Krp1, a novel kelch related protein that is involved in pseudopod elongation in transformed cells

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Abstract

We have previously shown that the transcription factor AP-1 regulates the expression of genes which allow neoplastically transformed rat fibroblasts to become invasive. Searches for further AP-1 target genes led to the identification of a gene encoding a novel rat kelch family member, named kelch related protein 1 (Krp1). Kelch family members are characterized by a series of repeats at their carboxyl terminus and a BTB/POZ domain near their amino terminus. Rat Krp1 has a primarily cytoplasmic localization, and a small fraction appears to accumulate and co-localize with F-actin at membrane ruffle-like structures in the tips of pseudopodia. Overexpression of Krp1 in transformed rat fibroblasts led to the formation of dramatically elongated pseudopodia, while expression of truncated Krp1 polypeptides resulted in a reduction in the length of pseudopodia. We propose that the transformation-specific expression of Krp1 is required for pseudopod elongation, which are structures that are required for cell motility and invasion.

Introduction

One of the major issues of cancer progression is the complex process that results in a benign tumour becoming malignant. The final change in tumours during this process is that they acquire the ability to invade, which allows them to penetrate the basement membrane and migrate into neighbouring tissues. Such processes require alterations in the following: cell–cell interactions; cell–extracellular matrix (ECM) adhesion; cytoskeleton; and cell motility (reviewed in Liotta et al., 1991a). We have recently proposed that the transcription factor AP-1 in v-fos transformed 208F rat fibroblasts (FBR cells) co-ordinates the changes in gene expression that allow the cells to become invasive (Hennigan et al., 1994; Lamb et al., 1997a,b).

V-fos encodes the transforming agent of the FBR murine sarcoma virus, and forms part of the transcription factor AP-1 (Finkel et al., 1975; van Beveren et al., 1984; Curran and Verma, 1984; Curran and Franza, 1988). AP-1 is a heterodimeric protein composed of various combinations of the Jun and Fos family proteins, and this flexibility permits transactivation or transrepression of gene expression (Bushel et al., 1995; De Cesare et al., 1995; reviewed in Karin et al., 1997). Upon transformation of 208F cells with v-fos, the cells show extensive reorganization in their cytoskeleton and become invasive (Hennigan et al., 1994; Lamb et al., 1997a). These cytoskeletal rearrangments result in a striking change in the morphology of the cells: they change from being flat with F-actin stress fibres to being bipolar with long pseudopodia, which are lined with cortical F-actin cables that have dynamic actin-rich membrane ruffle-like structures at their tips. Pseudopodia have previously been shown to be implicated in motility and invasion of tumour cells, highlighting the importance of cytoskeletal reorganization in invasion (Guirguis et al., 1987; Hay et al., 1989; Lamb et al., 1997b; Liotta et al., 1991b; Nabi et al., 1999).

We have previously shown that AP-1 is essential for invasion of FBR cells and 208F cells treated with EGF (Hennigan et al., 1994; Lamb et al., 1997a). This was achieved using antisense oligonucleotides to c-jun, and the expression of a c-Jun dominant negative mutant (TAM-67) to inhibit invasion. Studies in c-fos null mice showed that Fos was needed for premalignant papillomas to progress to malignant skin carcinomas, when subjected to chemical carcinogens or an activated ras oncogene (Saez et al., 1995). Transformation of fibroblasts by oncogenes, such as raf and ras, have also been shown to be dependent on AP-1 activity (Johnson et al., 1996; Lloyd et al., 1991; Rapp et al., 1994; Suzuki et al., 1994). The role of AP-1 in transformation is not limited to fibroblasts, since mouse and human squamous cell carcinomas expressing the dominant negative Jun mutant have reduced tumourigenicity and in vitro invasiveness (Domann et al., 1994; Dong et al., 1997; Malliri et al., 1998).

Studies of AP-1 target genes in oncogenically transformed cells have also highlighted the role of AP-1 in invasion. Examples of proteins expressed are type 1 collagenase and stromelysin 1, both of which are proteases that are involved in the degradation of the ECM (Kerr et al., 1988; Schonthal et al., 1988). In order to identify more AP-1 regulated genes that are needed for FBR invasion, we have used differential screening and subtractive suppression hybridization (Diatchenko et al., 1996; Hennigan et al., 1994; Johnston et al., 1999, unpublished). These searches have revealed genes which encode proteins that have previously been implicated in invasion, as well as numerous novel genes that may play a role in transformation. An example of the genes whose expression are up-regulated is that encoding the cell surface hyaluronan receptor CD44, which is localized at the tips of pseudopodia in v-fos and EGF transformed 208F cells (Lamb et al., 1997a). CD44 antisense oligonucleotides caused a reduction in invasiveness in both of these cells. The expression of ezrin, which is a member of the ezrin/radixin/moesin (ERM) family and is thought to link CD44 to the cell’s cytoskeleton, was also shown to be upregulated (Lamb et al., 1997b; Jooss and Muller, 1995; Tsukita et al., 1994). Ezrin is located at the tips of pseudopodia in FBR cells and EGF-treated 208F cells, and removal of it from pseudopodia using the micro-CALI technique resulted in retraction of pseudopodia, suggesting that ezrin has a role in pseudopod extension (Lamb et al., 1997b).

