There is a direct interaction between the carboxy-terminus of LPP and the PDZ domains of Scrib
To further assess the binding between LPP and Scrib, we investigated whether there is a direct interaction between these two proteins. For this, we performed GST pull-down experiments. In vitro translated full length Scrib was tested for binding with glutathione beads, which were coupled with GST-LPP-LTWT, GST-LPP-LTL612A, or GST alone. GST-LPP-LTWT contains 40 amino acids of the pre-LIM region, the three LIM domains, and the wild-type carboxy-terminal tail of human LPP. GST-LPP-LTL612A is identical to GST-LPP-LTWT except for a point mutation to alanine introduced at leucine612 (position 0). All GST-fusion proteins as well as GST alone were expressed well in E. coli (Fig. 6A). As shown in Fig. 6B, Scrib interacted specifically with the wild-type LPP protein but not with its mutated form, GST-LPP-LTL612A or with GST alone. These results indicate that there is a specific and direct interaction between LPP and Scrib.
Figure 6  Direct interaction between the carboxy-terminus of LPP and the PDZ domains of Scrib. (A) GST fused to either wild-type LPP (40 amino acids of the pre-LIM region, the three LIM domains and the tail), or a similar LPP molecule with a mutated carboxy-terminus (L612A) and GST alone were expressed in E. coli, purified and analyzed by SDS-PAGE and Coomassie Blue staining. All proteins were expressed well. Protein markers are as indicated. (B) In vitro synthesized [35S]-methionine-labelled full length Scrib was incubated with immobilized GST or with either one of the above-described GST fusion proteins and allowed to interact over night at 4°C. After extensive washing, bound proteins were eluted in sample buffer, separated by SDS-PAGE and visualized by autoradiography. The amount of synthesized protein loaded as a reference on the gel corresponds to 10% of the input used in each binding experiment. (C) All four PDZ domains of Scrib (amino acids 616 – 1490), either wild-type or mutated as indicated, were synthesized in vitro and [35S]-methionine-labelled. these labelled proteins were incubated with immobilized GST or with GST-LPP-LTWT and allowed to interact over night at 4°C. Bound proteins were eluted in sample buffer, separated by SDS-PAGE and visualized by autoradiography. The amount of synthesized protein loaded as a reference on the gel corresponds to 10% of the input used in each binding experiment.   To further investigate the requirements in the Scrib protein for binding to LPP, we performed additional GST pull-down experiments. From our previously described experiments (yeast and mammalian two-hybrid), it was clear that the PDZ domains of Scrib bind to LPP. These findings were confirmed by using GST pull-down: as shown in Fig. 6C, upper panel, a portion of the Scrib protein encompassing all four PDZ domains was efficiently pulled down by GST-LPP-LTWT. To find out which of the four PDZ domains of Scrib was responsible for the observed interaction with LPP, we mutated the PDZ domains of Scrib, one at the time, by destroying their carboxylate binding loop (LG → AE), and tested how efficiently these mutated proteins were pulled down by GST-LPP-LTWT. From the results, which are presented in Fig. 6C, we can conclude that all four PDZ domains of Scrib more or less contribute to the binding to LPP, but that PDZ 3 is most important, since binding to GST-LPP-LTWT was almost completely abolished when the carboxylate binding loop of this PDZ domain was destroyed.