Research Article
Characterization of RSF-1, the Caenorhabditis elegans homolog of the Ras-association domain family protein 1

https://doi.org/10.1016/j.yexcr.2012.10.008Get rights and content

Abstract

Mammals have 10 RASSF proteins, which are characterized by the Ras-association (RA) domain. Among them, RASSF1 to RASSF6 have the Salvador/RASSF/Hippo (SARAH) domain and form the subclass C-terminal RASSF proteins. Drosophila genome has a single C-terminal RASSF, dRASSF. All these RASSF proteins are related to the tumor suppressive Hippo pathway, and are considered to function as tumor suppressors. Caenorhabditis elegans T24F1.3 encodes a protein with the RA and the SARAH domains. The amino acid sequences are 40% and 55% similar to those of RASSF1 in the RA and the SARAH domains, respectively. We have characterized T24F1.3 gene product and named it RSF-1 as RASSF1 homolog. RSF-1 is widely expressed in epithelial cells. About 14% rsf-1 mutants exhibit defects in embryonal morphogenesis and the actin disorganization. The combinatorial synthetic lethal analysis demonstrates that the lethality is enhanced to more than 80% in rsf-1 mutants with the WASP-family verprolin homologous protein complex-related gene depletions and corroborates the implication of RSF-1 in the regulation of actin cytoskeleton. In rsf-1 mutants, the structures of muscle actin are preserved, but the swimming ability is impaired. Although we could not detect the direct physical interaction of LET-60 with RSF-1, rsf-1 mutants suppress the multivulva phenotype of the active let-60 mutants, suggesting that rsf-1 genetically interacts with the Ras signaling.

Highlights

► We named Caenorhabditis elegans T24F1.3 rsf-1 as a putative RASSF1 homolog. ► RSF-1 is important for embryonal morphogenesis and the regulation of actin cytoskeleton. ► rsf-1 genetically interacts with let-60.

Introduction

Mammalian genome has 10 RASSF proteins, RASSF1 to RASSF10 [1], [2]. RASSF1 to RASSF6, which are classified as the C-terminal RASSF proteins, harbor the Ras-association (RA) domain in the middle region and the Salvador/RASSF/Hippo (SARAH) domain in the C-terminal region, while RASSF7 to RASSF10 have the RA domain in the N-terminal region and lack the SARAH domain. RASSF1 and RASSF5 (also called Nore1/RAPL) have a conserved phorbol ester/diacylglycerol-binding (C1) domain in the N-terminal region. These C-terminal RASSF proteins regulate apoptosis and cell cycle, and function as tumor suppressors. Drosophila genome has one C-terminal RASSF, dRASSF [3]. dRASSF interacts with a serine/threonine-kinase, Hippo, which is a core kinase of the tumor suppressive Hippo pathway. dRASSF inhibits the interaction between Hippo and Salvador, and antagonizes the Hippo pathway. This finding is apparently inconsistent with the tumor suppressive functions of mammalian RASSF proteins. Additional twists make a story more complicated. Hippo mutants that lack the SARAH domain exhibit the uncontrolled tissue overgrowth, and this phenotype is enhanced by loss of dRASSF, suggesting that dRASSF suppresses the tissue overgrowth in these mutants. In contrast, dRASSF loss-of-function has no effect on the overgrowth in the kinase-dead Hippo mutant bearing the intact SARAH domain. Moreover, Drosophila imaginal tissues poorly grow in Ras1 loss-of-function mutants, and this phenotype is rescued by loss of dRASSF, suggesting that dRASSF antagonizes Ras1. These findings rather support that dRASSF possesses a tumor suppressor function like mammalian RASSF proteins under some condition. Mammalian RASSF1A and RASSF5 interact with and inhibit Hippo homolog, mammalian Ste20-like kinase (MST) 1, but the coexpression of Ras enhances MST1 activity [4]. RASSF1A interacts with MST2 to release it from Raf1-mediated inhibition and enhances MST2 activity to mediate apoptosis through the Hippo pathway [5], [6]. RASSF2 and RASSF5 are also reported to activate MST kinases [4], [7], [8]. These findings are consistent with the tumor suppressive roles of these RASSF proteins. On the contrary, RASSF6 inhibits MST1/2 and induces apoptosis independently of the Hippo pathway, while MST kinases reciprocally inhibit RASSF6-mediated apoptosis [9]. We previously proposed the model that RASSF6 and MST1/2 form a complex under the basal condition to inhibit each other through the interaction via the SARAH domains and that upon the activation of the Hippo pathway, the complex is dissociated, so that the Hippo pathway and RASSF6-mediated apoptosis are simultaneously activated. This model, if it is true of dRASSF and Hippo, can explain why dRASSF suppresses tissue overgrowth in the Hippo mutants lacking the SARAH domain but not in the Hippo mutants with the intact SARAH domain.

Based on the sequence homology, Caenorhabditis elegans (C. elegans) T24F1.3 was regarded as a C-terminal RASSF homolog but it has not yet been extensively studied [10]. In this study, we studied the expression pattern of RSF-1 using a RSF-1::green fluorescent protein (GFP) reporter transgene and analyzed the phenotypes of rsf-1 deficiencies using RNAi and the loss-of-function mutation. We have shown that RSF-1 plays a role in the regulation of embryonal morphogenesis and of actin cytoskeleton. We have also demonstrated that rsf-1 genetically interacts with let-60.

Section snippets

Strains and nematode growth

Nematodes were grown on nematode growth media (NGM) plates streaked with Escherichia coli OP50-1 (a streptomycin-resistant derivative of OP50) at 20 °C as described [11]. Strains used in this study include: let-60(n1046), lin-15(n765); ihIs04[rsf-1::gfp, lin-15(+)], ihIs04; let-60(n1046), rsf-1(tm5002), rsf-1(tm5002); let-60(n1046) and N2 wild type. We outcrossed rsf-1(tm5002) six times with N2. The rsf-1(tm5002) allele was tracked and/or sequenced by PCR amplification of genomic sequence

RSF-1 is the C. elegans RASSF1 homolog

T24F1.3 gene has been registered as a putative homolog of RASSF1 in the C. elegans genome database (http://www.wormbase.org/) [10]. The BLASTP program picked up pig roundworm RASSF1 (e-value=2e−96), zebrafish RASSF1 (e-value=1.9e−44), human RASSF1 (e-value=4e−52), mouse RASSF1 (e-value=7.8e−50), and rat RASSF1 (e-value=3e−37) as proteins similar to the T24F1.3 gene product. The Pfam database [18] demonstrates that the T24F1.3 gene product contains a C1 domain and an RA domain (Fig. 1A). Given

Discussion

In this study, we have characterized T24F1.3 as C. elegans C-terminal RASSF gene and named it rsf-1. RSF-1 has a C1 domain and an RA domain. The presence of the C1 domain suggests that RSF-1 is similar to RASSF1 and RASSF5 than to other RASSF proteins. The C-terminal region of RSF-1 is significantly similar to the SARAH domains of RASSF proteins. We confirmed that RSF-1 interacts with CST-1 and CST-2 in vitro. RSF-1 induces the phosphorylation of CST-1 via the SARAH domain. These findings

Acknowledgments

We thank the National BioResource Project at Tokyo Women’s Medical University School of Medicine and the Caenorhabditis Genetic Center supported by NIH NCRR for the strains. We also thank A. Fire for plasmid vectors and J. Ahringer for the RNAi library. This work was supported by research grants from the Ministry of Education, Sports, Science, and Technology (17081008), Japan Society for the Promotion of Science (22790275, 22590267), Takeda Science Foundation, Suzuken Memorial Foundation, and

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