Here we present data on a novel protein, named Kelch-related protein 1 (Krp1), which was isolated due to its upregulation upon transformation of 208F cells with v-fos and v-ras. We show that Krp1 is a member of a family of proteins that is defined by a carboxyl terminal series of approximately 50 amino acid repeats (the kelch repeats), and a protein–protein interaction domain, named the BTB/POZ domain, near its amino terminus (Xue and Cooley, 1993; Hernandez et al., 1997; Kim et al., 1998). Members of this family have been shown to be involved in cytoskeleton organization and function (Xue and Cooley, 1993; Hernandez et al., 1997; Kim et al., 1998; Robinson and Cooley, 1997). We report that rat Krp1 is cytoplasmically localized in FBR cells, and accumulates at the tips of pseudopodia, where it co-localizes with F-actin. In addition, we show through overexpression studies of the entire rat Krp1 protein in FBR and Ras cells, and the BTB/POZ domain or the Kelch repeats in FBR cells, that Krp1 is required for pseudopod elongation.

Results

Isolation of a cDNA which is upregulated in 208F cells transformed with v-fos and v-ras

A cDNA library representing the mRNAs which are upregulated in FBR cells compared to 208F cells was prepared using the suppression subtractive hybridization method (Diatchenko et al., 1996), and 500 random clones were sequenced (Johnston et al., 1999, unpublished). The open reading frame (ORF) from one 400 bp clone, designated krp1, showed 31% amino acid sequence identity to a Drosophila kelch protein, which is involved in crosslinking actin filaments in the ring canals during development (Xue and Cooley, 1993; Robinson and Cooley, 1997). As transformation by v-fos results in reorganization of the cytoskeleton, we predicted that such a protein may play a role in this remodelling, and the cDNA clone was chosen for further investigation.

To determine the expression pattern of krp1, Northern blots were prepared with total RNA isolated from the following cell lines: 208F, FBR, 208F transformed with ki-ras (Ras), and FBR expressing a dominant negative c-Jun mutant (FBR-TAM67). Upon hybridization with krp1 an RNA species of approximately 2.4 kbp was detected in FBR cells but not in 208F cells, indicating that it is upregulated upon v-fos transformation (Figure 1a). In addition, its expression was reduced in FBR-TAM67 cells. Since TAM67 is a dominant negative Jun mutant and blocks AP-1 function, this again confirms that krp1 expression is AP-1 dependent. A longer exposure of the blot revealed that krp1 was also upregulated in the Ras cells, although to a lesser extent than in the FBR cells (Figure 1b). These data illustrate that increased krp1 expression correlates with the transformation of 208F cells, and its expression is dependent on AP-1.

Figure 1
figure1

krp1 upregulation in FBR and Ras cells and expression in rat tissue. Northern blot analysis of total RNA isolated from FBR-TAM67, Ras, FBR and 208F cell lines. The blot was probed with the krp1 cDNA clone and left for a 3 h exposure (a) and a 3 day exposure (b). The blot was stripped and then probed with s7 as a loading control (c). A Rat poly A+ tissue blot from Clontech was probed with krp1 (d) and then stripped and probed with actin as a loading control (e)

To examine whether or not krp1 is expressed in a tissue-specific manner, we performed Northern blots of poly A+ RNA from various rat tissues, and found that krp1 is expressed primarily in muscle tissue (Figure 1d,e).

Isolation and characterization of rat and human krp1 cDNAs

To isolate a full-length cDNA clone of krp1, a library derived from FBR mRNA was screened. Six clones were isolated, and two of these, designated pkrp14 and pkrp16, contained an identical ORF of 1821 bp. The ORF encoded a putative polypeptide with a molecular weight of 68 212 Da, which we named Kelch-related protein 1 (Krp1; Figure 2a). Five non-identical repeats of approximately 50 amino acids are apparent at the carboxyl terminus, which are characteristic of proteins belonging to the kelch family (see Figure 2b). At the amino terminus of the putative polypeptide (amino acid residues 5–122) there is a domain that shows 42% sequence similarity to the BTB/POZ domain. This domain is thought to be involved in protein–protein interactions and is found in many of the kelch family of proteins, as well as in some DNA binding proteins such as the Drosophila Broad Complex (reviewed in Albagli et al., 1995). A schematic representation of some members of the kelch family, highlighting the positions of the kelch domains and BTB/POZ domains, is shown in Figure 2c.

