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¹ Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Harima Science Garden City, Hyogo 678-1297, Japan
² Department of Biomolecular Sciences, Institute of Biomedical Sciences, Fukushima Medical College, Hikariga-oka, Fukushima 960-1295, Japan

	Abstract

Top Abstract Introduction Results Discussion Experimental procedures References

 
A GTPase activating protein (GAP), p122, has previously been cloned as a phospholipase C (PLC)₁-interacting protein. p122 shows a specific GAP activity for Rho and enhances the enzyme activity of PLC₁. In this study, we examined the localization and functions of p122/RhoGAP, using enhanced green fluorescent protein (EGFP)-tagged proteins. EGFP-p122 was observed as punctate structures at the plasma membrane of BHK (fibroblastic) cells and MDCK (epithelial) cells. This patchy distribution depended on membrane cholesterol levels and the C-terminal region of p122 containing the GAP domain was responsible for it. Sucrose density gradient centrifugation and immunostaining of caveolin-1 revealed that p122 was localized in caveolin-enriched membrane domains mainly via its GAP domain. We demonstrated that transient expression of EGFP-p122 caused internalization of caveolin-1. Moreover, when the EGFP-tagged GAP domain was introduced in another fibroblastic cell line, NRK cells, punctate fluorescent structures were co-localized with caveolin-1. In this case, caveolin-1-positive structures were found in patches of F-actin, unlike those of untransfected cells that formed linear arrays along with actin stress fibres. These results suggest that p122 is localized in caveolae and plays an important role in caveolin distribution through reorganization of the actin cytoskeleton.

	Introduction

Top Abstract Introduction Results Discussion Experimental procedures References

 
Rho, a member of small GTPases, is involved in the formation of actin stress fibres and focal adhesions (Ridley & Hall 1992). The Rho small GTPases rotate between the GDP-bound inactive form and the GTP-bound active form and act as a molecular switch in various signalling pathways (Bourne et al. 1991; Takai et al. 1995). Guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) participate in the regulation of this cycle and play essential roles in the regulation of the Rho small GTPases signalling pathways. GEFs accelerate the change from GDP to GTP and activate the Rho small GTPases (Nobes & Hall 1994). Conversely, GAPs facilitate the hydrolysis of GTP to GDP and inactivate the Rho small GTPases (Bourne et al. 1990).

A RhoGAP, p122, has been cloned as a phospholipase C (PLC)₁-interacting protein from rat brain expression library, which exhibits a specific GAP activity on Rho to enhance the phosphatidylinositol(4,5)-bisphosphate-hydrolysing activity of PLC₁in vitro (Homma & Emori 1995). Over-expression of the C-terminal region of p122 alone can inhibit the lysophosphatidic acid (LPA)-induced formation of actin stress fibres and focal adhesions and immediately induces elevation of intracellular Ca²⁺ levels (Sekimata et al. 1999). The GAP domain (647–796 amino acids) in the C-terminal region mediates the responses (Sekimata et al. 1999). Recently, a human homologue of the p122 gene, the deleted in liver cancer (DLC)-1 gene, has been isolated as a tumour suppressor gene; the DLC-1 gene inhibits human cancer cell growth and the in vivo tumorigenicity in nude mice (Yuan et al. 1998, 2003). Therefore, it is highly plausible that the p122 family molecules exert the anti-oncogenic function by modulating the Rho–actin cytoskeleton interaction and/or the phosphoinositide-Ca²⁺ signalling pathway.

Despite the potential importance of p122 as an anti-oncogenic protein, its intracellular localization has not been explored in detail. Caveola, which is a lipid raft coated by the particular protein, caveolin, and involved in intracellular cholesterol trafficking and Ca²⁺ mobilization (Harder & Simons 1997; Isshiki & Anderson 1999), is one of the potential sites for its residency. Many proteins participating in signal transduction, such as Src-like kinase, H-Ras, endothelial nitric oxide synthase, heterotrimeric G proteins, epidermal growth factor (EGF) receptor and Rho, have been found in caveolae (Anderson 1998; Gingras et al. 1998; Michaely et al. 1999; Smart et al. 1999). Caveolae are known as regions of more ordered membrane structure compared with other part of the plasma membrane and are resistant to solubilization with non-ionic detergents at low temperatures (Brown & London 1998). Cholesterol is a major lipid component of caveolae and other membrane rafts (Smart et al. 1999). Caveolae are confined to the cell surface by the actin cytoskeleton and rearrangement of the actin network controls intracellular caveolar trafficking, distribution and functions (Mundy et al. 2002; Pol et al. 2000; Stahlhut & van Deurs 2000).

An examination of the structure of p122 indicated that there are essentially no apparent functional motifs or domains in the N-terminal region, whereas there are two domains, the GAP domain and the steroidogenic acute regulatory protein (StAR)-related lipid-transfer (START) domain (878–1079 amino acids) (Ponting & Aravind 1999), in the C-terminal region (Fig. 1A top). The START domains of StAR and its homologue, MLN64, bind directly to cholesterol (Tsujishita & Hurley 2000). To dissect how these regions or domains contribute to the cellular localization of p122 could bring new insights into physiological functions of the molecule.

