We have investigated the cellular distribution of p122RhoGAP, a GTPase-activating protein of Rho small GTPase and an activator of phospholipase C-δ1. Immunofluorescence studies demonstrated that endogenous p122 is localized at the tips of actin stress fibres and co-localizes with vinculin in normal rat kidney cells. In immunoprecipitation studies, p122 co-precipitated with vinculin, indicating that p122 is localized at the sites of focal adhesion. We have also shown that the N-terminal half of p122 is responsible for this localization. It is conceivable, therefore, that p122 is involved in the reorganization of the actin cytoskeleton and focal adhesions that regulate cell–substratum adhesion and cell migration.
p122RhoGAP (p122) (where GAP stands for GTPase-activating protein) is a dual functional molecule. It was cloned from a rat cDNA library and demonstrated to interact with PLCδ1 (phospholipase C-δ1) in vitro . p122 consists of 1083 amino acid residues and contains a GAP domain and a START (StAR-related lipid-transfer) domain in the C-terminal region. The START domain is found in proteins that transfer lipids, such as cholesterol, phosphatidylcholine and ceramide between organelles [2,3]. p122 activates the PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate)-hydrolysing activity of PLCδ1in vitro . Overexpression of the C-terminal region of p122 abrogates the formation of actin stress fibres and focal adhesions by inhibiting the GTP-bound active form of Rho and resulting in a concomitant increase in intracellular Ca2+ levels . We have recently shown that p122 is localized in the caveolin-enriched membrane domains in fibroblastic and epithelial cells . Since the activation mechanisms of PLCδ1 are not clear yet, it is intriguing to explore where, when and how p122 interacts with and activates PLCδ1 in cells.
Recently, a gene encoding a human orthologue of p122 was identified as a tumour suppressor gene by a reverse genetic approach using loss of heterozygosity in human liver cancer cells . The gene was named DLC-1 (deleted in liver cancer-1). It has become clear that this gene inhibits the cell growth of various carcinoma cells and in vivo tumorigenicity [7,8].
The intracellular localization and function of endogenous p122 or the DLC-1 gene product, however, have not been explored in detail. In the present study, we examined the localization of endogenous p122 in NRK (normal rat kidney) cells by immunofluorescent staining. We also overexpressed EGFP (enhanced green fluorescent protein)-fused p122 in NRK and HeLa cells.
Localization of endogenous p122 in focal adhesions
To detect endogenous p122, we raised a polyclonal antibody against the N-terminal region of p122. Figure 1(A) shows a typical distribution pattern of the actin filaments visualized by Texas-Red phalloidin and p122 visualized by anti-p122 and then FITC-anti-rabbit IgG as a second antibody under a confocal microscope. Endogenous p122 was observed at the tips of actin stress fibres. Since the tips of actin stress fibres link to focal adhesions, it is highly probable that p122 is localized in the focal adhesions in NRK cells. Generally, the focal adhesion complex is composed of many adhesion and cytoskeletal proteins including integrins, focal adhesion kinases, paxilin, vinculin and talin. It has been reported that the binding of PtdIns(4,5)P2 to vinculin causes its conformational change. Activated vinculin then binds to actin filaments and other cytoskeletal proteins, leading to the formation of focal adhesions. We, therefore, explored the possibility of an interaction between p122 and vinculin in the focal adhesions.
Immunofluorescent images of NRK cells
Co-localization of endogenous p122 with vinculin
The distribution of endogenous p122 was compared with that of vinculin in NRK cells by immunofluorescent staining (Figure 1B). Both p122 and vinculin were stained as dots and almost completely overlapped in the merged picture, indicating that p122 co-localized with vinculin in focal adhesions. Furthermore, an immunoprecipitation assay was performed to check whether p122 actually interacts with the proteins in focal adhesion complexes. When p122 was immunoprecipitated from NRK cell lysates using anti-p122 antiserum, vinculin was found in the immune complex. Similarly, p122 was found in the complex when vinculin was immunoprecipitated using an anti-vinculin antibody. These results suggest that p122 is a member of the focal adhesion complex.
Necessity of the N-terminal region for the localization in focal adhesions
It was previously reported that overexpression at high levels of the EGFP-tagged p122 induced immediate and significant morphological changes in cells . Nevertheless, the effect of a low-level expression of EGFP–p122 has not been examined. Cells expressing EGFP–p122 at low levels also show a disappearance of actin stress fibres and changes in morphology to some extent, but the fluorescence at the tips of actin stress fibres, in the same pattern shown by endogenous p122, can still be observed.
To determine which region of p122 participates in the localization in focal adhesions, we made four mutant constructs of p122 fused with EGFP: EGFP–p122–117ΔC that contains only a small stretch of the N-terminal region; EGFP–p122–534ΔC that has the N-terminal half of p122; EGFP–p122-ΔGAP that lacks the entire GAP domain (residues 633–797); and EGFP–p122-R668E that cannot induce morphological changes because of a loss-of-function mutation in the GAP domain. We expressed each construct in NRK cells. EGFP–p122–117ΔC was localized in the cytosol, whereas other constructs were localized in the focal adhesions similar to the EGFP–p122 wild-type (results not shown). The result indicates that the N-terminal region of p122, especially the section that contains residues from 117 to 533, participates in the localization in focal adhesions. Since we have previously demonstrated that the C-terminal half of p122 is important for the interaction with caveolin and, therefore, for the localization of p122 in caveolae in BHK cells, it seems that the N-terminal half and the C-terminal half of p122 have different targeting characteristics. When EGFP–p122–534ΔC was expressed in HeLa cells, it localized in the focal adhesions, whereas expression of EGFP–p122–617ΔN showed a pattern with random spots.
p122 as a dual negative regulator of cell adhesions
As reported previously, Rho activates PtdIns(4)P 5-kinases and thereby stimulates PtdIns(4,5)P2 production . PtdIns(4,5)P2 then binds to actin-associated cytoskeletal proteins, including vinculin and talin, to cause their conformational change. For example, activated vinculin and talin expose binding sites for the actin filament and other cytoskeletal proteins necessary for the formation of focal adhesions . p122 may, therefore, modulate the formation of focal adhesions by reducing the amount of PtdIns(4,5)P2 directly through activation of PLCδ1 and indirectly through inactivation of Rho. Thus p122 should be recognized as a dual negative regulator of cell-adhesion formation (Figure 2).
A model of the dual negative regulation of p122
In conclusion, the results demonstrate that p122 is localized in focal adhesions through its N-terminal region. p122 may exert its function through negative regulation of Rho and activation of PLCδ1 in focal adhesions. Many cancer cells need to form focal adhesions to migrate or to maintain their morphology. p122 can, therefore, act as an anti-carcinogenic factor, as has been reported.
Research Colloquia: Research Colloquia at BioScience2004, held at SECC Glasgow, U.K., 18–22 July 2004. Edited by M. Bouvier (Montreal, Canada), G. Milligan (Glasgow, U.K.), V. O'Donnell (Cardiff, U.K.), M. Brand (MRC-Dunn Human Nutrition Unit, Cambridge, U.K.), M. Schweizer (Heriot-Watt University, Edinburgh, U.K.), R. Insall (Birmingham, U.K.), A. Ridley (Ludwig Institute for Cancer Research, London, U.K.) and M. Sutcliffe (Leicester, U.K.). The first eight papers featured in this Section were presented as a part of the GPCR Regulation and Signalling Research Colloquium, incorporating the GPCR–Ion Channel Interactions Pfizer-Sponsored Research Colloquium.
This work was supported in part by Hyogo Science and Technology Association, Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology of Japan.