Figure 2
figure2

Amino acid sequence of rat and human Krp1. (a) Shows the complete amino acid sequence of rat Krp1. Also shown are the amino acids which differ between rat and human Krp1. The BTB/POZ domain is underlined and the kelch repeats of Krp1 are shown in bold. (b) Alignment of the rat Krp1 kelch repeats. The bottom line shows the consensus sequence. White letters on the black background represent conserved residues, while white letters on a grey background represent similar amino acids. K1-5 represents the five kelch repeats. (c) Schematic representation of some of the kelch family members

To isolate the human homologue of krp1, a human skeletal muscle cDNA library was screened with the rat krp1 full-length cDNA. This library was used because rat krp1 is expressed primarily in muscle tissue (Figure 1d,e). Four clones were isolated, two of which were predicted to contain the complete ORF. The human gene encodes a putative polypeptide with a molecular weight of 68 151 Da, which shows 95% amino acid sequence identity to rat Krp1, confirming we have isolated a human gene which is homologous to rat Krp1 (Figure 2a). The amino acid sequence identity of 95% between rat and human Krp1 is higher than the 41% identity observed between rat Krp1 and another human kelch family member, NRP/B (Kim et al., 1998). These data suggest that within the kelch family there are sub-families which are more closely related.

Rat Krp1 co-localizes with F-actin at the tips of pseudopodia in FBR cells

To analyse the subcellular localization of endogenous Krp1 in FBR cells, polyclonal rabbit antiserum was raised to bacterially-expressed full-length rat Krp1 fused to a 6 Histidine epitope. A Western blot was performed with FBR, Ras and 208F cell lysates and was probed with the anti-Krp1 antiserum. A band of approximately 68 kDa was detected in the FBR and Ras cell lysates, corresponding to the predicted size of the Krp1 polypeptide, while nothing was detected in the 208F cell lysate (Figure 3a). These data, along with the Northern analysis (above), confirm that Krp1 expression is specifically upregulated upon transformation of 208F cells.

Figure 3
figure3

Krp1 localization in FBR, FBR-Tam67 and 208F cells with anti-Krp1 antiserum. (a) Western blot analysis of Ras, 208F and FBR cell lysates probed with anti-Krp1 antiserum. The blot was stripped and reprobed with anti-ERK2 antisera as a loading control. (b) Double immunofluorescence staining of FBR cells with Krp1 antiserum with a FITC-labelled anti-rabbit secondary antibody and tubulin antiserum with TRITC-labelled anti-mouse secondary antibody. Double immunofluoresence of FBR cells (c–e), 208F cells (f) and FBR-TAM67 cells (g) with Krp1 antiserum with a FITC-labelled anti-rabbit secondary antibody and TRITC-phalloidin for F-actin staining. (d) Shows tips of a FBR psuedopod with the same labelling, but at a higher magnification. (e,1) Shows immunofluoresence staining without pre-permeablization of the cells and in (e,2 and 3) with pre-permeablization of the cells in 0.02% Triton x100/PBS for 1 min. Actin is shown in red and Krp1 in green. The figure also shows the merged images where yellow represents where both Krp1 and F-actin co-localize. Arrows highlight the localization of Krp1 at the tips of pseudopodia. Bars=25 μm