View larger version (49K): [in this window] [in a new window]   Figure 1  Intracellular localization of EGFP-p122-WT and its mutants in BHK and MDCK cells. (A) Schematic representation of EGFP-p122-WT, EGFP-p122–617 N and EGFP-p122–534C. In the C-terminal region, p122 has a RhoGAP domain and a START domain. The numbers above each construct refer to the amino acid numbers. (B) BHK cells were transfected with EGFP-p122-WT and its mutants for transient expression. The cell lysates were subjected to SDS-PAGE followed by immunoblotting analysis using an anti-GFP monoclonal antibody. Positions of EGFP-tagged proteins were denoted by asterisks. (C) Plasmid DNA encoding EGFP or EGFP-p122-WT (100 ng/µL) was microinjected into the nuclei of BHK (upper panels) and MDCK (lower panels). Two hours after microinjection, the cells were fixed and fluorescent images of cells expressing EGFP alone (a and c) or EGFP-p122-WT (b and d) indicated by arrows were observed using a confocal laser microscopy system. (D) Plasmid DNA encoding EGFP-p122–617 N (a) and EGFP-p122–534C (b) (100 ng/µL) was microinjected into the nuclei of BHK cells. After 2 h, the cells were fixed and analysed using a confocal laser microscopy system. (E) BHK (a) and MDCK (b) cells were pretreated with 5 mM MßCD at 37 °C. After 30 min, plasmid DNA encoding EGFP-p122-WT was microinjected into the nuclei of BHK and MDCK cells. After 2 h, the cells were fixed and analysed using a confocal laser microscopy system. Arrowheads indicate the cells in which EGFP-p122-WT is diffusely localized in the cytoplasm. The scale bar corresponds to 10 µm.

	Results

Top Abstract Introduction Results Discussion Experimental procedures References

 
EGFP-p122 is localized as patches in BHK and MDCK cells

To examine the localization of p122, EGFP-tagged p122 wild-type (EGFP-p122-WT) (Fig. 1A top) was expressed in BHK and MDCK cells. Since EGFP-p122-WT immediately and dramatically induces morphological changes in cells when over-expressed at high levels (Sekimata et al. 1999), it has been difficult to determine the localization of EGFP-p122-WT. Therefore, to restrict the expression level of EGFP-p122-WT to minimum levels, plasmid DNA encoding EGFP-p122-WT was microinjected into the nuclei of BHK and MDCK cells. Two hours after microinjection, the cells were fixed and fluorescent images of EGFP-p122 were observed under confocal laser microscopy. The cells expressing EGFP-p122-WT retracted and their shape became round. EGFP-p122-WT was observed as scattered patches (or dots) in BHK cells (Fig. 1C, b) and in MDCK cells (Fig. 1C, d). Similar punctate structures were also seen in NRK cells (data not shown). Since EGFP alone did not form such patchy structures in these cells (Fig. 1C, a & c), this kind of localization was specific for p122.

The C-terminal region of p122 is involved in the patch-like localization of EGFP-p122

Next, to determine the region of p122 that participates in this patch-like localization, we examined the intracellular localization of EGFP-fused C-terminal and N-terminal region of p122, EGFP-p122–617 N (617–1083 amino acids Fig. 1A middle) and EGFP-p122–534C (1–534 amino acids Figure 1A bottom), respectively. Although there was a slight degradation, a majority of each fusion protein remained intact in BHK cells and the expression level was essentially the same as that of EGFP-p122-WT (Fig. 1B). EGFP-p122–534C, which lacked both the RhoGAP and START domains, showed a diffuse distribution in the cytoplasm (Fig. 1D, b). On the other hand, EGFP-p122–617 N, which contains the RhoGAP and START domains, formed patches as was observed for EGFP-p122-WT (Fig. 1D, a). Similar results were obtained with MDCK cells expressing these mutants (data not shown). These results indicate that the C-terminal region of p122 participates in the patch-like localization of EGFP-p122.

Cholesterol is necessary for the patch-like localization of EGFP-p122

Since the START domain is a cholesterol-binding domain, we examined whether cholesterol is involved in the patch-like localization of EGFP-p122. To investigate the effect of cholesterol depletion on this patch-like localization of EGFP-p122-WT, BHK and MDCK cells were treated with methyl-ß-cyclodextrin (MßCD), which is a cholesterol-binding reagent and removes cholesterol from intact cells (Kilsdonk et al. 1995; Ohtani et al. 1989). It has been reported that depletion of cellular cholesterol by MßCD caused a loss of compartmentalization of signalling molecules in lipid rafts and caveolae (Pike & Miller 1998). Thirty minutes after treatment with or without 5 mM MßCD, a plasmid encoding EGFP-p122-WT was microinjected into cell nuclei and the cells were then observed under confocal microscopy. In cells treated with MßCD the fluorescence was diffusely localized in the cytoplasm (Fig. 1E, a for BHK cells and Fig. 1E, b for MDCK cells), the pattern clearly different from that of MßCD-untreated cells (Fig. 1C, b & d, respectively). These results indicate that the patch-like localization of EGFP-p122 depends on the presence of membrane cholesterol.

