FRL, a novel formin-related protein, binds to Rac and regulates cell motility and survival of macrophages

Abstract

We have isolated a cDNA, frl (formin-related gene in leukocytes), a novel mammalian member of the formin gene family. The frl cDNA encodes a 160-kDa protein, FRL, that possesses FH1, FH2, and FH3 domains that are well conserved among other Formin-related proteins. An FRL protein is mainly localized in the cytosol and is highly expressed in spleen, lymph node, and bone marrow cells. Formin-related genes and proteins have been reported to play crucial roles in morphogenesis, cell polarity, and cytokinesis through interaction with Rho family small GTPases. FRL binds to Rac at its N-terminal region including the FH3 domain and associates with profilin at the FH1 domain. In a macrophage cell line, P388D1, overexpression of a truncated form of FRL containing only the FH3 domain (FH3-FRL) strongly inhibited cell adhesion to fibronectin and migration upon stimulation with a chemokine. Moreover, expression of the truncated FH3-FRL protein resulted in apoptotic cell death of P388D1 cells, suggesting that the truncated FH3-FRL protein may interfere with signals of FRL. Overexpression in the P388D1 cells of full-length FRL or of the truncated protein containing the FH3 and FH1 domains, with simultaneous expression of the truncated FH3-FRL protein, blocked apoptotic cell death and inhibition of cell adhesion and migration. These results suggest that FRL may play a role in the control of reorganization of the actin cytoskeleton in association with Rac and also in the regulation of the signal for cell survival.

FIG. 1

Structure of protein and cDNA of FRL and other formin-related proteins. (A) Deduced amino acid sequence of FRLα. An FH1 domain is indicated by a bold underline, and FH2 by a bold broken line. An FH3 domain is indicated by a thin underline. Coiled-coil regions are indicated by a thin broken line. The accession numbers of the nucleotide sequences in GenBank are AF215666 for frlα and AF006466 for frlβ. (B) Isoforms of frl cDNA. frlα encodes a protein of 1,094 amino acids, and frlβ encodes a protein of 1,064 amino acids. frlα differs from frlβ at its 5′ end and has an in-frame deletion of 99 bp at its 3′ terminus. (C) Schematic presentation of FRL and other formin-related proteins. The homology was calculated by the ratio of the number of identical amino acids to the number of corresponding amino acids of FRL and is expressed as a percentage in each indicated region.

FIG. 2

Expression of the frl gene and protein in various mouse tissues and cell lines. (A) Northern blot analysis of frl expression in various mouse tissues (left) and in a mouse embryo (right). (B) Northern blot analysis of frl in mouse hematopoietic cell lines. Total RNAs were probed with a common part of frlα and frlβ cDNAs. The amounts of loaded RNA were normalized by the level of β-actin mRNA. (C) Western blot analysis of various mouse tissues. Total tissue extracts (100 μg of protein/lane) from each organ were blotted with a rabbit anti-FRL antibody and a horseradish peroxidase-labeled anti-rabbit IgG antibody. (D) FRL localization in P388D1 cells. P388D1 cells were lysed and separated into three fractions: nuclei, cytosol (S100), and membrane (P100). Each sample was subjected to Western blotting with a rabbit anti-FRL antibody.

FIG. 3

Association of FRL and truncated FRL proteins with Rac1 in vitro and in vivo. (A) Precipitation of FRL by the GTPγS- or GDP-bound form of Rac1. P388D1 cell lysates were incubated with GST-RhoA, GST-Rac1, GST-Cdc42Hs, or GST, each of which was preloaded with either GTPγs or GDP. GST fusion proteins were precipitated by glutathione-Sepharose 4B beads, and the pellets were analyzed by immunoblotting with an anti-FRL antibody. (B) In vivo association of FRL with Rac1. P388D1 cell lysates were immobilized on protein A-agarose beads without (lane 1) or with (lane 2) anti-Rac1. The precipitates were analyzed by Western blotting with an anti-FRL antibody and an anti-Rac1 antibody. (C) Structure of truncated FRLs. FH3-FRL contains only the FH3 domain, FH1+FH3-FRL contains the FH1 and FH3 domains, and FH2-FRL contains only the FH2 domain. N1 consists of the N-terminal 80 amino acids, and N2 consists of the N-terminal 216 amino acids. (D) FRL associates with Rac1 at its N-terminal region containing the FH3 domain. COS7 cells transfected with each expression vector (FH3-pEGFP-C1, FH1+FH3-pEGFP-C1, FH2-pEGFP-C1, N1-pEGFP-C1, or N2-pEGFP-C1) were lysed, incubated with GST-Rac1, and precipitated by glutathione-Sepharose 4B. The pellets were analyzed by immunoblotting with an anti-GFP antibody.

