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• W2091210876 abstract "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 α-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 α-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. 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 α-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 α-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. LIM-containing lipoma-preferred partner focal adhesion nuclear export signal vasodilator-stimulated phosphoprotein high mobility group AT-hook 2 myeloid/lymphoid leukemia or mixed lineage leukemia green fluorescent protein thyroid hormone receptor-interacting protein 6 β-galactosidase phosphate-buffered saline leptomycin B chromosomal region maintenance 1 In recent years, it has become clear that compartmentalization within mammalian cells is a key factor for the correct functioning of the complex network of signaling pathways in these cells. Trafficking of signaling molecules between the cytoplasmic and nuclear compartments, for instance, has important implications for the magnitude and specificity of gene expression (1Aplin A.E. Juliano R.L. J. Cell Biol. 2001; 155: 187-191Crossref PubMed Scopus (62) Google Scholar). An interesting recent development is the realization that adhesion receptors and their cytoskeletal partners can regulate this nucleocytoplasmic trafficking of signaling proteins. Specialized cell adhesion sites not only play an architectural role in organizing cell structure and polarity but also are dynamic units directly involved in communication via the nuclear trafficking capability of several adhesion site-associated proteins (1Aplin A.E. Juliano R.L. J. Cell Biol. 2001; 155: 187-191Crossref PubMed Scopus (62) Google Scholar). One such protein that may play a role in this process is the LIM-containing lipoma-preferred partner (LPP).1 LPP is a protein that is composed of an extensive proline-rich N-terminal region and three C-terminal LIM domains (Fig. 1 A) (2Petit M.M.R. Mols R. Schoenmakers E.F. Mandahl N. Van de Ven W.J. Genomics. 1996; 36: 118-129Crossref PubMed Scopus (186) Google Scholar). LIM domains are cysteine- and histidine-rich double zinc finger protein motifs that comprise ∼55 residues, with the primary sequence CX 2CX 16–23HX 2CX 2CX 2CX 16–23CX 2C (where X is any amino acid) (3Bach I. Mech. Dev. 2000; 91: 5-17Crossref PubMed Scopus (483) Google Scholar, 4Dawid I.B. Breen J.J. Toyama R. Trends Genet. 1998; 14: 156-162Abstract Full Text Full Text PDF PubMed Scopus (518) Google Scholar) (Fig. 1 C). The LPP protein localizes in focal adhesions, which are membrane attachment sites of cells to the extracellular matrix (5Petit M.M. Fradelizi J. Golsteyn R.M. Ayoubi T.A. Menichi B. Louvard D. Van de Ven W.J. Friederich E. Mol. Biol. Cell. 2000; 11: 117-129Crossref PubMed Scopus (120) Google Scholar). In addition, LPP can be transiently translocated to the nucleus (5Petit M.M. Fradelizi J. Golsteyn R.M. Ayoubi T.A. Menichi B. Louvard D. Van de Ven W.J. Friederich E. Mol. Biol. Cell. 2000; 11: 117-129Crossref PubMed Scopus (120) Google Scholar). The nucleocytoplasmic distribution of this protein involves a nuclear export signal (NES) that resides in the proline-rich region (Fig. 1 A). At cell adhesions, LPP interacts with VASP (vasodilator-stimulated phosphoprotein) via its proline-rich region that contains two VASP-binding (FP4) motifs (Fig. 1 A) (5Petit M.M. Fradelizi J. Golsteyn R.M. Ayoubi T.A. Menichi B. Louvard D. Van de Ven W.J. Friederich E. Mol. Biol. Cell. 2000; 11: 117-129Crossref PubMed Scopus (120) Google Scholar). In addition, LPP also interacts with α-actinin at these sites via its α-actinin binding site located near its N terminus in the proline-rich region (Fig. 1 A). 