Screening for LPP-interacting proteins by yeast two-hybrid
In a previous study [13], we showed that the LIM domains of LPP are the major units for targeting LPP to focal adhesions. LIM domains are cysteine- and histidine-rich domains that form two zinc fingers capable of mediating protein-protein interactions [20,21]. However, the protein(s) that is/are responsible for the targeting of LPP to focal adhesions, i.e. protein(s) that bind(s) to the LIM domains of LPP, are not yet known. To identify protein binding partners of the LIM domains of LPP, we performed a yeast two-hybrid screening experiment. We made use of a yeast two-hybrid system that is based on transcriptional activation of two reporter genes HIS3 and LacZ whose expression is driven by upstream GAL4 DNA-binding sites. Because all three LIM domains of LPP cooperate to target LPP to focal adhesions [13], we initially focused on a screening using a bait that contained all three LIM domains. Unlike in mammalian cells, where we have shown that the three LIM domains of LPP have transcriptional activation capacity [11], this bait, although well expressed, did not activate the reporter genes in yeast cells (results not shown). This is similar to what has been found for zyxin's LIM domains [22], but in contrast to what has been found for the three LIM domains of TRIP6 that do activate reporter genes in yeast [22]. However, the bait containing all three LIM domains of LPP appeared to be very sticky since thousands of yeast colonies were obtained in which both reporter genes were activated. In an effort to reduce background activity, we deleted the first LIM domain, or the first and the second LIM domain, in the bait, leaving the two most carboxy-terminal, or the most carboxy-terminal LIM domain(s) intact, respectively. These deletions completely abolished all background activity making these baits the baits of choice to perform a library screening. Here, we report about the screening that was performed with the bait containing only the most carboxy-terminal LIM domain of LPP. As described before [13], we showed that the third LIM domain of LPP only has a very weak targeting capacity for focal adhesions. This makes it very unlikely that, by using this bait, we would pick up a protein that targets LPP to these structures, which was the initial goal of our studies. Indeed, our screening did not reveal any focal adhesion binding partners of LPP, however, in stead, we found another very interesting LPP-interacting protein as will be outlined in the following sections.
A mouse embryonal cDNA library was screened using a bait (pGBT9-LPPWT) containing the third LIM domain and carboxy-terminus of human LPP (amino acids 531–612). Among ~1.0 × 106 yeast cotransformants (Leu+ and Trp+), 56 clones were His+ of which 23 were LacZ+ too. PCR analysis of these His+/LacZ+ clones, using prey-specific insert-flanking primers, revealed that 21 of the 23 obtained clones, contained a prey-construct having a 2 kb cDNA insert (results not shown). Subsequent fragmentation of the obtained 2 kb PCR products, representing the cDNA inserts of the prey-constructs, using the HaeIII restriction enzyme (frequent cutter), indicated that all 21 isolated prey-constructs, having a 2 kb insert, were identical. The 2 kb cDNA insert of one representative prey-construct was completely sequenced and the sequence was submitted to the NCBI database (Genbank accession no. AF271735).
A BLAST (Basic Local Alignment Search Tool)-search revealed that this mouse prey-construct encoded an amino- and carboxy-terminally truncated protein comprising four PDZ domains that was almost identical to the human Scrib protein (Fig. 1B), indicating that the prey-construct represented mouse Scrib. The Scrib protein contains a set of 16 leucine-rich repeats (LRRs) near its amino-terminus and four PDZ (PSD-95, Discs large, ZO-1) domains distributed throughout the remainder of the protein (Fig. 1B). The partial mouse Scrib protein, expressed by the prey-construct, corresponded to amino acids 709 – 1242 of human Scrib (Fig. 1B).
Further analysis indicated that the isolated prey-construct, which was named pACT2-mScrib, activated the HIS3 and LacZ reporter genes of the yeast only in the presence of pGBT9-LPPWT, identifying pACT2-mScrib as a true positive (Table 1, upper three rows).
Table 1  Interaction of LPP with Scrib in the yeast two-hybrid system  Yeast cells (CG-1945), cotransformed with a bait and a prey as indicated, were selected on medium containing 5 mM 3-AT, lacking Trp, Leu and His. Yeast colonies were tested for the expression of β-galactosidase.
+ indicates strong positive interaction; - indicates no interaction.