotide binding domains I–IV are indicated above the sequences. The proline residues in the unique N-terminal domain of Wrch-1, that are predicted to form PXXP SH3 binding motifs, are indicated by asterisks. The conserved cysteine residues at the C-termini of these proteins that serve as the prenylation site are indicated.

Figure 4

Figure 4

Expression of the Wrch-1 mRNA in adult human tissues and mouse embryos. (A) Poly(A)+ RNA blots containing 2 μg of poly(A)+ RNA per lane from multiple human tissues were hybridized with a human Wrch-1 cDNA probe (containing codons for amino acids 81–207). The same filter was reprobed with human β-actin as a loading control. (B) Expression of the Wrch-1 mRNA in mouse embryos. In situ hybridization was performed on serial sections from various developmental stages using either a mouse Wrch-1 antisense or sense probe. Sections were also stained with hematoxylin/eosin (H&E) to visualize the tissue. Representative images are shown. Panels A, D, and G show Wrch-1 expression in E12 spinal cord, E15 dorsal root ganglion of the spinal cord, and E15 trigeminal ganglion, respectively. Sites of Wrch-1 expression are indicated by arrows.

Figure 4

Figure 4

Expression of the Wrch-1 mRNA in adult human tissues and mouse embryos. (A) Poly(A)+ RNA blots containing 2 μg of poly(A)+ RNA per lane from multiple human tissues were hybridized with a human Wrch-1 cDNA probe (containing codons for amino acids 81–207). The same filter was reprobed with human β-actin as a loading control. (B) Expression of the Wrch-1 mRNA in mouse embryos. In situ hybridization was performed on serial sections from various developmental stages using either a mouse Wrch-1 antisense or sense probe. Sections were also stained with hematoxylin/eosin (H&E) to visualize the tissue. Representative images are shown. Panels A, D, and G show Wrch-1 expression in E12 spinal cord, E15 dorsal root ganglion of the spinal cord, and E15 trigeminal ganglion, respectively. Sites of Wrch-1 expression are indicated by arrows.

Figure 5

Figure 5

Wrch-1 activates PAK-1 and JNK-1. (A) COS-7 cells were cotransfected with the HA-PAK-1 plasmid (3 μg) and either the empty vector or one of the plasmids (3μg) encoding Wrch-1, Q107L, and T63N, or a combination of plasmids encoding Wrch-1 and T63N (1.5 μg each). 36 h after transfection, HA-PAK1 was immunoprecipitated, and the kinase activity of PAK-1 was determined by assessing autophosphorylation of PAK-1 using the immunocomplex kinase assay. Radiolabeled products of the assay were separated by SDS-PAGE and visualized by autoradiography (upper panel). Phosphorylated PAK-1 (P-PAK-1) is indicated. Levels of PAK-1 in each sample were determined by Western blotting and shown to be similar (lower panel). (B) COS-7 cells were cotransfected with a HA-JNK-1 plasmid and the plasmid(s) as described above. 24 h later, cells were serum-starved for 16 h, and then HA-JNK-1 was immunoprecipitated. The activity of JNK-1 was determined by the immunocomplex kinase assay using GST-C-Jun as a substrate. Products of the kinase assay were separated by SDS-PAGE and revealed by autoradiography (upper panel). Phosphorylated C-Jun is indicated (P-C-Jun). Levels of HA-JNK-1 were assessed by Western blot analysis and shown to be similar (lower panel).

Figure 6

Figure 6

Wrch-1 induces filopodium formation and stress fiber dissolution in Swiss 3T3 cells. Swiss 3T3 cells were infected with the recombinant adenoviruses expressing β-galactosidase (β-gal), Q107L, or T63N. All of these viruses express GFP. Twelve hours after infection, cells were serum-starved for 24 h, and then fixed and stained for F-actin. The structure of F-actin and expression of GFP were detected by confocal immunofluorescent microscopy. A scale bar is shown in the image.

Figure 7

Figure 7

Wrch-1 stimulates quiescent Swiss 3T3 cells to reenter the cell cycle. Swiss 3T3 cells were rendered quiescent by culturing in the absence of serum for 48 h. They were then infected with recombinant adenoviruses expressing β-galactosidase, E2F-1, Q107L, or T63N. Mock-infected cells were included as a control. After culturing without serum for 48 h, cells were fixed, stained with P.I., and analyzed by flow cytometry. The histogram plots of cell number versus DNA content are presented. The percentages of cells in different phases of cell cycle based on their DNA contents are indicated.

Figure 8

Figure 8

Wrch-1 morphologically transforms C57MG cells. C57MG cells were infected with either a retrovirus expressing Wrch-1Q107L or Wrch-1T63N, or an empty retroviral vector. Cells stably expressing each of these proteins were selected for 4 d. The cell morphology was examined by microscopy.

Similar articles

Cited by

References

    1. Behrens J, von Kries JP, Kuhl M, Bruhn L, Wedlich D, Grosschedl R, Birchmeier W. Functional interaction of β-catenin with the transcription factor LEF-1. Nature. 1996;382:638–642. - PubMed
    1. Cadigan KM, Nusse R. Wnt signaling: A common theme in animal development. Genes & Dev. 1997;11:3286–3305. - PubMed
    1. Chan EF, Gat U, McNiff JM, Fuchs E. A common human skin tumour is caused by activating mutations in β-catenin. Nat Genet. 1999;21:410–413. - PubMed
    1. Clark EA, Golub TR, Lander ES, Hynes RO. Genomic analysis of metastasis reveals an essential role for RhoC. Nature. 2000;406:532–535. - PubMed
    1. Coso OA, Chiariello M, Yu JC, Teramoto H, Crespo P, Xu N, Miki T, Gutkind JS. The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell. 1995;81:1137–1146. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources