PP2A binds to the LIM domains of lipoma-preferred partner through its PR130/B″ subunit to regulate cell adhesion and migration.

Here, we identify the LIM protein lipoma-preferred partner (LPP) as a binding partner of a specific protein phosphatase 2A (PP2A) heterotrimer that is characterised by the regulatory PR130/B″α1 subunit (encoded by PPP2R3A). The PR130 subunit interacts with the LIM domains of LPP through a conserved Zn²âº-finger-like motif in the differentially spliced N-terminus of PR130. Isolated LPP-associated PP2A complexes are catalytically active. PR130 colocalises with LPP at multiple locations within cells, including focal contacts, but is specifically excluded from mature focal adhesions, where LPP is still present. An LPP-PR130 fusion protein only localises to focal adhesions upon deletion of the domain of PR130 that binds to the PP2A catalytic subunit (PP2A/C), suggesting that PR130-LPP complex formation is dynamic and that permanent recruitment of PP2A activity might be unfavourable for focal adhesion maturation. Accordingly, siRNA-mediated knockdown of PR130 increases adhesion of HT1080 fibrosarcoma cells onto collagen I and decreases their migration in scratch wound and Transwell assays. Complex formation with LPP is mandatory for these PR130-PP2A functions, as neither phenotype can be rescued by re-expression of a PR130 mutant that no longer binds to LPP. Our data highlight the importance of specific, locally recruited PP2A complexes in cell adhesion and migration dynamics.

Smooth muscle expression of lipoma preferred partner is mediated by an alternative intronic promoter that is regulated by serum response factor/myocardin.

Lipoma preferred partner (LPP) was recently recognized as a smooth muscle marker that plays a role in smooth muscle cell migration. In this report, we focus on the transcriptional regulation of the LPP gene. In particular, we investigate whether LPP is directly regulated by serum response factor (SRF). We show that the LPP gene contains 3 evolutionarily conserved CArG boxes and that 1 of these is part of an alternative promoter in intron 2. Quantitative RT-PCR shows that this alternative promoter directs transcription specifically to smooth muscle containing tissues in vivo. By using chromatin immunoprecipitation, we demonstrate that 2 of the CArG boxes, including the promoter-associated CArG box, bind to endogenous SRF in cultured aortic smooth muscle cells. Electrophoretic mobility-shift assays show that the conserved CArG boxes bind SRF in vitro. In reporter experiments, we show that the alternative promoter has transcriptional capacity that is dependent on SRF/myocardin and that the promoter associated CArG box is required for that activity. Finally, we show by quantitative RT-PCR that the alternative promoter is strongly downregulated in SRF-deficient embryonic stem cells and in smooth muscle tissues derived from conditional SRF knockout mice. Collectively, our data demonstrate that expression of LPP in smooth muscle is mediated by an alternative promoter that is regulated by SRF/myocardin.

Lpp is involved in Wnt/PCP signaling and acts together with Scrib to mediate convergence and extension movements during zebrafish gastrulation.

The zyxin-related LPP protein is localized at focal adhesions and cell-cell contacts and is involved in the regulation of smooth muscle cell migration. A known interaction partner of LPP in human is the tumor suppressor protein SCRIB. Knocking down scrib expression during zebrafish embryonic development results in defects of convergence and extension (C&E) movements, which occur during gastrulation and mediate elongation of the anterior-posterior body axis. Mediolateral cell polarization underlying C&E is regulated by a noncanonical Wnt signaling pathway constituting the vertebrate planar cell polarity (PCP) pathway. Here, we investigated the role of Lpp during early zebrafish development. We show that morpholino knockdown of lpp results in defects of C&E, phenocopying noncanonical Wnt signaling mutants. Time-lapse analysis associates the defective dorsal convergence movements with a reduced ability to migrate along straight paths. In addition, expression of Lpp is significantly reduced in Wnt11 morphants and in embryos overexpressing Wnt11 or a dominant-negative form of Rho kinase 2, which is a downstream effector of Wnt11, suggesting that Lpp expression is dependent on noncanonical Wnt signaling. Finally, we demonstrate that Lpp interacts with the PCP protein Scrib in zebrafish, and that Lpp and Scrib cooperate for the mediation of C&E.

Targeted disruption of the mouse Lipoma Preferred Partner gene.