FIG. 4

FRL associates with profilin. (A) Interaction of FRL and profilin in vitro. P388D1 cell lysates were incubated with GST-profilin I or with GST-profilin II and then precipitated by glutathione-Sepharose 4B beads, and the pellets were analyzed by immunoblotting with anti-FRL, anti-GST, and anti-actin antibodies. (B) In vivo association of FRL with profilin. P388D1 cell lysates were immobilized on protein A-agarose beads without (lane 1) or with (lane 2) an anti-FRL antibody. The precipitates were analyzed by Western blotting with anti-FRL and anti-profilin antibodies. (C) The FH1 domain of FRL binds to profilin. COS7 cells transfected with each expression vector (FH3-pEGFP-C1, FH1+FH3-pEGFP-C1, and FH2-pEGFP-C1) were lysed, incubated with GST-profilin I, and precipitated by glutathione-Sepharose 4B beads. The pellets were analyzed by immunoblotting with anti-GFP and anti-actin antibodies.

FIG. 5

The inducible P388D1 transformants express various forms of FRL; effects of overexpression of the truncated FH3-FRL protein in P388D1 cells. (A) Cell lysates from doxycycline-inducible transformants of P388D1 (FH3/P388D1, FH1+FH3/P388D1, FH2/P388D1, and FULL/P388D1) were collected after incubation with doxycycline (1.0 μg/ml) at 0, 24, 48, or 72 h and subjected to Western blotting with an anti-Xpress antibody. FRL was strongly expressed from 24 to 72 h after the addition of doxycycline. (B) Inhibition of cell spreading and of formation of lamellipodia in P388D1 cells by doxycycline-inducible overexpression of FH3-FRL. Mock-transformed cells (a and c) and FH3/P388D1 cells (b and d) pretreated with doxycycline were starved for 15 h and then incubated without (a and b) or with (c and d) SDF-1 (100 ng/ml). The cells expressing FH3-FRL protein did not spread well or show the formation of membrane ruffles (d), compared to mock-transformed cells (c). (C) Expression of the truncated FH3-FRL protein strongly inhibited P388D1 cell adherence to fibronectin. After 24 h of incubation with doxycycline, each transformant of P388D1 cells was examined for adhesion to fibronectin-coated wells. Error bars represent standard deviations in triplicate cultures. Similar results were obtained in three separate experiments (P < 0.01 in Student's t test for FH3. (D) Reduction of the chemotaxis of P388D1 cells upon SDF-1 by overexpression of FH3-FRL truncated protein. Inducible P388D1 transformants expressing full-length FRL or each truncated form of FRL and mock-transformed cells were examined for chemotactic activity in response to SDF-1 (100 ng/ml). Random fields of transwell membranes were counted for the number of migrating cells. Similar results were obtained in three separate experiments (P < 0.01 by Student's t test for FH3).

FIG. 6

Expression of the truncated FH3-FRL protein inhibited cell growth and induced apoptotic cell death in P388D1 cells. (A) [³H]thymidine incorporation of inducible P388D1 transformants at 72 h after the addition of doxycycline (1.0 μg/ml). Overexpression of the truncated FH3-FRL protein strongly inhibited proliferation of P388D1 cells (clone FH3-1 and clone FH3-2). Similar results were obtained in three separate experiments (P < 0.01 by Student's t test for FH3-1 and -2. (B) DNA fragmentation in FH3/P388D1 and mock-transformed cells at 0, 24, 48, and 72 h after the addition of doxycycline (1.0 μg/ml). FH3, FH3/P388D1 cells incubated with doxycycline. (C) FH3/P388D1 and mock-transformed cells were stained with Annexin-V at 0, 24, 48, and 72 h after the addition of doxycycline (1.0 μg/ml). Error bars represent standard deviations in triplicate samples. Similar results were obtained in three separate experiments.

FIG. 7

Overexpression of FH1+FH3-FRL or FULL-FRL protein restored the cell adhesion, migration, and proliferation abilities of doxycycline-treated FH3/P388D1 cells. FH3/P388D1 cells were transfected with FH1+FH3-pEGFP-C1 (FH1+FH3), FULL-pEGFP-C1 (FULL), and pEGFP-C1 vector only (control), and then cultured with doxycycline for 24 h. (A) The cells expressing FH1+FH3-FRL or FULL-FRL together with FH3-FRL truncated protein spread and formed ruffles upon stimulation with SDF-1. In the panels on the left, cells were stained with rhodamine-conjugated phalloidin for actin staining. Right panels show GFP-positive cells. Note that the cell without expression of transfected FRL-GFP fusion protein does not show spreading or the formation of membrane ruffles. (B) Adhesion assay. Expression of FH1+FH3 or FULL-FRL also rescued the failure of FH3/P388D1 cells to adhere to fibronectin. (C) Migration of FH3/P388D1 against SDF-1 after expression of FH1+FH3-FRL or FULL-FRL protein together with FH3-FRL truncated protein. (D) [³H]thymidine incorporation of FULL-FRL- or FH1+FH3-FRL-transfected FH3/P388D1 cells at 72 h after the addition of doxycycline (1.0 μg/ml). Proliferation of FH3/P388D1 cells was restored by expression of FH1+FH3-FRL or FULL-FRL protein, even in the presence of the truncated FH3-FRL induced by doxycycline.