2B. Li, L. Zhuang, M. Reinhard, and B. Trueb, submitted for publication. Although the molecular function of LPP is not yet known in detail, several characteristics of this protein suggest it has multiple functions in different compartments of the cell. LPP binding to VASP and α-actinin suggests that it has a role in certain aspects of cell motility and actin dynamics. VASP appears to have a universal role in the control of these processes (6Reinhard M. Jarchau T. Walter U. Trends Biochem. Sci. 2001; 26: 243-249Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 7Machesky L.M. Cell. 2000; 101: 685-688Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). α-Actinin is a cross-linker of filamentous actin and a dynamic constituent of focal adhesions (8Djinovic-Carugo K. Young P. Gautel M. Saraste M. Cell. 1999; 98: 537-546Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 9Edlund M. Lotano M.A. Otey C.A. Cell Motil. Cytoskeleton. 2001; 48: 190-200Crossref PubMed Scopus (112) Google Scholar). In this regard, the cytoskeletal role of LPP may be quite similar to that of zyxin, which is a family member of LPP that also localizes to focal adhesions and binds to VASP and α-actinin (10Beckerle M.C. J. Cell Biol. 1986; 103: 1679-1687Crossref PubMed Scopus (104) Google Scholar, 11Crawford A.W. Michelsen J.W. Beckerle M.C. J. Cell Biol. 1992; 116: 1381-1393Crossref PubMed Scopus (200) Google Scholar, 12Drees B. Friederich E. Fradelizi J. Louvard D. Beckerle M.C. Golsteyn R.M. J. Biol. Chem. 2000; 275: 22503-22511Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 13Reinhard M. Jouvenal K. Tripier D. Walter U. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7956-7960Crossref PubMed Scopus (162) Google Scholar). Several lines of evidence implicate zyxin in actin assembly and organization, and in cell movements that are known to depend on actin (14Nix D.A. Fradelizi J. Bockholt S. Menichi B. Louvard D. Friederich E. Beckerle M.C. J. Biol. Chem. 2001; 276: 34759-34767Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). In the nucleus, LPP harbors a significant transcriptional activation capacity residing in the proline-rich region as well as in the LIM domains suggesting that LPP is directly involved in the regulation of gene transcription (5Petit M.M. Fradelizi J. Golsteyn R.M. Ayoubi T.A. Menichi B. Louvard D. Van de Ven W.J. Friederich E. Mol. Biol. Cell. 2000; 11: 117-129Crossref PubMed Scopus (120) Google Scholar). In addition, LPP may play a role in the development of some benign and malignant tumors. In a subgroup of lipomas, which are benign tumors of adipose tissue, the LPP gene acts as the preferred translocation partner of HMGA2 and in these tumors, HMGA2/LPP fusion transcripts are expressed (2Petit M.M.R. Mols R. Schoenmakers E.F. Mandahl N. Van de Ven W.J. Genomics. 1996; 36: 118-129Crossref PubMed Scopus (186) Google Scholar, 15Schoenmakers E.F. Wanschura S. Mols R. Bullerdiek J. Van den Berghe H. Van de Ven W.J. Nat. Genet. 1995; 10: 436-444Crossref PubMed Scopus (532) Google Scholar, 16Ashar H.R. Fejzo M.S. Tkachenko A. Zhou X. Fletcher J.A. Weremowicz S. Morton C.C. Chada K. Cell. 1995; 82: 57-65Abstract Full Text PDF PubMed Scopus (412) Google Scholar). Identical fusion transcripts have also been found in a subgroup of pulmonary chondroid hamartomas (17Rogalla P. Kazmierczak B. Meyer-Bolte K. Tran K.H. Bullerdiek J. Genes Chromosomes Cancer. 1998; 22: 100-104Crossref PubMed Scopus (33) Google Scholar) as well as in a parosteal lipoma (18Petit M.M. Swarts S. Bridge J.A. de Ven W.J. Cancer Genet. Cytogenet. 