LPP (Lipoma Preferred Partner) is a zyxin-related cell adhesion protein that is involved in the regulation of cell migration. We generated mice with a targeted disruption of the Lpp gene and analysed the importance of Lpp for embryonic development and adult functions. Aberrant Mendelian inheritance in heterozygous crosses suggested partial embryonic lethality of Lpp(-/-) females. Fertility of Lpp(-/-) males was proven to be normal, however, females from Lpp(-/-) x Lpp(-/-) crosses produced a strongly reduced number of offspring, probably due to a combination of female embryonic lethality and aberrant pregnancies. Apart from these developmental and reproductive abnormalities, Lpp(-/-) mice that were born reached adulthood without displaying any additional macroscopic defects. On the other hand, Lpp(-/-) mouse embryonic fibroblasts exhibited reduced migration capacity, reduced viability, and reduced expression of some Lpp interaction partners. Finally, we discovered a short nuclear form of Lpp, expressed mainly in testis via an alternative promoter.

The LIM domain protein LPP is a coactivator for the ETS domain transcription factor PEA3.

PEA3 is a member of a subfamily of ETS domain transcription factors which is regulated by a number of signaling cascades, including the mitogen-activated protein (MAP) kinase pathways. PEA3 activates gene expression and is thought to play an important role in promoting tumor metastasis and also in neuronal development. Here, we have identified the LIM domain protein LPP as a novel coregulatory binding partner for PEA3. LPP has intrinsic transactivation capacity, forms a complex with PEA3, and is found associated with PEA3-regulated promoters. By manipulating LPP levels, we show that it acts to upregulate the transactivation capacity of PEA3. LPP can also functionally interact in a similar manner with the related family member ER81. Thus, we have uncovered a novel nuclear function for the LIM domain protein LPP as a transcriptional coactivator. As LPP continually shuttles between the cell periphery and the nucleus, it represents a potential novel link between cell surface events and changes in gene expression.

HMGA2 regulates transcription of the Imp2 gene via an intronic regulatory element in cooperation with nuclear factor-kappaB.

IMP2 (insulin-like growth factor-II mRNA binding protein 2) is an oncofetal protein that is aberrantly expressed in several types of cancer. We recently identified the Imp2 gene as a target gene of the architectural transcription factor HMGA2 (high mobility group A2) and its tumor-specific truncated form HMGA2Tr. In this study, we investigated the mechanism via which HMGA2 regulates Imp2 gene expression. We show that HMGA2 and HMGA2Tr directly regulate transcription of the Imp2 gene by binding to an AT-rich regulatory region located in the first intron. In reporter experiments, we show that this AT-rich regulatory region mimics the response of the endogenous Imp2 gene to HMGA2 and HMGA2Tr. Furthermore, we show that a consensus nuclear factor-kappaB (NF-kappaB) binding site located immediately adjacent to the AT-rich regulatory region binds NF-kappaB and that NF-kappaB and HMGA2 cooperate to regulate Imp2 gene expression. Finally, we provide evidence that there is a strong and statistically significant correlation between HMGA2 and IMP2 gene expression in human liposarcomas.

Transactivation functions of the tumor-specific HMGA2/LPP fusion protein are augmented by wild-type HMGA2.

The gene encoding the architectural transcription factor HMGA2 is frequently rearranged in several benign tumors of mesenchymal origin. The lipoma preferred partner (LPP) gene is the most frequent translocation partner of HMGA2 in a subgroup of lipomas, which are benign tumors of adipose tissue. In these lipomas, HMGA2/LPP fusion transcripts are expressed, which encode for the three AT-hooks of HMGA2 followed by the two most carboxyl-terminal LIM domains (protein-protein interaction domains) of LPP. Identical fusion transcripts are also expressed in other benign mesenchymal tumors. Previous studies revealed that the LIM domains of LPP have transcriptional activation capacity in GAL4-based luciferase reporter assays. Here, we show that the HMGA2/LPP fusion protein retains the transactivation functions of the LPP LIM domains and thus functions as transcription factor. The HMGA2/LPP fusion protein activates transcription from the well-characterized PRDII element, which is a part of the IFN-beta enhancer and which is known to bind to HMGA2. We also show that HMGA2/LPP activates transcription from the BAT-1 element of the rhodopsin promoter, a HMGA1-binding element. HMGA1 is a closely related family member of HMGA2. Finally, in a number of lipomas, HMGA2/LPP and HMGA2 are coexpressed, and HMGA2 augments the transactivation functions of HMGA2/LPP. These results support the concept that the transactivation functions of the novel HMGA2/LPP transcription factor contribute to lipomagenesis.

The tumor suppressor Scrib interacts with the zyxin-related protein LPP, which shuttles between cell adhesion sites and the nucleus.