p122 is localized in low-density caveolin-enriched membrane fractions

It has been well documented that cholesterol is a major component of lipid rafts and caveolae (Smart et al. 1999). Therefore, we next examined biochemically whether endogenous p122 exists in the lipid microdomain-like structure. To isolate caveolin-enriched membrane domains, we carried out sucrose density gradient centrifugation in the absence of detergent (Brown & Rose 1992) using BHK cells. The distribution of endogenous p122 was analysed by immunoblotting the fractions collected using an anti-p122 antiserum. Caveolin-enriched membrane fractions were determined by immunoblotting using an anti-caveolin-1 antibody. Caveolin-1, a major marker of caveolae, was enriched in the low-density fractions that corresponded to the 5%/35% sucrose interface of the gradient (Fig. 2A, fractions 6–8, bottom panel). Endogenous p122 was detected in the low-density caveolin-1-enriched membrane fractions (Fig. 2A, fractions 6–8, upper panel), although it was also detected in fractions containing cytosolic proteins (Fig. 2A, fractions 12–15, upper panel). These results strongly suggest that endogenous p122 is localized in lipid rafts or caveolae.

View larger version (27K): [in this window] [in a new window]   Figure 2  p122 interacts with caveolin-1 and is localized in caveolae. (A) Sucrose density gradient centrifugation was performed with BHK cell lysates. The discontinuous sucrose gradient (45% : 35% : 5% = 1 : 1 : 1) was centrifuged at 200 000 g for 18 h at 4 °C. Fractions were then collected from the top. The pellets were resuspended in MES buffer. Each fraction was subjected to SDS-PAGE followed by immunoblotting analysis using an anti-p122 antiserum or an anti-caveolin-1 polyclonal antibody. Fractions 6–8 corresponded to the low-density caveolin-enriched membrane fractions. (B) BHK cells were pretreated with 5 mM MßCD for 30 min at 37 °C to deplete cholesterol from the cells. After treatment, the cells were harvested and subjected to sucrose density gradient centrifugation followed by SDS-PAGE and immunoblotting analysis using an anti-p122 antiserum or an anti-caveolin-1 polyclonal antibody. Sucrose densities corresponding to each fraction are denoted under the fraction numbers. (C) BHK cells expressing EGFP-p122-WT were fixed and stained with an anti-caveolin-1 monoclonal antibody, followed by Cy3-conjugated anti-mouse-IgG antibody. Fluorescence images of EGFP (a) and caveolin-1 visualized by Cy3 (b) were taken using a confocal laser microscopy system. The merged images are shown in the right panels (c). (D) BHK cell lysates were incubated in the presence of a control rabbit preimmune serum or an anti-p122 antiserum. Then, the immune complexes were precipitated with protein A-Sepharose 4B. The immune complexes were subjected to immunoblotting analysis using an anti-caveolin-1 antibody. The arrowhead denotes the position of caveolin-1 and an arrow denotes the position of p122.

p122 interacts with caveolin-1 in vivo and may be localized in caveolae

To reveal that p122 is localized in caveolae, we next investigated the localization of EGFP-p122-WT and caveolin-1 in BHK cells. Cells expressing EGFP-p122-WT were immunostained with an anti-caveolin-1 antibody. As shown in Fig. 2C, most of the punctate structures of EGFP-p122-WT were co-localized with caveolin-1, suggesting that p122 is localized in caveolae. Similar results were also obtained in MDCK cells (data not shown). Since most of the proteins existing in caveolae are associated with caveolin-1, we next examined the interaction of endogenous p122 with caveolin-1. An immunoprecipitation assay was performed using an anti-p122 antiserum. When p122 was immunoprecipitated from BHK cell lysates, caveolin-1 was co-immunoprecipitated with p122 (Fig. 2D). Endogenous p122 and caveolin-1 were co-immunoprecipitated only when cells were lysed with 60 mMß-octylglucoside that can solubilize caveolae. When cells were solubilized with 0.5% Triton X-100 that cannot solubilize caveolae, caveolin-1 could not be co-immunoprecipitated with p122 (data not shown). These data indicate that p122 interacts with caveolin-1 in vivo and suggest that it is localized in caveolae.

