Analysis of chromosomal aberrations occurring in lipomas led to the identification of the LPP
gene as the preferred translocation partner of HMGIC
, a gene encoding an architectural factor. The deduced LPP amino acid sequence (Petit et al., 1996
) revealed that LPP belongs to a family of LIM domain proteins, including transcription factors and components of cell adhesion sites (Beckerle, 1997
). Our results show that despite their low sequence identity (41%), LPP and zyxin likely share overlapping functional properties, but they also have distinct features suggesting that each protein may fulfill specific roles in a cell. Moreover, we obtained evidence that LPP, a member of this new family, may participate in the control of gene transcription.
Targeting of proteins to distinct intracellular domains plays an essential role in the control of their activity. Although the localization of LPP in focal adhesions was similar to that of zyxin, LPP was not localized along stress fibers, in contrast to zyxin. Because overproduction of LPP in transfected cells did not result in its codistribution with stress fibers, it is unlikely that competition between LPP and zyxin for common binding sites would account for this difference in localization. Although zyxin and LPP share a similar domain organization, their sequences differ considerably. Therefore, it is conceivable that the ligands responsible for the targeting of LPP and zyxin, proteins that are not yet identified, are different. In the case of paxillin, an adapter protein with multiple binding motifs, it has been shown that the last LIM domain is responsible for its targeting to focal adhesions (Brown et al., 1996
). Interestingly, whereas the HMGIC/LPP-short fusion localized solely to the nucleus, HMGIC/LPP-long could, in addition, localize to focal contacts when overexpressed in transfected cells. This observation opens the possibility that a small portion of the proline-rich domain (amino acids 372–413) and/or the first LIM domain of LPP may, at least partially, be responsible for directing LPP to focal adhesions.
LPP contains two FPPPP repeats, motifs that have been shown to be important for the interaction with proteins of the Ena/VASP family (Niebuhr et al., 1997
; Fedorov et al., 1999
; Prehoda et al., 1999
). Using an overlay assay with a VASP-enriched cell fraction, we found that the N-terminal proline-rich region of LPP interacts directly with this protein in vitro. This finding suggests that at least one of the two potential VASP-binding motifs of LPP is functional, validating the consensus sequence for Ena/VASP binding determined with peptides (Niebuhr et al., 1997
). The weaker binding signal obtained with LPP compared with ActA is most likely due to the fact that LPP contains only two VASP/Mena-binding sites, whereas ActA has four. Furthermore, our coimmunoprecipitation data, together with the observation that the proline-rich domain of LPP recruits VASP in vivo when targeted to an ectopic location on mitochondria, suggest that LPP and VASP are present in a complex in cultured cells.
By virtue of binding profilin, an actin monomer-binding protein that increases the rate of actin polymerization in vitro, Ena/VASP family members may control actin dynamics (Theriot and Mitchison, 1993
; Gertler et al., 1996
; Reinhard et al., 1996
; Golsteyn et al., 1997a
). The activity of Ena/VASP may be spatially controlled by scaffold proteins that recruit them to specific subcellular domains. Quantitation of the amount of LPP and zyxin revealed that these two proteins coexist in primary cells, as has been shown in platelets and HFF cells (Golsteyn et al., 1997b
; this report) and suggested by Northern blot analysis of tissues (Macalma et al., 1996
; Petit et al., 1996
). In addition to zyxin and LPP, the focal adhesion protein vinculin is also able to interact with Ena/VASP family members (Reinhard et al., 1996
). The biological significance of this partial functional redundancy remains unknown. Regulation by distinct signaling pathways as well as differences in the binding specificities or affinities for their ligands (Reinhard et al., 1996
; Niebuhr et al., 1997
) may allow each of these proteins to play a specific role in the fine tuning of actin dynamics in cells.