Immunofluorescence experiments performed on FBR cells using anti-Krp1 antiserum revealed that Krp1 has a primarily cytoplasmic localization, and a small fraction accumulates at the tips of transformation-specific pseudopodia (Figure 3b–d). Cell fractionation experiments confirmed that Krp1 is primarily found in the cytoplasm, since the majority of it was present in the soluble fraction (data not shown). Examination of Krp1 expression in pseudopod tips at a higher magnification revealed that it was present in membrane ruffle-like structures that appeared to be forming a looping conformation (Figure 3d). Because it is known that some kelch family proteins interact with actin (Xue and Cooley, 1993; Hernandez et al., 1997; Soltysik-Espanola et al., 1999), and that the yeast Tea1 kelch family member localizes at the ends of microtubules (Mata and Nurse, 1997), we performed two double immunofluorescence experiments on FBR cells: anti-Krp1 antiserum and phalloidin to detect F-actin; anti-Krp1 and tubulin antisera (Figure 3b–d). Both tubulin and Krp1 were present in the cytoplasm, but while Krp1 extended into the loop-like structures at the tips of pseudopodia, tubulin stopped just short of these structures (Figure 3b). Krp1 and F-actin co-localized at the tips of pseudopodia, but not in the cytoplasm, where F-actin was found as long cables (Figure 3c,d). To confirm that Krp1 does not co-localize with F-actin cables in the cytoplasm, live cells were treated with triton X-100 to remove diffusable proteins before fixing for immunofluorescence (Figure 3e). This revealed that Krp1 remained at the tips of pseudopodia with F-actin, but did not associate with the F-actin cables. Double immunofluorescence with anti-Krp1 antiserum and phalloidin was also performed on 208F cells. Krp1 was not expressed in these cells (Figure 3f), confirming the Northern and Western data. The same experiment performed on the FBR-TAM67 cells, which have a reverent phenotype in that they have a flattened morphology, and showed loss of pseudopodia and a gain of stress fibres, indicated that Krp1 had a cytoplasmic localization (Figure 3g). It should also be noted that in FBR-TAM67 cells Krp1 does not co-localize with F-actin in the stress fibres or in the membrane ruffle structures (Figure 3g). These data confirm that Krp1 expression and localization is transformation-specific, and that the protein only co-localizes with F-actin at the membrane ruffle-like structures at the tips of pseudopodia.

Rat Krp1-myc localizes at the tips of pseudopodia in transformed 208F cells

To confirm the localization of Krp1 at the tips of pseudopodia, the full-length rat krp1 ORF was cloned into the mammalian expression vector pcDNA3.1 Myc-His, creating a translation fusion in which the 9 amino acid myc epitope was placed at the carboxyl terminus of Krp1 (Krp1-myc). The construct was then transfected into FBR cells and the distribution of Krp1-myc assessed 24 h later using immunofluorescence with an anti-myc monoclonal antibody. The subcellular localization of Krp1-myc appeared identical to that of endogenous Krp1, confirming that Krp1 is primarily cytoplasmic and that a small fraction localizes at the tips of pseudopodia in FBR cells (Figure 4a).

Figure 4
figure4

Localization of Krp1-myc in FBR, 208F, EGF treated 208F and Ras cells. Transfection of Krp1-myc into FBR (a), 208F (b), 208F treated with 100 ng/ml of EGF for 5 min, 2, 4 and 24 h (c) and Ras (d). Cells were viewed in (a) and (d) by immunofluorescence with myc monoclonal antibody followed by a TRITC-labelled anti-mouse secondary antibody, and in (b) and (c) by double immunofluorescence with monoclonal antibody to myc-epitope followed by a TRITC-labelled anti-mouse secondary antibody and FITC-phalloidin for F-actin. Arrows point to the accumulation of Krp1-myc at the tips of pseudopodia and at the leading edge of the cells. Size bars=25 μm

The Krp1-myc construct was next transfected into 208F cells to determine its effect and localization in untransformed cells. Krp1-myc was distributed throughout the cytoplasm, and did not appear to associate with the membrane (Figure 4b). Notably, Krp1-myc did not co-localize with the cytoplasmic F-actin, which form stress fibres in these cells. In addition, expression of krp1-myc did not appear to have any effect on the morphology of 208F cells. To test whether this localization was specific for v-fos transformed 208F cells, we next expressed Krp1-myc in 208F cells which were subsequently treated with EGF and in Ras cells. Treatment of 208F cells with 100 ng/ml of EGF results in membrane ruffling after 4 h, and removal of stress fibres and the formation of pseudopodia after 24 h (Kaplan and Ozanne, 1983). We determined the localization of Krp1-myc in 208F cells after 5 min, 2 h, 4 h and 24 h of EGF treatment. At both 5 min and 2 h of EGF treatment Krp1 remained cytoplasmic, while at 4 h Krp1 was found to localize at membrane ruffles (Figure 4c). After 24 h a small fraction was localized at the tips of pseudopodia (Figure 4c). It should be noted that not all the cells after 24 h had long pseudopodia, but were instead only partially transformed, as they had small pseudopodia with ruffling tips where Krp1 localized. Ras cells have a distinct morphology to FBR cells in that they have many short pseudopodia with actin-rich structures at their tips, while FBR cells have only two long pseudopodia. Despite this, Krp1-myc was still localized in the cytoplasm and accumulated at the tips of all pseudopodia (Figure 4d). These data, together with the localization of endogenous Krp1 in FBR and FBR-TAM67 cells, suggest that the localization of Krp1 in the tips of pseudopodia is transformation-specific.