The GAP domain is important for the patch-like localization of p122

We then investigated a detailed mechanism of the localization of p122. In the C-terminal region of p122, on which the patch-like localization of p122 depends, the GAP domain and the START domain are identified. To examine which domains of p122 is important for interaction with caveolin, we constructed two mutants, EGFP-p122–798 N, which practically consists of the START domain only, and EGFP-p122-GAP, which corresponds to the GAP domain (617–797 amino acids) of p122 (Fig. 3A). A majority of each mutant expressed in BHK cells was intact and the expression level was essentially the same (Fig. 3B). EGFP-p122–534C (Fig. 1A bottom), which possesses the N-terminal half of p122 but lacks the RhoGAP and START domain, was localized diffusely in the cytoplasm and was not co-localized with caveolin-1 in BHK cells (Fig. 3C, a–c). On the other hand, EGFP-p122-GAP was localized as patches and co-localized with caveolin-1 (Fig. 3C, g–i). The nuclear fluorescence could be due to degraded forms of EGFP-p122-GAP that show nonspecific intracellular localization. Alternatively, EGFP-p122–798 N also formed patch-like structures, but was only partially co-localized with caveolin-1 (Fig. 3C, d–f), indicating that the START domain alone is not sufficient for the specific localization to caveolin-1 enriched membrane domains.

View larger version (32K): [in this window] [in a new window]   Figure 3  The GAP domain is sufficient for caveolae-localization of p122. (A) Schematic representation of EGFP-p122–798 N and EGFP-p122-GAP. (B) BHK cells were transfected with EGFP-p122–798 N and EGFP-p122-GAP for transient expression. The cell lysates were subjected to SDS-PAGE followed by immunoblotting analysis using an anti-GFP monoclonal antibody. The positions of EGFP-tagged proteins were denoted by asterisks. The other bands may be degradations. (C) BHK cells expressing EGFP-p122–534C (a–c), EGFP-p122–798 N (d–f) or EGFP-p122-GAP (g–i) were subjected to immunostaining using an anti-caveolin-1 antibody, followed by Cy3-conjugated anti-mouse-IgG antibody. Fluorescence images of EGFP (left panels, green) and caveolin-1 visualized by Cy3 (middle panels, red) were taken using a confocal laser microscopy system. The merged images are shown in the right panels (c, f and i). The scale bar corresponds to 10 µm (D) BHK cells were transfected with EGFP-p122–534C, EGFP-p122–798 N, EGFP-p122-GAP and EGFP for transient expression. After 24 h, the cells were harvested and subjected to sucrose density gradient centrifugation followed by SDS-PAGE and immunoblotting analysis using an anti-GFP monoclonal antibody. Low-density caveolae-enriched membrane fractions correspond to fractions 6–8.expressing EGFP-p122–534C (a–c), EGFP-p122–798 N (d–f) or EGFP-p122-GAP (g–i) were subjected to immunostaining using an anti-caveolin-1 antibody, followed by Cy3-conjugated anti-mouse-IgG antibody. Fluorescence images of EGFP (left panels, green) and caveolin-1 visualized by Cy3 (middle panels, red) were taken using a confocal laser microscopy system. The merged images are shown in the right panels (c, f and i). The scale bar corresponds to 10 µm (D) BHK cells were transfected with EGFP-p122–534C, EGFP-p122–798 N, EGFP-p122-GAP and EGFP for transient expression. After 24 h, the cells were harvested and subjected to sucrose density gradient centrifugation followed by SDS-PAGE and immunoblotting analysis using an anti-GFP monoclonal antibody. Low-density caveolae-enriched membrane fractions correspond to fractions 6–8.

Transient expression of EGFP-p122-WT induces internalization of caveolin-1 or caveolin-1-enriched membrane domains

We then examined temporal changes in the distribution of p122 and that of caveolin-1 in EGFP-p122-WT-expressing BHK cells. At 60 min after microinjection of a plasmid DNA encoding EGFP-p122-WT, the fluorescence was observed mainly at the cell surface. Caveolin-1 was also found at the cell surface and was co-localized with EGFP-p122-WT (Fig. 4a–c). Patches of EGFP-p122-WT then started to translocate into the cytoplasm. At 90 min after microinjection, caveolin-1-positive structures also moved towards the perinuclear regions and were co-localized with the GFP fluorescence (Fig. 4d–f). By 120 min after microinjection, apparent accumulation of both EGFP-p122-WT and caveolin-1 were observed in the perinuclear regions, although they partly remained at the cell surface (Fig. 4g–i). These results suggest that p122 induces internalization of caveolin-1 itself or caveolin-enriched membrane domains in BHK cells.

View larger version (55K): [in this window] [in a new window]   Figure 4  Effect of transient expression of EGFP-p122-WT on distribution of caveolin-1 in BHK cells. Sixty (a–c), 90 (d–f) and 120 (g–i) min after microinjection of the plasmid DNA encoding EGFP-p122-WT into the nuclei of BHK cells, the cells were fixed and stained with an anti-caveolin-1 monoclonal antibody, followed by Cy3-conjugated anti-mouse-IgG antibody. Fluorescence images of EGFP (left panels, green) and caveolin-1 visualized by Cy3 (middle panels, red) were taken using a confocal laser microscopy system. The merged images are shown in the right panels (c, f and i). Caveolin-1 was co-localized with EGFP-p122-WT in the plasma membrane (arrows) and perinuclear region (arrowheads). The scale bar corresponds to 10 µm.