The accumulation of LPP in the nucleus of leptomycin B–treated cells suggests that nuclear export of LPP relies on a CRM1-dependent mechanism. Indeed, we found that an N-terminally located leucine-rich stretch of residues sharing sequence homology with well-characterized NES sequences is essential for nuclear export of LPP. Chicken zyxin contains another NES, located at the hinge between the proline-rich region and the LIM domains, that is partially conserved in LPP (Nix and Beckerle, 1997
). However, the deletion of this sequence in LPP had no effect on its intracellular distribution, confirming that the NES in LPP is different from that in chicken zyxin. Differences of intramolecular location between these NES sequences may reflect a different regulation of the export of zyxin and LPP. The observation that LPP is transiently located in the nucleus raises the question of how this protein is imported into this compartment. Because LPP does not present a consensus nuclear localization signal, it may be imported via an interaction with a nuclear localization signal–containing protein.
In agreement with the observation that LPP is able to shuttle between the cytoplasm and the nucleus, we found that LPP has significant transcriptional activation capacity in a GAL4 transactivation assay in mammalian cells. While this article was under review, transcription activation capacity, measured in a similar assay, was reported for TRIP6, a protein sharing 53% sequence identity with LPP (Wang et al., 1999
). This finding further emphasizes that LPP belongs to a new family, the members of which are involved in cell surface–nucleus communication. Interestingly, GAL4-LPP fusions that were undetectable in the nucleus by immunofluorescence still retained significant activity in the GAL4 assay. The activity of these fusion proteins was increased upon deletion of the NES sequence and their accumulation in the nucleus. These results suggest, first, that small amounts of endogenous LPP present in the nucleus at steady state may be sufficient for transcription activation, and second, that the spatial control of LPP and its transcription activation capacity are closely linked.
To date, the mechanism by which LPP activates transcription remains unknown. Although LIM domains have a similar structural organization as zinc-finger DNA-binding sequences, only LIM domains of hic5 have been shown to interact directly with DNA (Nishiya et al., 1998
). Because there is substantial evidence that LIM domains function in protein-protein interactions, it is more likely that LPP interacts with a component (or components) of a transcriptional complex (Wadman et al., 1997
). Because the activity of LPP depends on its intracellular distribution, it is possible that LPP modulates the activity of transcription factors by controlling their spatial distribution.
We found that the proline-rich domain and LIM domains contribute to the transcription activation capacity of LPP. The LPP sequence 248–413 is essential for this activity and has an ~40-fold higher activity than the full-length GAL4-LPP fusion. Thus, it is possible that intramolecular interactions may regulate the activity of the full-length protein. Alternatively, the N-terminal portion of LPP (amino acids 1–247), which has no activity per se, may be involved in the binding of this protein to the cell surface, thereby decreasing the pool of LPP available for nuclear function.
HMGIC/LPP fusion proteins may interfere with the normal function of the HMGIC and/or LPP proteins in tumors. No transcriptional activation could be detected in the GAL4 assay for the nuclear HMGIC/LPP fusion proteins, suggesting that the transcriptional activation capacity of the LIM domains of LPP is negatively influenced by fusion to the DNA-binding domains of HMGIC in this assay. Wild-type HMGIC is thought to act as an architectural factor that binds to AT-rich sequences, thereby modulating the activity of other transcription factors (Goodwin, 1998
; Mantovani et al., 1998
). Thus, it cannot be excluded that if the DNA-binding domains of HMGIC bind to specific chromosomal structures in a physiological situation, the transcription activation capacity of the LIM domains is exposed, a possibility that our assay did not test.
Signaling pathways that control the organization of the actin cytoskeleton and cell proliferation are closely linked. Here we showed that LPP
, a gene that is targeted by the preferential t(3;12)(q27-q28;q15) chromosomal translocation in lipomas, encodes a protein with multiple functional domains that are important for its interaction with proteins of focal adhesions and cell-to-cell contacts as well as for its transcription activation capacity. Although the physiological role of LPP is still unknown, our findings open the possibility that LPP may serve as a scaffold on which distinct protein complexes are assembled in the cytoplasm and the nucleus. Similar to the adherens junction protein β-catenin, LPP may participate in the organization of the cortical actin cytoskeleton at cell adhesion sites and in the regulation of gene transcription (Behrens et al., 1996
). Knowledge obtained in the present study will constitute the basis for further investigations of the biological function of LPP and of its role as well as that of HMGIC/LPP fusions in tumorigenesis.