These studies were driven by the desire to understand the mechanism of action of the class II tumor suppressor gene H-REV107-1. Since the rat H-rev107 gene has been cloned, several reports were published about H-REV107-1 and its related genes in different species (Hajnal et al., 1994; Kuchinke et al., 1995; Sers et al., 1997; Husmann et al., 1998; Di Sepio et al., 1998; Akiyama et al., 1999; Huang et al., 2000; Ito et al., 2001; Sers et al., 2002). The main common features of these genes are that they are down-regulated in tumors and tumor derived cell lines, and possess growth suppressive properties. The primary sequence of the H-REV107-1 product did not provide any clues to the mechanism of its function as a growth suppressor. In order to elucidate the mechanism of human H-REV107-1 mediated growth inhibition, we performed a search for interacting proteins using a yeast two-hybrid assay.
Yeast two hybrid systems were developed in 1990 by Stanley and his colleagues, and provided a novel way of detecting protein-protein interactions in vivo. One of the first examples of its application was the identification of the Raf Kinase as a Ras interactor and a downstream effector of Ras function (Vojtek et al., 2003).
We have used a LexA-based yeast two-hybrid system supposed to produce fewer false positives as compared to the GAL4-based system. This proposition is based on the fact that in the LexA system the DNA–binding domain (BD) and the activation domain (AD) are provided entirely by the prokaryotic LexA, and B42 E. coli proteins, respectively. The prokaryotic proteins are supposed to interact with very few of yeast or mammalian proteins over-expressed in yeast cells. In contrast, the GAL4 system contains plasmids with eukaryotic GAL4-DNA binding and activation domains which are supposed to be less selective. Nevertheless, some interactions can be recognised only by using a LexA-based yeast two hybrid system, and other interactions can be identified only with the help of the GAL4-based system (Golemis et al., 1994). The reason for this observation is unknown.
Prior to a large scale transformation for a library screening, the human H-REV107-1 protein was kindly tested by E. Cuppen (Department of Cell Biology, Institute of Cellular Signalling, University Nijmegen, The Netherlands) in an established yeast two hybrid approach. This test demonstrated that for a successful application of the approach, the truncated form of the H-REV107-1 protein without the membrane binding domain has to be used. Expression of the full length protein in yeast did not resulted in colony growth. We supposed that the membrane binding domain hampered a transport of the protein into the yeast nucleus where the interactions with library proteins take place. Therefore, we performed the entire screen using a truncated form of the H-REV107-1 protein, lacking the 27 C-terminal aminoacids.
The use of a truncated protein bears the risk of loosing some interactors, which bind to the deleted region. However, it was rather doubtful that a transmembrane domain will participate in protein-protein binding. Most investigations of protein-protein interactions demonstrated that membrane bound domains do not participate in interaction with cytoplasmic proteins.
There are only a few examples demonstrating that such domains can be involved in interaction with other proteins, or in the formation of homodimers within a membrane (Langosch et al., 2002). The bacterial protein glycophorin is known to interact with other proteins via its transmembrane domain, although the mechanism of this protein-protein recognition is unclear (Gerber and Shai, 2002). In eukaryotes, large transmembrane proteins participating in transmembrane channel organisation were demonstrated to form homo- and heterodimers via the transmembrane regions (Nakayama et al, 2002).
Although the LexA-and GAL4-based yeast two hybrid systems are powerful tools for detecting protein-protein interactions, they have several disadvantages. They produce false positives, when library proteins intrinsically modulate transcription, for example when they function as transcriptional activators or repressors. In addition, the interaction between bait and pray takes place in the yeast nucleus, where proteins do not undergo posttranslational modifications like terminal glycosylation, sulfation, phosphorylation or methylation.
Interactions that require protein posttranslational modifications in the cytoplasm can not be detected by these systems, as well as interactions which require modulating factors present in the cytoplasm or the cell membrane. These aspects were very important for our assay, because we supposed that the human H-REV107-1 protein is subject to posttranslational modification. Five potential tyrosine phosphorylation sites and one serine site were identified with a score more than 0,6 in the H-REV107-1 protein sequence (Fig. 31) using the NetPhos 2.0 program developed by the Technical University of Denmark (http://www.cbs.dtu.dk/services/NetPhos/). An additional search for potential myristoylation, glycosylation or tyrosine sulfation sites of the H-REV107-1 protein revealed negative results.
Furthermore, a Western blot analysis of COS-7 cells transiently transfected with an H-REV107-1 expression vector revealed two bands of a slightly different mobility in SDS-PAGE (Fig. 12), which also suggests some kind of posttranslational modification.
Thus, the Lex-A yeast two hybrid assay identified only putative interactors of H-REV107-1 which can bind to the non-modified protein.
Fig. 36 Prediction of protein phosphorylation sites of the H-REV107-1 protein Five tyrosine and one serine phosphorylation sites were identified using NetPhos program. These residues are shown in red.
Unfortunately, after we had established the LexA yeast hybrid system, several new two-hybrid approaches were developed, allowing an improved search for interacting proteins.