The actin cytoskeleton anchors to caveolin and arrangement of caveolae depends on association with the cortical actin filaments particularly in fibroblastic cells (Stahlhut & van Deurs 2000). We examined the relationship of actin filaments with p122 and caveolin-1 using NRK cells, that show well-developed actin fibres among fibroblastic cell lines. As shown in Fig. 5a–f, caveolin-1 forms linear arrays on actin filaments. Since p122 regulates the actin cytoskeleton by inhibiting Rho (Sekimata et al. 1999), we thought that it could control the caveolar localization by depolymerization of actin filaments by its Rho GTPase activity. To prove this, we examined the effect of expression of EGFP-p122-GAP on the distribution of caveolin-1 and the structure of actin filaments in NRK cells. The GFP fluorescence was observed as patch-like structures and co-localized with caveolin-1 in cells expressing EGFP-p122-GAP (Fig. 5g–i). In this case, caveolin-1-positive structures did not form the linear arrays on the actin filaments anymore, but were still co-localized with the patchy structures of EGFP-p122-GAP. In the cells expressing EGFP-p122-GAP, however, caveolin-1 appeared to be localized in less-ordered structures. Similar results were observed in BHK cells (Fig. 3C, h). Interestingly, a part of actin fibres were observed as patch-like structures with which EGFP-p122-GAP is co-localized, although the rest remained filamentous, showing no co-localization with the GAP domain (Fig. 5j–l). These results therefore indicate that expression of the GAP domain of p122 converts a part of the cortical F-actin from filamentous to dot-like structures and also affects the distribution of caveolin-1-containing membranes.

View larger version (73K): [in this window] [in a new window]   Figure 5  Effects of transient expression of EGFP-p122-GAP on distribution of caveolin-1 and the structure of actin filaments in NRK cells. NRK cells (a–f) or EGFP-p122-GAP expressing NRK (g–l) cells were subjected to immunostaining using an anti-caveolin-1 antibody, followed by FITC-conjugated anti-mouse-IgG antibody (a and d, green) or Cy3-conjugated anti-mouse-IgG antibody (h, red). Actin filaments were visualized with Texas Red-X phalloidin (b, e and k, red). Fluorescence images of EGFP-p122-GAP are shown in g and j (green). Fluorescence images were taken using a confocal laser microscopy system. The merged images are shown in the right panels (c, f, i and l). Arrowheads denote regions where caveolin-1 distributes along actin stress fibres. The regions in white boxes are shown magnified below (d–f). The scale bar corresponds to 10 µm.

	Discussion

Top Abstract Introduction Results Discussion Experimental procedures References

Using deletion mutants fused with EGFP we found the C-terminal half of p122 was responsible for the patch-like distribution of p122. The region contains the GAP domain and the START domain, and we found the GAP domain alone is sufficient for the patchy localization in caveolin-1-enriched membrane domains (Fig. 3C, g–i, D the second panel from the bottom). We found amino acid sequences that resemble putative caveolin-binding motifs (XXXXX, XXXXXX or XXXXXXX), where is an aromatic residue and X is any amino acid (Couet et al. 1997) in the N-terminal region (¹⁴WLRVTGFPQY²³ and ⁹³WTFQRDSKRWSRLEEFDVF¹¹¹) and in the GAP domain (⁶⁹⁰YVNYEGQSAY⁶⁹⁹ and ⁷²⁵FLQIYQY⁷³¹) of p122. Since EGFP-p122–534C does not seem to reside in caveolin-1-containing membranes (Fig. 3C, a–c and Fig. 3D top panel), the sequences in the N-terminal region may not function as caveolin-binding motifs. Either (or both) the sequence(s) in the GAP domain, however, could be important for caveolin-1 binding and therefore responsible for the distribution of p122 (Fig. 3). It is therefore possible that the RhoGAP domain of p122 contributes not only to the catalytic activity but also to the intracellular localization of p122. The exact roles of the N-terminal region have to be clarified.

EGFP-tagged START domain (EGFP-p122–798 N) was also observed as patches, but unlike EGFP-p122-WT or EGFP-p122-GAP, it was just partially co-localized with caveolin-1 in BHK cells (Fig. 3C, d–f) and in MDCK cells (data not shown). The patch-like localization of EGFP-p122–798 N was similar to the distribution of cholesterol visualized by filipin, a fluorescent molecule that specifically binds to free cholesterol (data not shown). The result suggests that the START domain can associate with cholesterol but it is not sufficient to recruit p122 to caveolin-1-enriched membrane domains. Moreover, the result of sucrose density gradient centrifugation, that only a small amount of EGFP-p122–798 N was detected in low-density membrane fractions (Fig. 3D), also supports the idea of weak interaction between the START domain and caveolin-1-containing structures. It is highly possible that EGFP-p122–798 N resides not only in caveolae but also in other membrane regions to which cholesterol is distributed.

