LPP interacts with Scrib PDZ domains in mammalian cells
We verified the Scrib-LPP interaction, which was identified in yeast cells, in mammalian two-hybrid experiments. Doing the assay in mammalian cells rather than in yeast cells, provides a more physiological environment: proteins are more likely to be in their physiological configuration, i.e. appropriately folded and modified posttranslationally, etc. Interaction between bait- and prey-proteins in a mammalian two-hybrid assay takes place in the nucleus. For an accurate performance of this assay, this means that bait- and prey-proteins should be localized in the nucleus. In contrast to the yeast assays, where we used partial bait-proteins, we wanted to use full length bait-proteins in the mammalian assay. However, since LPP contains a nuclear export signal (NES) (amino acids 117–128) in its pre-LIM region [11], we used bait-proteins in which this NES had been deleted. To verify whether deletion of the NES in LPP induced nuclear accumulation of the bait-proteins that were used in the mammalian two-hybrid assay, we introduced wild-type and mutated LPP-bait-proteins in 293T cells. While pM-LPPWT, containing GAL4-fused full length wild-type human LPP with an intact NES, was excluded from nuclei, pM-LPPdNESWT, containing GAL4-fused full length human LPP with a deletion of the NES, was accumulating in the nuclei of the cells (results not shown). These results indicate that deletion of the NES in the LPP bait proteins used in this study indeed induce nuclear accumulation of these proteins. To perform the mammalian two-hybrid experiments, we used as baits: pM-LPPdNESWT, containing full length human LPP with a deletion of its NES, and pM-LPPdNEST610A and pM-LPPdNESL612A, which are identical to pM-LPPdNESWT except for a point mutation to alanine introduced at threonine610 (position -2) and leucine612 (position 0), respectively. As determined in the yeast two-hybrid assay, each of the threonine610 and leucine612 residues is critical for the interaction with Scrib. As prey-protein, we used pSNATCH-hScribPDZ containing a part of the human Scrib protein (amino acids 669–1233) encompassing all four PDZ-domains. As summarized in Fig. 2, the interaction between wild-type full length LPP and Scrib PDZ domains resulted in high levels of luciferase reporter activity. These high levels dropped to background levels when pM-LPPdNEST610A or pM-LPPdNESL612A were used as baits in combination with Scrib as prey. The "background" levels of luciferase that were detected when pM-LPP-baits were used in combination with pSNATCH (empty prey-vector) as prey, are due to the intrinsic transcriptional activation activity of the LPP protein [11].
Figure 2  Scrib interacts with LPP in mammalian cells. pM-bait- and pSNATCH-prey-constructs were cotransfected into 293 cells in the combination indicated, together with a GAL4-regulated luciferase reporter and a CMV-β-galactosidase internal control. Cell lysates were assayed for luciferase activity 18–24 hours after transfection. Relative luciferase activity is reported as the average of three independent duplo experiments (with standard error).   These results indicate that LPP binds to Scrib PDZ domains and that this binding is abolished when amino acids at position 0 or -2 are mutated.