Overexpression of rat Krp1-myc and dominant negative Krp1 mutants suggest that Krp1 has a role in pseudopod elongation

To determine whether overexpression of rat Krp1-myc in FBR and Ras cells had any effect on the morphology of the cells, transfections were performed and the phenotypes of Krp1-myc positive cells analysed. Thirty-six per cent of FBR cells and 25% of RAS cells showed elongated pseudopodia (Figure 5b–e). Elongated pseudopodia were particularly apparent in Ras cells, as they normally have many short pseudopodia. To verify that Krp1-myc overexpression was responsible for this phenotypic change, two Krp1 variants were cloned into the pcDNA3.1 Myc-His vector. The first construct contained amino acids 1–181, encompassing the BTB/POZ domain, and the second contained the kelch repeats (amino acids 335–606). To confirm that these constructs were expressing products of the expected sizes, they were transfected into COS-7 cells and Western blots were performed on the cell lysates. Bands of 68, 20 and 30 kDa were detected by an anti-myc monoclonal antibody, which were the expected sizes for the Krp1-myc, Krp1poz-myc and Krp1repeats-myc polypeptides respectively (Figure 5a). Transfection of these constructs into FBR and Ras cells revealed that elongated pseudopodia were seen more frequently in cells expressing the entire Krp1 protein, indicating that the whole protein is needed to exert this effect. In FBR cells transfected with Krp1poz-myc or Krp1 repeats-myc, 54 and 55% of the cells respectively displayed pseudopodia shortened by up to 80% (Figure 5f–h). A control construct expressing Beta-galactosidase fused to the myc epitope did not cause any significant lengthening or shortening of pseudopodia, indicating that these effects are Krp1 specific. It is likely that the truncated Krp1 polypeptides are acting as dominant negative mutants, which are interfering with wild type Krp1 function in pseudopod elongation.

Figure 5
figure5

Effects of overexpression of Krp1-myc and the variant Krp1 constructs in FBR and Ras cells. (a) The Krp1-myc, Krp1repeats-myc and Krp1poz-myc constructs were transfected into COS-7 and the cell lysates were used in a Western blot analysis. Blots were probed with anti-myc monoclonal antibody. (b) and (d) Illustrates elongated pseudopodia produced when Krp1-myc is overexpressed in both FBR and Ras cells respectively. Cells were visualized using anti-myc monoclonal antibody and secondary TRITC-labelled anti-mouse antibody. (c) and (e) Quantitative analysis of the average percentage of FBR and Ras cells respectively that showed elongated pseudopodia. The average length of pseudopodia in FBR cells was approximately 30 μm and in RAS cells 10 μm, anything more than two times the length of the average was classified as elongated. The experiment was performed on three separate occasions on FBR cells and two times on Ras cells, on each occasion the experiments were performed in duplicate. (f) and (g) Show the reduced pseudopodia size when FBR cells are transfected with either the Krp1poz-myc and Krp1repeats-myc. (h) Quantitative analysis of the average percentage of FBR cells showing a reduction in pseudopodia length. Shortened pseudopodia were classified as anything smaller than 15 μm. The experiment was repeated on three separate occasions, and was performed in duplicate each time. Bars=25 μm

Discussion

Through our investigation of genes which are upregulated as a consequence of v-fos transformation of rat fibroblasts, we identified a new member of the kelch family of proteins, which are characterized by a BTB/POZ domain at the amino terminus and a series of repeats at their carboxyl terminus. We have shown that the expression and co-localization of Krp1 with F-actin at membrane ruffle-like structures at the tips of pseudopodia is dependent upon transformation of rat fibroblasts. In addition, overexpression of full-length Krp1 results in dramatic elongation of extending pseudopodia, while expression of either the BTB/POZ domain or the carboxyl terminal repeats in isolation results in truncated pseudopodia. These data suggest that Krp1 has a role in pseudopod elongation.

Krp1 is a member of the kelch family of proteins, which have been found in many different species. It has become apparent that the kelch family members have many diverse functions. Some of them are thought to interact with actin through their kelch repeats. An example is Drosophila kelch (Xue and Cooley, 1993), which is found in the ring canals (actin-rich structures that link the nurse cells to the developing oocyte) where it is thought to stabilize actin filaments. Another protein, alpha-scruin of Limulus, which has kelch repeats present at both its amino and carboxyl termini but lacks a BTB/POZ domain, cross-links actin filaments within the acrosomal processes of sperm (Way et al., 1995a). Further kelch proteins are also thought to interact with actin: murine and human ENC1 (Hernandez et al., 1997, 1998); spe-26 of C. elegans (Varkey et al., 1995); and human Mayven (Soltysik-Espanola et al., 1999). Many other family members are thought not to interact with actin; beta-scruin of Limulus, for instance, is found in sperm acrosomal vesicles, which do not contain actin (Way et al., 1995b). In addition, Schizosaccharomyces pombe kelch family member Tea1 is found at the tips of growing cells and is thought to interact with the ends of microtubules rather than with actin (Mata and Nurse, 1997). Several other family member have been characterized, but it is not known what, if any, cytoskeletal components they interact with: Kel1p and Kel2p of Saccharamyces cerevisiae (Philips and Herskowitz, 1998); human NRP/B (Kim et al., 1998); and mouse Muskelin (Adams et al., 1998). It is apparent from the above that we can not assign a specific function to Krp1 based upon its sequence similarity to the kelch family, as different members appear to have diverse functions. It is conceivable, however, that within the whole kelch-family there are sub-families with the same or related functions, and it is therefore interesting to note the high level of sequence identity (95%) between rat and human Krp1.