1998; 106: 18-23Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In a case of acute monoblastic leukemia, the LPP gene acts as translocation partner of the MLL gene, and the tumor expresses MLL/LPP fusion transcripts (19Daheron L. Veinstein A. Brizard F. Drabkin H. Lacotte L. Guilhot F. Larsen C.J. Brizard A. Roche J. Genes Chromosomes Cancer. 2001; 31: 382-389Crossref PubMed Scopus (43) Google Scholar). All tumor-specific fusion transcripts that are expressed in the above mentioned tumors encode similar LPP fusion proteins containing AT-hooks (DNA binding domains) of the HMGA2 or MLL proteins followed by LIM domains of LPP. As we have shown before, these fusion proteins are mainly expressed in the nucleus (5Petit M.M. Fradelizi J. Golsteyn R.M. Ayoubi T.A. Menichi B. Louvard D. Van de Ven W.J. Friederich E. Mol. Biol. Cell. 2000; 11: 117-129Crossref PubMed Scopus (120) Google Scholar). As LPP appears to execute different functions depending on its intracellular localization, in focal adhesions or in the nucleus, targeting of LPP to these intracellular compartments is expected to be crucial for the differential functioning of this protein. It is possible that LPP may contribute to the tumorigenic process when its targeting is deregulated. To date however, little is known about the parts of LPP that target the protein to focal adhesions or to the nucleus. To obtain more insight into this matter, we made a variety of GFP-LPP fusion constructs containing either full-length LPP molecules or a number of mutated forms there off. We investigated the intracellular distribution of these chimeras. In this way, we were able to identify distinct regions in the LPP protein as key regulators of the subcellular distribution of LPP. Plasmids for the expression of GFP (green fluorescent protein)-tagged parts of the human LPP protein were made by subcloning the appropriate PCR products in the EcoRI andPstI sites of the pEGFP-C2 vector (Clontech). The plasmid for the expression of GFP-tagged full-length LPP was described before (5Petit M.M. Fradelizi J. Golsteyn R.M. Ayoubi T.A. Menichi B. Louvard D. Van de Ven W.J. Friederich E. Mol. Biol. Cell. 2000; 11: 117-129Crossref PubMed Scopus (120) Google Scholar). Plasmids for the expression of GFP-tagged human zyxin, GFP-tagged human TRIP6, and GFP-tagged mouse ajuba were made by cloning the appropriate cDNAs into the EcoRI and BamHI sites of the pEGFP-C1 vector (for zyxin), in the SmaI site of the pEGFP-C2 vector (for TRIP6), or in the BglII and EcoRI sites of the pEGFP-C1 vector (for ajuba). Plasmids for the expression of N-terminal GFP-tagged and C-terminal β-galactosidase (βGAL)-tagged parts of LPP were made by subcloning the appropriate PCR products in the NheI and XbaI (or SacII) sites of the pHM830 vector (20Sorg G. Stamminger T. BioTechniques. 1999; 26: 858-862Crossref PubMed Scopus (62) Google Scholar). The pHM840 construct has been described before (20Sorg G. Stamminger T. BioTechniques. 1999; 26: 858-862Crossref PubMed Scopus (62) Google Scholar). LPP constructs containing mutations or small internal deletions were made by site-directed mutagenesis using the QuikChangeTM site-directed mutagenesis kit (Stratagene) according to the supplier's instructions. All PCR amplifications were done with the Pwo DNA Polymerase (Roche Molecular Biochemicals). All synthetic mutations and PCR-amplified regions were verified by sequencing. Cell lines used in this work included CV-1 (African green monkey kidney fibroblast cells; ATCC CCL-70), NIH/3T3 (mouse embryonal fibroblast cells; ATCC CRL-1658), and 293T (human embryonal kidney epithelial cells containing SV40 T-antigen). Cell lines were grown in Dulbecco's