Since the START domain is a protein motif that binds cholesterol and phosphatidylcholine and has been found in a wide range of proteins involved in diverse cellular functions (Ponting & Aravind 1999), it is possible that the START domain changes its conformation by binding cholesterol and regulates the activity of the adjacent GAP domain through an inter-domain interaction. To reveal this possibility, detailed characterization of the START-GAP domain structures and interaction would be important.

It is well documented that caveolin species participate in various events that occur in caveolae by acting as scaffolding proteins (Smart et al. 1999). It has also been reported that Rho is found in caveolin-enriched membrane domains and binds directly to caveolin-1 (Gingras et al. 1998; Michaely et al. 1999). Therefore, it is possible that by binding to both p122 and Rho, caveolin-1 facilitates the GAP activity of p122 on Rho, leading to inactivation of Rho.

Although caveolae, flask-shaped membrane invaginations, are not the sites of constitutive endocytosis, they are dynamically translocated into the cell interior under special conditions, such as infection of simian virus 40 (Pelkmans et al. 2001) and stimulation by growth factors such as EGF (Pol et al. 2000). Regulation mechanisms and physiological implications of caveolar internalization, however, have not been well understood. It was reported that the actin cytoskeleton associates with caveolin and stabilizes caveolae at the plasma membrane (Stahlhut & van Deurs 2000; Thomsen et al. 2002). A recent study showed that in Chinese hamster ovary cells stably expressing caveolin-1-GFP, the GFP fluorescence moved to the cell interior and accumulated in the perinuclear regions by treatment with latrunculin A, an actin depolymerization reagent (Mundy et al. 2002). We found that transient expression of EGFP-p122-WT also induced internalization and the perinuclear accumulation of caveolin-1 in BHK cells (Fig. 4). Since p122 is a RhoGAP and depolymerizes actin filaments by inactivating Rho, the effect of EGFP-p122-WT on distribution of caveolin-1-positve structures could be similar to that of latrunculin A. Our results suggest that Rho signalling pathway regulated by p122 is one of potential mechanisms for internalization of caveolae. When EGFP-p122-WT was transiently expressed in BHK cells, the GFP fluorescence was found at the cell surface caveolar-like structures first, but gradually appeared in the cytoplasm (Fig. 4). It is important to note that disruption of actin stress fibres and focal adhesions also occurs (Sekimata et al. 1999) almost concomitantly with this internalization. The function of p122 as a whole therefore may not be limited only to the caveolin-1 internalization.

Interestingly, expressed EGFP-GAP domain of p122 alone, which contains putative caveolin-binding motifs, appeared to be strongly recruited to and confined in the caveolin-1-enriched membrane domains (Figs 3 & 5) in both BHK and NRK cells. Actin filaments co-localized with EGFP-p122-GAP were observed in patchy cavelin-1-positive structures and no longer formed fibres in NRK cells, whereas those that were not associated with EGFP-p122-GAP maintained intact fibrous structures and cell shapes remained unchanged (Fig. 5g–l), suggesting the GAP domain functions only in caveolae. In normal NRK cells caveolin-1-positive structures form linear arrays along with actin filaments (Fig. 5a–f). In EGFP-p122-GAP expressing cells, however, they did not form linear arrays but were found in patches where both EGFP-p122-GAP and F-actin are co-localized (Fig. 5g–l). It is therefore conceivable that p122 plays an important role in the regulation of intracellular caveolar traffic through the GAP domain. The GAP domain alone can reorganizes the local actin cytoskeleton and changes the distribution of caveolin-1, but unlike full-length p122, its over-expression is not sufficient to cause drastic changes in cell shapes. The difference could be due to the difference in protein–protein interaction. In the N-terminal region, p122 has the sterile alpha motif (SAM)-like domain (3–70 amino acids). It have been reported that the SAM domain functions as a protein–protein interaction module and forms homo- or hetero-origomers (Schultz et al. 1997). It is possible therefore that full-length p122, but not the GAP domain, forms homo-origomers or associates with other proteins containing the SAM domain. These interactions would endow p122 with a functional variation in the cell.

It would be most important to identify the physiological functions of p122 in caveolae. In this study, we show that p122 is involved in regulation of intracellular distribution of caveolae through reorganization of the actin cytoskeleton. Although the physiological meaning of this regulation has been unclear, it has been reported that the distribution of caveolae is closely related to intracellular events, such as control of intracellular Ca²⁺ levels (Lockwich et al. 2001) or activation of mitogen-activated protein (MAP) kinase (Pol et al. 2000). Sekimata et al. (1999) previously showed that over-expression of the C-terminal region of p122 caused an increase in Ca²⁺ levels. Although caveolae play an important role in Ca²⁺ entry via the plasma membrane (Fujimoto 1993), mechanisms for the regulation of the Ca²⁺ influx has not been well elucidated. Recent studies have demonstrated that transient receptor potential protein (Trp) cation channels are localized in caveolae (Lockwich et al. 2000, 2001; Torihashi et al. 2002). Trp has been proposed to be a component of the store-operated Ca²⁺ channel (SOC) (Liu et al. 2000). When cells are stimulated by agonists, inositol(1,4,5)-trisphosphate (Ins1,4,5P₃)-dependent release of Ca²⁺ from internal Ca²⁺ stores via the Ins(1,4,5)P3 receptors (IP₃R) occurs. Depletion of Ca²⁺ from internal Ca²⁺ stores activates SOC and induces Ca²⁺ influx via the Trp channel. The direct interaction between Trp in the caveola membrane and the IP₃R in the ER membrane is necessary for this SOC activation (Kiselyov et al. 1998; Patterson et al. 1999). Additionally, stabilization of cortical actin inhibits SOC-mediated Ca²⁺ entry (Lockwich et al. 2001). Since p122 is a specific GAP for Rho small GTPase and inhibits the formation of actin fibres, it is possible that p122 cancels the inhibition of SOC-mediated Ca²⁺ entry, inducing the increase in intracellular Ca²⁺ levels (Fig. 6).