The sub-cellular localization studies presented reveal that Krp1 co-localizes with F-actin in a dynamic membrane ruffle-like structure, which forms looping conformations at the tips of pseudopodia in FBR cells. Although Krp1 co-localizes with F-actin within this structure, it does not do so in the cytoplasm: here F-actin appears as large cables that run the length of the cell body, while Krp1 appears to be dispersed throughout the cytoplasm. In Ras cells Krp1-myc also only co-localized with F-actin at the tips of pseudopodia. It should also be noted that when Krp1-myc was expressed in 208F cells, and when we examined the low level of endogenous Krp1 that is present in FBR-TAM67 cells, no co-localization with F-actin containing stress fibres was detected. This result contradicts work presented by Hernandez et al., (1997) and Soltysik-Espanola et al. (1999), who reported that two other kelch family members, ENC1 and Mayven, interact with stress fibres containing F-actin. Together these data suggest that Krp1 only co-localizes with F-actin when it is in the looped membrane ruffle-like structure at the tips of pseudopodia. Krp1 may not directly interact with actin, but instead with unidentified proteins that are required for actin to adopt this conformation. It is interesting to note in this regard that we have been unable to show any direct biochemical interactions between actin and Krp1.

The presence of Krp1 in the cytoplasm raises the question of what, if any, function it has here. One possibility is that this simply represents a cytoplasmic pool of Krp1 that is constantly being moved to the dynamic processes at the tips of pseudopodia. Preliminary two-dimensional SDS–PAGE data suggest that Krp1 in FBR cells is phosphorylated (data not shown). Perhaps Krp1 is phosphorylated when present at the tips of pseudopodia, while cytosolic Krp1 is inactive and not phosphorylated.

The localization of Krp1 at the tips of pseudopodia in FBR cells, and the recruitment of Krp1-myc to this structure in 208F cells treated with EGF for 24 h and Ras cells, is reminiscent of the products of two other AP-1 regulated genes: CD44 and ezrin. Both of these proteins are up-regulated in FBR cells and EGF-treated 208F cells and appear to cluster at the tips of pseudopodia (Lamb et al., 1997a,b). This raises the question of whether these proteins might be interacting at the looped structure. It has been reported that ezrin links the cytoplasmic domain of CD44 to the actin cytoskeleton in baby hamster kidney cells and mouse L fibroblasts (Tsukita et al., 1994; Yonemura et al., 1998, 1999; Legg and Isacke, 1998), but we do not know if this interaction occurs in FBR cells. None of our data allow us to conclude that Krp1 interacts with these proteins, but the presence of all of them at pseudopod tips upon transformation of 208F cells supports the hypothesis that AP-1 regulates the expression of these genes, and that they are recruited to a structure that is likely to allow transformed cells to become invasive (Guirguis et al., 1987; Hay, 1989; Lamb et al., 1997b; Liotta et al., 1991b; Nabi et al., 1999). Further evidence for this proposal is seen when Krp1-myc, ezrin or CD44 were individually expressed in 208F cells, since they did not result in the formation of large pseudopodia (Lamb et al., 1997a,b). These data imply that AP-1 must regulate the expression of numerous genes which are all needed for the cells to become invasive. Further analysis of genes isolated from the subtractive suppression hybridization libraries representing genes up-regulated in v-fos transformed cells may give us a better understanding of the range of genes needed for invasion.