BACKGROUND: At sites of cell adhesion, proteins exist that not only perform structural tasks but also have a signaling function. Previously, we found that the Lipoma Preferred Partner (LPP) protein is localized at sites of cell adhesion such as focal adhesions and cell-cell contacts, and shuttles to the nucleus where it has transcriptional activation capacity. LPP is a member of the zyxin family of proteins, which contains five members: ajuba, LIMD1, LPP, TRIP6 and zyxin. LPP has three LIM domains (zinc-finger protein interaction domains) at its carboxy-terminus, which are preceded by a proline-rich pre-LIM region containing a number of protein interaction domains. RESULTS: To catch the role of LPP at sites of cell adhesion, we made an effort to identify binding partners of LPP. We found the tumor suppressor protein Scrib, which is a component of cell-cell contacts, as interaction partner of LPP. Human Scrib, which is a functional homologue of Drosophila scribble, is a member of the leucine-rich repeat and PDZ (LAP) family of proteins that is involved in the regulation of cell adhesion, cell shape and polarity. In addition, Scrib displays tumor suppressor activity. The binding between Scrib and LPP is mediated by the PDZ domains of Scrib and the carboxy-terminus of LPP. Both proteins localize in cell-cell contacts. Whereas LPP is also localized in focal adhesions and in the nucleus, Scrib could not be detected at these locations in MDCKII and CV-1 cells. Furthermore, our investigations indicate that Scrib is dispensable for targeting LPP to focal adhesions and to cell-cell contacts, and that LPP is not necessary for localizing Scrib in cell-cell contacts. We show that all four PDZ domains of Scrib are dispensable for localizing this protein in cell-cell contacts. CONCLUSIONS: Here, we identified an interaction between one of zyxin's family members, LPP, and the tumor suppressor protein Scrib. Both proteins localize in cell-cell contacts. This interaction links Scrib to a communication pathway between cell-cell contacts and the nucleus, and implicates LPP in Scrib-associated functions.

The tumor suppressor Scrib selectively interacts with specific members of the zyxin family of proteins.

The zyxin family of proteins consists of five members, ajuba, LIMD1, LPP, TRIP6 and zyxin, which localize at cell adhesion sites and shuttle to the nucleus. Previously, we established that LPP interacts with the tumor suppressor Scrib, a member of the leucine-rich repeat and PDZ (LAP) family of proteins. Here, we demonstrate that Scrib also interacts with TRIP6, but not with zyxin, ajuba, or LIMD1. We show that TRIP6 directly binds to the third PDZ domain of Scrib via its carboxy-terminus. Both proteins localize in cell-cell contacts but are not responsible to target each other to these structures. In the course of our experiments, we also characterized the nuclear export signal of human TRIP6, and show that LIMD1 is localized in focal adhesions. The binding between two of zyxin's family members and Scrib links Scrib to a communication pathway between cell-cell contacts and the nucleus, and implicates these zyxin family members in Scrib-associated functions.

Differential regulation of the insulin-like growth factor II mRNA-binding protein genes by architectural transcription factor HMGA2.

The developmentally regulated architectural transcription factor, high mobility group A2 (HMGA2), is involved in growth regulation and plays an important role in embryogenesis and tumorigenesis. Little is known, however, about its downstream targets. We performed a search for genes of which expression is strongly altered during embryonic development in two HMGA2-deficient mouse strains, which display a pygmy-phenotype, as compared to wild-type mice. We found that the insulin-like growth factor II mRNA-binding protein 2 gene (IMP2), but not its family members IMP1 and IMP3, was robustly downregulated in mutant E12.5 embryos. Furthermore, we show that wild-type HMGA2 and its tumor-specific truncated form have opposite effects on IMP2 expression. Our results clearly indicate that HMGA2 differentially regulates expression of IMP family members during embryogenesis.

The focal adhesion and nuclear targeting capacity of the LIM-containing lipoma-preferred partner (LPP) protein.

Targeting of proteins to a particular cellular compartment is a critical determinant for proper functioning. LPP (LIM-containing lipoma-preferred partner) is a LIM domain protein that is localized at sites of cell adhesion and transiently in the nucleus. In various benign and malignant tumors, LPP is present in a mutant form, which permanently localizes the LIM domains in the nucleus. Here, we have investigated which regions in LPP target the protein to its subcellular locations. We found that the LIM domains are the main focal adhesion targeting elements and that the proline-rich region of LPP, which harbors binding sites for alpha-actinin and vasodilator-stimulated phosphoprotein (VASP), has a weak targeting capacity. All of the LIM domains of LPP cooperate in order to provide robust targeting to focal adhesions, and the linker between LIM domains 1 and 2 plays a pivotal role in this targeting. When overexpressed in the cytoplasm of cells, the LIM domains of LPP can deplete endogenous LPP and vinculin from focal adhesions. The proline-rich region of LPP contains targeting sites for focal adhesions and stress fibers that are distinct from the alpha-actinin and VASP binding sites, and the LPP LIM domains are dispensable for targeting LPP to the nucleus. Our studies have defined novel functional domains in the LPP protein.