View larger version (24K): [in this window] [in a new window]   Figure 6  A model for localization and functions of p122 in caveolae. p122 binds caveolin-1 through the RhoGAP domain. The START domain may bind cholesterol. These interactions confine p122 in caveolae. In caveolae p122 inactivates Rho, resulting in the depolymerization of actin filaments that are associated with caveolin. Caveolae participate in the regulation of SOC-mediated Ca2+ influx that is controlled by the actin cytoskeleton (Lockwich et al. 2001; Ma et al. 2000). It is conceivable that p122 regulates the Ca2+ influx in caveolae through interactions with Rho and PLC1, which is not present in caveolae but somehow capable of interacting with p122. The site of p122 to interact with PLC1 still remains unknown. Reorganization of the actin cytoskeleton induces redistribution of caveolae, followed by down-regulation of the MAP kinase signalling. See Discussion for details.

Pol et al. (2000) reported that EGF-mediated MAP kinase activation, which is a downstream of Ras signalling pathway, requires the intact actin cytoskeleton and recruitment of caveolin to early endosomes in NRK cells. They showed that treatment with disruptive reagents of the actin cytoskeleton inhibited both caveolin-sorting and MAP kinase activation. Interestingly, DLC-1, a human homologue of p122, has been reported to be a product of tumour suppressor gene (Yuan et al. 1998). Recently, it has been reported that over-expression of the GAP domain of DLC-2, a homologue of DLC-1, inhibits Ras-induced cellular transformation in NIH3T3 fibroblasts (Ching et al. 2003). It is therefore attractive to speculate that regulation of caveolar trafficking by the Rho signalling pathway, in addition to the drastic reorganization of the actin cytoskeleton that leads to changes in cell morphology, constitutes at least in part the molecular mechanisms for the anti-tumorigenic function of the p122/DLC family molecules.

Although lipid rafts have been recognized as platforms for a variety of cellular functions, the regulation of events that occur in caveolae has not been clarified. Our findings therefore may provide important insights into these regulation mechanisms including the physiological roles of p122.

	Experimental procedures

Top Abstract Introduction Results Discussion Experimental procedures References

 
Materials and chemicals

BHKC13 cells were kindly provided by Dr F. Tokunaga (Osaka City University, Japan). MDCK cells and NRK cells were obtained from the Riken Cell Bank (Wako, Japan). An anti-p122 antiserum was raised against a synthetic peptide, IRDSFSNQSTESKDTRSR, corresponding to residues 1066–1083 of rat p122. An anti-caveolin-1 rabbit polyclonal and mouse monoclonal (clone C060) antibodies were purchased from Transduction Laboratory (Lexington, USA). An anti-GFP mouse monoclonal antibody (clone JL-8) was purchased from BD Biosciences Clontech (Palo Alto, USA). MßCD was purchased from Aldrich (Milwaukee, USA). Other materials and chemicals were obtained from commercial sources.

Plasmid constructions

Mammalian expression plasmids for EGFP fusion proteins, pEGFP-p122-WT, pEGFP-p122–534C, pEGFP-p122–617 N and pEGFP-p122–798 N, were produced as previously described (Sekimata et al. 1999). A plasmid DNA for expressing the RhoGAP domain (617–797 amino acids) of p122 alone, pEGFP-p122-GAP, was constructed from pEGFP-p122–617 N, which was generated by inserting the XhoI/EcoRI fragment of pEGFP-p122–617 N into the XhoI and EcoRI sites of pEGFP-C1 (BD Biosciences Clontech, Palo Alto, USA). To confirm expression of EGFP-tagged proteins, the BHK cells were transfected with each plasmid DNA encoding EGFP-tagged protein using Lipofectamine^TM 2000 (Invitrogen Corp., Carlsbad, USA). Then the cells were harvested and lysed with a lysis buffer (buffer A: 10 mM Tris/HCl (pH 7.5), 1 mM EDTA, 10 µM[p-amidinophenyl]-methanesulphonyl fluoride, 10 µg/mL leupeptin, 1.0% Triton X-100, 0.5% NP-40, 60 mMß-octylglucoside). The cell lysates were subjected to immunoblotting analysis using an anti-GFP mouse monoclonal antibody.

