In the course of our studies of chromosomal aberrations in benign tumors, we have previously discovered the LPP
gene as being rearranged in certain subtypes of these tumors [2
], and identified the LPP protein as a member of the zyxin family of proteins [11
]. In this study, we report that LPP specifically interacts with Scrib. We provide evidence that this interaction is mediated by the carboxy-terminus of LPP on the one hand, and the PDZ domains of Scrib on the other hand. Futhermore, we show that Scrib is not necessary for targeting LPP to focal adhesions, and that Scrib and LPP are dispensable to target each other to cell-cell contacts.
Scrib is a member of the LAP (LRR (leucine-rich repeat) and PDZ (PSD-95/Discs-large/ZO-1)) family of membrane-associated proteins that play a role in the regulation of cell polarity [16
]. LAP family members have been identified in mammals (Erbin, Densin-180, Lano, and Scrib) [29
], in Caenorhabditis elegans
], and in Drosophila melanogaster
]. LAP proteins contain a set of leucine-rich repeats (LRRs) at their amino-terminus, and either four (Scrib and Scribble), one (Erbin, Densin-180 and LET-413) or no (Lano) copies of the PDZ domain. A specific characteristic of these proteins are the LAP-specific domains (LAPSa and b), which are located carboxy-terminally of the LRRs [34
Most information regarding the function of Scrib comes from studies in Drosophila melanogaster
Scribble was identified as being required for the apical confinement of polarity determinants in epithelia [18
]. Mutations in Scribble cause aberrant cell shapes and loss of the monolayer organization in embryonic epithelia. Scribble is localized in septate junctions and loss of Scribble function results in the misdistribution of apical proteins and adherens junctions to the basolateral cell surface. Subsequent studies in Drosophila
provided evidence that Scribble is a tumor suppressor and cooperates with two other tumor suppressors, Lethal giant larvae (Lgl) and Discs-large (Dlg) to regulate cell polarity and growth control [19
]. Recently, these three tumor suppressors were shown to regulate cell size and mitotic spindle asymmetry in Drosophila
]. The role of Scribble in tumorigenesis was further supported by the discovery that Scribble mutants cooperate with oncogenic Ras or Notch to cause neoplastic overgrowth of the eye disc [36
], and that cooperation between oncogenic Ras and inactivation of Scribble leads to metastatic behavior [37
]. Additional studies in Drosophila
implicate Scribble in the regulation of synaptic plasticity and synaptic vesicle dynamics [38
], and show that Scribble is essential for olfactory behavior in Drosophila
As for mammalian Scrib, little information is available at the moment. Relating to the control of cell polarity and proliferation, human Scrib was found to be a functional homologue of the Drosophila scribble
]. Polarity defects and tumorous overgrowth of Scribble-mutant flies are rescued by human Scrib predicting an important role for human Scrib in the suppression of mammalian tumorigenesis. Further support for this hypothesis, was obtained by the fact that human and mouse Scrib are targeted for degradation by high-risk papillomavirus E6 proteins [32
]. Human papilloma viruses cause papillomas or warts on skin, genital tissues, and the upper respiratory tract, and high-grade lesions progress to carcinomas at a high frequency. The high-risk subgroup of human papilloma viruses detected in these lesions have been causally linked to the development of over 90% of uterine cervical carcinomas, the second leading cause of cancer-related deaths among women world-wide. High-risk papilloma virus E6 proteins direct Scrib for degradation by directly binding to the PDZ-domains of Scrib.
In this regard, it is noteworthy that we observed a remarkable aspect regarding the expression levels of Scrib in a number of mammalian cell lines. As already mentioned before (Fig. ), we noticed that 293T cells expressed much lower levels of Scrib as compared to 293 cells. 293T cells are derived from 293 but, in contrast, these cells stably express Simian Virus 40 largeT antigen. SV40 large T is a powerful oncoprotein capable of transforming a variety of cell types [42
]. Its transforming activity is attributed to its binding and manipulation of the function of certain key tumor suppressors and cell regulatory proteins such as retinoblastoma and p53. However, certain factors that contribute to its full transformation potential are not yet completely understood. We hypothesize that large T induces the downregulation of Scrib expression, and that Scrib contributes to the transformation potential of SV40 large T.