To gain further insight into the function of Krp1, we presented data from overexpression studies in FBR and Ras cells. Dramatically elongated pseudopodia were seen in both FBR and Ras transformed cells when they were subjected to overexpression of a Krp1-myc construct, suggesting that Krp1 may have a direct role in pseudopod elongation. This is supported by the result of overexpressing the BTB/POZ domain or kelch repeats in isolation, which resulted in shortening of the pseudopodia, presumably because these Krp1 variants are acting as dominant negative mutants which interfere with endogenous Krp1 function. A comparison between Krp1 and ezrin function can again be made here, since ablation of endogenous ezrin led to the loss of pseudopodia. We are not the first to draw a comparison between ezrin and kelch proteins, since Vega and Solomon (1997) reported the similarity between ezrin and the yeast kelch protein, Tea1. They reported that both proteins are localized at tips of cells, Tea1 being present at the tips of growing yeast cells and ezrin and other ERM proteins in growth cones, and that they both depend on the integrity of microtubules for localization. We have also shown that the positioning of Krp1 at the tips of pseudopodia is dependent on the integrity of the microtubules (data not shown). These data, together with the localization data, suggest that Krp1 may function with the ERM proteins at the tips of cells, but as stated above our data does not allow us to conclude that these proteins interact directly. What this does suggest, however, is that Krp1, ezrin and Tea1 are related in defining the structures and processes that occur at cell tips. Overexpression of other kelch family members (Tea1, Kel1p, Kel2p and NRP/B) in their respective cell types led to various changes in cell morphology (Mata and Nurse, 1997; Philips and Herskowitz, 1998; Kim et al., 1998). This suggests that the broad function of the kelch family proteins may be in creating and modulating various aspects of cell shape and structure.

Materials and methods

Cell lines

208F is a subclone of the Rat-1 fibroblast cell line originally obtained from K Quade. FBR cell lines were originally obtained from Tom Curran and are transformed non-producer 208Fs infected with FBR-MuSv. The Ras transformants in this study were generated by transfections of 208F with a ki-ras expressing construct. All cell lines were routinely passaged before confluence and maintained in Dulbecco’s modified Eagle medium (DMEM; Life Technologies) supplemented with 10% foetal calf serum (Advanced Protein Products Ltd) at 37°C in 5% CO2.

Isolation of rat and human krp1 cDNAs

A λZap11 FBR cDNA library was produced using the protocols described by Stratagene. Approximately 106 plaque forming units from the library were screened with the 32P-labelled cDNA clone krp1. After three rounds of screening six clones were isolated and excised from lambda phage in the form of the plasmid pBluescript, following the protocols described by Stratagene. The clones were sequenced on both strands using the ABI automatic sequencer 373A and 377. Human krp1 was isolated by screening approximately 106 plaque forming units from a λZap11 human skeletal muscle library (Stratagene), with 32P-labelled rat krp1 cDNA. Four positive clones were isolated and were characterized using the methods described above.

While this work was in progress, data base searches revealed that a number of muscle specific cDNAs have been identified (Sarcosin, AA192617, AA22114096, AA196024) which show high levels of sequence similarity to human krp1 (Hillier et al., 1996; Taylor et al., 1998). Analyses of these sequences revealed that none of them contain as long an open reading frame as the one predicted from clones isolated in this work, suggesting that they represent partial cDNAs.

Northern blot analysis

Total RNA was isolated using RNAzol B according to the manufacturer’s protocol (Biogenesis). For Northern blot analysis, 10 μg of total RNA was separated electrophoretically as described by Sambrook et al. (1989). The RNA was stained with ethidium bromide, photographed, and then transferred on to Hybond N (Amersham) as described by Sambrook et al. (1989). Membranes were UV cross-linked with a Stratalinker 1800 (Stratagene). The krp1 cDNA probe was 32P-labelled with a random priming kit from Pharmacia. Hybridization and stringency washes were performed as previously described (Sambrook et al., 1989).

Preparation of Anti-Krp1 antiserum

To prepare the His-Krp1 tagged recombinant protein, the complete open reading frame of krp1 was amplified by PCR with pkrp14 as a template using the following primers: 5k, 5’-GTC TCG AGG TAC CTA AGC AGC GG-3’ and the T7 primer of pBluescript. The PCR product was cloned into pCR-SCRIPT (Stratagene) then subcloned into the KpnI site of pTrcHisC (Invitrogen). Sequencing was performed to confirm that no errors had been introduced by the Pfu polymerase and to determine the correct orientation of the insert. Recombinant His-Krp1 was overexpressed in E. coli strain DH5α and affinity purified on a Ni column (Invitrogen). The purified protein was then used to raise polyclonal rabbit antisera to Krp1. To determine the specificity of the anti-Krp1 antiserum, Western blot analysis of the bacterially produced His-Krp1 was performed and was probed with this antibody. A single band of 68 kDa was detected which is the predicted size of the Krp1 polypeptide, confirming that the antibody recognized bacterially produced His-Krp1.