Cell culture

BHKC13 cells, NRK cells and MDCK cells were grown in Dulbecco's modified Eagles medium (DMEM) containing foetal bovine serum (FBS) 10%, 7% and 5%, respectively, at 37 °C in an air-5% CO2 atmosphere at constant humidity.

Microinjection of plasmid DNA

BHK cells were seeded at a density of 1 x 10⁶ cells/13-mm cover glass coated with 50 µg/mL fibronectin (Sigma, St Louis, USA) in 6-cm dishes. MDCK cells were seeded at a density of 1.5 x 10⁵ cells/13-mm cover glass in 6-cm dishes. At 24 h after seeding, plasmid DNA encoding EGFP-p122-WT, EGFP-p122–534C, EGFP-p122–617 N, EGFP-p122–798 N or EGFP-p122-GAP (100 ng/µL) was microinjected (Injection pressure: 120 hPa, Compensation pressure: 30 hPa, Injection duration: 0.3 s) into the nuclei of MDCK or BHK cells using sterile Femtotips (Eppendorf, Hamburg, Germany) held in a Micromanipulator 5171 (Eppendorf, Hamburg, Germany) with a pressure supply from Trancejector 5246 (Eppendorf, Hamburg, Germany). Two hours after microinjection, the cells were fixed with 3% formaldehyde in PBS for 10 min at room temperature. Fluorescent images were taken with a confocal laser microscopy system (Carl Zeiss LSM 510) built around a Zeiss Axioplan 2 microscope (Carl Zeiss, Oberkochen, Germany).

Detergent-free sucrose density gradient centrifugation

BHK cells were seeded at a density of 1 x 10⁶ cells/6-cm dish (three dishes). The cells were treated with or without 5 mM MßCD for 30 min at 37 °C and then scraped in 300 µL of 500 mM Na₂CO₃ (pH 11). The cell suspension was homogenized with 20 strokes of a Teflon-glass homogenizer and three sets of 15-s sonication. Then, 300 µL of cell lysates were adjusted to 45% sucrose by the addition of an equal volume of 90% sucrose in MES buffer (25 mM MES at pH 6.5, 150 mM NaCl, 1 mM EDTA) and placed in a centrifugation tube. Then, 35% sucrose and 5% sucrose in MES buffer were layered on top of the lysates (45% : 35% : 5% = 1 : 1 : 1). The discontinuous gradient was centrifuged at 200 000 g for 18 h at 4 °C in a TLS55 rotor (Beckman Coulter, Fullerton, USA). The sample was fractionated into 15 fractions. The pellets were resuspended in MES buffer.

Immunoflorescence analysis

MDCK and BHK cells were seeded and EGFP-p122-WT, EGFP-p122–534C, EGFP-p122–798 N or EGFP-p122-GAP was expressed in the cells by microinjection of plasmid DNA as described above (see ‘Microinjection of plasmid DNA’). Then, the cells were permeabilized with a permeabilizing and blocking buffer (0.1% Triton X-100, 2% FBS in PBS) for 10 min at room temperature. The permeabilized cells were incubated with an anti-caveolin-1 mouse monoclonal antibody, followed by Cy3-conjugated anti-mouse IgG (Amersham Biosciences Co., Piscataway, NJ) or fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (Kirkeggad & Perry Laboratories Inc., Gaithersburg, USA). Actin filaments were stained with Texas Red-X phalloidin (Molecular Probes Inc., Eugene, USA) for 20 min at room temperature. Fluorescent images were taken using a confocal laser microscopy system (Carl Zeiss LSM 510) built around a Zeiss Axioplan 2 microscope (Carl Zeiss, Oberkochen, Germany).

Co-immunoprecipitation assay of p122 with caveolin-1

Subconfluent BHK cells were harvested and lysed with buffer A, and then leaved on ice for 10 min, followed by sonication. The lysates were clarified by centrifugation at 100 000 g for 30 min at 4 °C. The soluble supernatants were incubated with an anti-p122 antiserum or a preimmune serum for 1 h at 4 °C. The immune complexes were then precipitated with 30 µL of protein A-Sepharose 4B. The immune complexes were washed four times with the lysis buffer, eluted by boiling in the SDS-PAGE sample buffer, and subjected to immunoblotting analysis with an anti-p122 antiserum and with an anti-caveolin-1 mouse monoclonal antibody.

	Acknowledgements

 
We thank Dr F. Tokunaga (Osaka City University) for providing the BHKC13 cells. This work was supported by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (#13033035 to H.Y.), and a Graduate Fellowship from the Japan Society for Promotion of Science to M.Y.

	Footnotes

 
Communicated by: Kozo Kaibuchi

*Correspondence: E-mail: yagisawa{at}sci.himeji-tech.ac.jp

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Received: 22 September 2003
Accepted: 24 October 2003

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