In addition to its role as a tumor suppressor, Scrib was also implicated in the regulation of planar cell polarity, a role that is not established for Drosophila
], and it was shown that disruption of Scrib is the causal factor for the severe neural tube defects that occur in the circletail
]. Disruption of neural tube closure leads to a group of disorders termed neural tube defects, which are one of the commonest causes of congenital malformation and lethality in humans. The most severe form of neural tube defect is craniorachischisis, in which almost the entire brain and spinal cord remain open. Craniorachischisis comprises 10–20% of human neural tube defects, and is caused by a failure to initiate neural tube formation at the start of neurulation. Circletail
is one of only two mouse mutants that exhibit craniorachischisis. The fact that Scrib was identified as the gene that was mutated in this mouse attributes an important role for Scrib in development [44
We show here that Scrib is expressed equally well in very different cell types, such as Jurkat cells, which are human T lymphocytes, epithelial cells such as 293 and MDCKII cells, and in fibroblasts such as CV-1 cells. As described above, the function of Scrib and its Drosophila ancestor Scribble have been mainly addressed in epithelial cells. To our knowledge, nothing is known yet about the function of Scrib in other cell types such as lymphocytes and fibroblasts.
We show here that LPP specifically binds to and partially co-localizes with Scrib in cell-cell contacts of epithelial and fibroblastic cell lines. Previous studies have shown that PDZ domain proteins play an important role in the targeting of proteins to specific membrane compartments and in the assembly of these proteins into supramolecular complexes [25
]. Therefore, we investigated whether Scrib was essential to localize LPP in cell-cell contacts. However, as demonstrated by these experiments, Scrib is not necessary to target LPP to these structures. These findings are similar to what has been found for targeting of zyxin family members to focal adhesions. Recently, zyxin and TRIP6 were shown to interact with members of the p130Cas
family of signal transducers, which are focal adhesion components [22
]. This interaction is primarily mediated by the LIM domains of zyxin and TRIP6. One specific function associated with the LIM domains of zyxin family members is targeting to focal adhesions. Despite this feature of the zyxin family LIM domains, and despite their interaction with p130Cas, it was shown that p130Cas is not required for focal adhesion localization of zyxin and TRIP6 [22
]. We also investigated whether LPP was responsible for targeting of Scrib to cell-cell contacts. However, as demonstrated by our experiments, also this appeared not to be the case. In fact, our results indicate that all of the PDZ domains of Scrib are dispensable for targeting the protein to cell-cell contacts. For epithelial cells, these results are in agreement to what has been published in the course of our investigations by Legouis and Jaulin-Bastard et al
], who have shown that a point mutation of a specific proline residue that is located at position 305 in LRR number 13 of human Scrib is enough to abolish membrane localization.
Taken into account that PDZ domains vary in their range and stringency of specificity [25
], it is not excluded that LPP might bind to other PDZ domains than the ones of Scrib. Concerning Scrib, to date, three other proteins have been described that bind to the PDZ domains of Scrib: as mentioned above, the high-risk human papillomavirus E6 protein [32
] interacts with the PDZ domains of human Scrib, whereas the GUKH (guanylate kinase holder) protein was shown to bind to the PDZ domains of Scribble at Drosophila
], and very recently, mammalian Scrib was shown to form a tight complex with the βPIX exchange factor at neuronal presynaptic compartments [46
]. These findings raise the possibility that different binding partners of the Scrib PDZ domains, including LPP, can compete with each other for binding to Scrib, and as such play a role in processes in which Scrib is involved.
In this regard, it is worth mentioning that the binding of LPP to Scrib appears to be regulated. In our mitochondrial targeting experiments (Fig. ), we noticed that the full length wild-type LPP-protein was not targeted to Scrib-coated mitochondria in all cells. In fact, in the majority of these cells, full length wild-type LPP was not recruited by Scrib. We hypothesize that the binding of LPP to Scrib is regulated by an intra- or intermolecular interaction of LPP, as a result of which the carboxy-terminal tail is hidden in such a way that it is not available anymore for binding to Scrib. One piece of information that supports this hypothesis is the observation that, in contrast to full length LPP, the carboxy-terminal region containing only the LIM-domains and the tail but lacking the pre-LIM region, was efficiently recruited to Scrib-coated mitochondria in nearly 100% of the cells examined while carboxy-terminally mutated versions were not recruited (our unpublished results). Our observations are similar to what has been reported for the binding of zyxin to the tumor suppressor warts/LATS1. In in vitro binding experiments, it was demonstrated that parts of the zyxin protein containing LIM domains 1 and 2 efficiently bind to warts/LATS1 while the full length protein does not bind [47
]. Based on these findings, the authors speculated that the LIM1/2 domains are masked in full-length zyxin, and that intramolecular and/or intermolecular modifications may regulate the interaction between zyxin and warts/LATS1.