Western blot analysis

Cells were washed twice in PBS and then lysed in EBC buffer (50 mM Tris-HCL pH 8, 120 mM NaCl, 0.5% Nonidet P-40, 1 mM EDTA) containing 10 μg/ml aprotinin, leupeptin and 1 mM PMSF (Hernandez et al., 1997). Protein concentrations were measured with a commercial reagent (Bio-Rad) and 50 μg of proteins were electrophoresed on either a SDS-7.5% or 10% polyacrylamide gel using standard methods. Proteins were transferred to polyvinylidene difluoride membranes (Immunobilon P:Millipore) with a transblot wet blotting apparatus (Bio-Rad). Membranes were blocked in blocking buffer (PBS containing 0.1% Tween 20 and 5% nonfat milk) for 1 h and probed with the following antibodies at the given dilution in blocking buffer: anti-Krp1 rabbit polyclonal antiserum at a 1 in 2500; anti-myc (9E10; Invitrogen) 1 in 100 dilution; anti-Erk2 (Transduction Laboratories) 1 in 5000. Membranes were then rinsed three times in blocking buffer followed by a 1 h incubation with the secondary antibodies, either peroxidase-labelled goat anti-rabbit or peroxibase-labelled goat anti-mouse at a 1 in 5000 dilution in blocking buffer. Three final washes were performed in PBS containing 0.1% Tween 20 and the blots were visualized using ECL (Amersham).

Construction of Krp1 fusions

The Krp1-myc construct was derived by PCR amplification with pkrp14 as a template using the following primers: 5k, 5’-AGA AGC TTA TGG ATT CCC AGC GG-3’ and 3k, 5’-CCA AGC TTT AGT TTA GAC AG-3’. This PCR product was cut with HindIII and ligated into the HindIII site of expression vector pcDNA3.1/Myc-HisA (Invitrogen) creating Krp1-myc. The Krp1 variants were also generated by PCR using pkrp14 as a template. Krp1poz-myc was generated using the following primers: p5, 5’-TAT CTA GAA TGG ATT CCC AGC GGG AGC TTG-3’ and p3, 5’-ATT CTA GAA TGA CAG AGA TCA GCT CCT GTG GGG AC-3’. The Krp1poz PCR product was cut with XbaI and ligated into the XbaI site of pcDNA3.1/Myc-His B (Invitrogen). To create Krp1repeat-myc the following primers were used: 5’ repeat, 5’-TAT CTA GAA TGA ATC ATT CCA GTA TTG TTA CC-3’ and 3k, 5’-CCA AGC TTT AGT TTA GAC AG-3’. This PCR product was cut with XbaI and HindIII and was cloned into XbaI and HindIII site of pcDNA3.1/Myc-HisA. To check the orientation of all constructs, as well as to determine whether errors had occurred during PCR, all the constructs were completely sequenced on both strands using ABI Automatic sequencer 373A or 377.

Transfectionos of Krp1 constructs

Cells were transfected with 1.5 μg of the relevant plasmid using Superfect lipid transfection reagent (Qiagen). Immunofluorescence of transfected cells was performed 24 h later by either pre-permeablizing the live cells in Triton X-100/PBS for 1 min before fixing them with 4% paraformaldehyde for 15 min at 37°C, or they were fixed without any pre-permeablization. After fixing, the cells were washed extensively in PBS and permeablized in PBS/20 mM glycine/0.05% triton X-100 for 5 min if they had not been pre-permeablized. Blocking of non-specific binding was performed for 20 min in blocking buffer (PBS/0.5% BSA/10% FCS), followed by a 1 h incubation at room temperature of the relevant primary antibody diluted in blocking buffer: a 1 in 400 dilution of anti-Krp1; 1 in 30 dilution of anti-tubulin monoclonal antibody (a gift from E Schieble); and 1 in 100 dilution of anti-myc monoclonal antibody. After extensive washing in blocking buffer, the relevant secondary antibody was added in blocking buffer and, when needed, either TRITC- or FITC-phalloidin was added and incubated at 45 min at room temperature. Three final washes in PBS were performed and then the cells were mounted with Vectashield (Vector Laboratories). Cells were visualized on a Bio-Rad MRC 600 confocal illumination unit attached to a Nikon Diaphot inverted microscope with various magnifications.

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Acknowledgements

We would like to thank Prof. JA Wyke and Drs MC Frame and G Stapleton for critical reading of the manuscript. We also thank P McHardy for assistance with microscopy. Thanks to E Schieble of the Beatson Institute for the tubulin monoclonal antibody. We gratefully acknowledge the Cancer Research Campaign for their financial support.

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Correspondence to Bradford W Ozanne.

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Keywords

  • Kelch
  • Krp1
  • v-fos
  • pseudopodia
  • cytoskeleton

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