One example is the CytoTrap two-hybrid system, which enables the discovery of protein-protein interactions in the cytoplasm of yeast cells. This system is based on the activation of Ras signaling pathways in a temperature-sensitive yeast cell line via the recruitment to the cellular membrane of human Sos protein (hSos) fused to the bait. The target proteins are anchored to the membrane. Therefore, interaction between bait and target leads to a close proximity of the hSos and Ras proteins. The hSos protein activates Ras by GDP/GTP exchange, and allows the yeast cells to grow at 37°C. The system was developed for the identification of interacting partners of the Jun protein (Aronheim et al., 1997). It is very well adapted for the identification of protein interactions which take place in the cytoplasm. An interesting modification of this system is, the so called “reverse Ras recruitment system”, in which the bait is anchored to the cellular membrane, and the protein partner (the prey) is fused to a cytoplasmic Ras mutant. Protein–proteininteraction between the prey and thebait results in Ras membrane translocation and activation of a viability pathway in yeast (Hubsman et al., 2001).
One of the possibilities to identify interactions requiring tyrosine phosphorylation is the so called “TK (tyrosine kinase) two-hybrid system”. This system is very similar to the standard LexA-based approach. The difference is that an additional tyrosine kinase domain is ligated into the LexA-vector, containing the bait. Expression of this vector in yeast leads to the expression of a bait, and a tyrosine kinase which then phosphorylates the bait protein. Thus, using this system, a specific search of interacting partners of the tyrosine phosphorylated protein can be performed (Vojtek and Hollenberg, 1995).
H-REV107-1
MRAPIPEPKPGDLIEIFRPFYRHWAIYVGDGYVVHLAPPSE 41
VAGAGAASVMSALTDKAIVKKELLYDVAGSDKYQVNNKHD 80
DKYSPLPCTKIIQRAEELVGQEVLYKLTSENCEHFVNELRY 122
GVARSDQVRDVIIAASVAGMGLAAMSLIGVMFSRNKRQKQ 162
We screened a human kidney cDNA library with a truncated form of the H-REV107-1 protein.
After rescue of the yeast plasmids from positive colonies, and the following sequencing analysis, we searched for homology of the isolated cDNA inserts with known genes in the NCBI database to chose targets for further investigation. This step of analysis is the most critical in the two-hybrid assay. Among the isolated 168 clones, only a few sequences were present more than one time. We excluded typical predicted false positives from further analysis, although it might lead to the loss of putative true positives. For example, heat shock proteins were described to bind many targets unspecifically in yeast (Golemis et al., 1994).
Therefore, we excluded the Hsp90 heat shock protein from further analysis. However in many publications it was demonstrated that Hsp90 is a molecular chaperone whose association is required for the stability and function of multiple mutated, and over-expressed signaling proteins that promote cancer growth and cell survival. Hsp90 client proteins include mutated p53, Bcr-Abl, Raf-1, c-Src, Akt, HER2 (Neckers, 2003). It participates in the prevention of tumor cells from apoptosis (Rashmi et al., 2003). Inhibition of Hsp90 results in induction of apoptosis through the activation of caspases–9 and -3 in AML HL-60 cells because of the attenuation of Hsp90-interacting proteins, including Akt, c-Raf-1, and c-Src protein kinases (Nimmanapalli et al., 2003). We demonstrated that ectopic expression and induction by IFNγ of H-REV107-1 in ovarian carcinoma cells leads to the induction of apoptosis through the activation of caspase-9 (Fig. 36). Therefore, in view of these findings, an interaction between Hsp90 and H-REV107-1 might also be plausible and should not entirely be excluded.
We also found a number of unknown genes, which we did not prove in a mating assay, and which were not further analysed in mammalian cells. Putative H-REV107-1 interacting partners might be among them. Therefore we recently repeated the BLAST search of sequences found three years ago. For most inserts still no homologies were found. However, the analysis revealed one novel gene, TSGA10 isolated in 2001 (Madarressi et al., 2001) without any known function. In addition two cDNA sequences encode proteins which were characterised in the meantime. One of them is the acid cluster protein ACP33, isolated as a novel intracellular binding partner of CD4, proposed to modulate a stimulatory activity of CD4 in T cells (Zeitlmann et al., 2001). The second cDNA insert encodes 100 amino acids at the C-terminus of the novel human protein Scribble, a homologue of the Drosophila protein scrib.
Scrib dysfunction results in the false distribution of apical proteins (Bilder and Perrimon, 2001). It has been demonstrated that scrib acts as a tumor suppressor and participates together with two other tumor suppressors, lethal giant larvae (lgl) and discs-large (dlg) in the regulation of cell polarity and growth control (Bilder et al., 2000). The human homologue, hScrib, was shown to be a target of the papillomavirus (HPV) E6 protein, which stimulates its
Based on the performed BLAST search analysis, and available literature about found potential interacting partners of H-REV107-1, we chose 11 clones for a mating test (Table 5).
The mating assay is a powerful supply for the verification of false positives from the yeast two-hybrid system. Using this assay, we tested whether induction of the reporter genes is a specific response based on the interaction between H-REV107-1 and the corresponding target protein. The assay resulted in the exclusion of 5 proteins from further analysis, because their ability to activate reporter genes was independent of the H-REV107-1 expression (Fig. 7).
For further conformation in the mammalian system, we chose 6 proteins, namely PC4, PR65, RARG, S100A6, ETF1, and P14.5. The aim of the investigation was to prove if the interactions identified in yeast also occur in mammalian cells, and to define a role of the confirmed protein-protein bindings in the H-REV107-1 - mediated growth suppression, and induction of cell death. We verified that H-REV107-1 protein interacts with PC4, PR65, RARG, S100A6, and ETF1 proteins, but not with the P14.5 protein in transfected COS-7 cells.
4.2 H-REV107-1 is a Target of IRF-1 and Modulates IFNγ - Dependent Inhibition of