modified Eagle's medium/F12 (1:1) (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone). All cells were cultured at 37 °C in a humidified CO2 incubator. Transient transfections were performed using FuGENETM 6 transfection reagent (Roche Molecular Biochemicals). Cells were grown on glass coverslides (coated with fibronectin in case of NIH/3T3 cells) to 50–60% confluency in 24-well plates. For each transfection 1.5 μl of FuGENETM 6 transfection reagent in 50 μl of serum-free Dulbecco's modified Eagle's medium (Invitrogen) was added to 0.5 μg of DNA and incubated at room temperature for 20 min after which the mixture was applied directly to the growth medium of the cells. Cells were incubated further at 37 °C for 18–24 h before analysis. Expression of GFP-tagged and GFP-β-galactosidase-tagged proteins was verified by Western blotting using a polyclonal rabbit anti-GFP antibody, dilution 1:5000 (Santa Cruz Biotechnology). Cell extracts from transfected cells in 24-well plates were prepared by washing the cells three times in phosphate-buffered saline (PBS) followed by direct lysis in 100 μl of SDS-PAGE sample buffer (60 mm Tris-HCl, pH 6.8, 12% glycerol, 4% SDS, 5% β-mercaptoethanol). 25 μl of each cell extract were heated at 95 °C for 5 min and were loaded onto a 7.5% SDS-polyacrylamide gel. After size-separation, proteins were electrophoretically transferred to PROTEAN nitrocellulose membranes (Schleicher and Schuell). ECL Western blotting was performed using Renaissance Western blotting detection reagents (PerkinElmer Life Sciences) according to the supplier's instructions. In Fig. 7 A, a rabbit polyclonal anti-LPP antibody MP2 was used for ECL Western blotting at a dilution of 1:3000. Cells were fixed in 4% formaldehyde for 20 min followed by three washes in PBS containing 0.9 mmCaCl2 and 0.5 mm MgCl2(PBS2+). Quenching was performed by incubating the cells for 10 min at room temperature in PBS2+ containing 50 mm NH4Cl. Cells were then permeabilized with 0.4% Triton X-100 for 10 min at room temperature. Subsequently, the slides were incubated with primary antibodies for 30 min at room temperature. After washing the cells three times in PBS2+, bound primary antibodies were detected with fluorescently labeled secondary antibodies (Molecular Probes) for 30 min at room temperature. Following three washes in PBS2+, slides were mounted in Vectashield mounting medium (Vector Laboratories). For detection of GFP fluorescence, cells were fixed and thereafter directly mounted. Slides were analyzed on a Zeiss Axiophot fluorescence microscope equipped with a cooled digital CCD camera system (Photometrics) using SmartCaptureTM software. Primary antibodies used included rabbit polyclonal anti-LPP antibody MP2, dilution 1:200 (5Petit M.M. Fradelizi J. Golsteyn R.M. Ayoubi T.A. Menichi B. Louvard D. Van de Ven W.J. Friederich E. Mol. Biol. Cell. 2000; 11: 117-129Crossref PubMed Scopus (120) Google Scholar) and a mouse monoclonal anti-vinculin antibody hVIN-1, dilution 1:400 (Sigma). As outlined above, certain benign tumors express HMGA2/LPP fusion transcripts encoding HMGA2/LPP fusion proteins. These proteins are mainly localized in the nucleus (5Petit M.M. Fradelizi J. Golsteyn R.M. Ayoubi T.A. Menichi B. Louvard D. Van de Ven W.J. Friederich E. Mol. Biol. Cell. 2000; 11: 117-129Crossref PubMed Scopus (120) Google Scholar). In lipomas, two different fusion transcripts are found encoding HMGA2/LPP fusion proteins composed of the three DNA binding domains of HMGA2 followed by either the two most C-terminal LIM domains of LPP (HMGA2/LPP-short) or a portion of the proline-rich region (amino acids 372–413) and all three LIM domains of LPP (HMGA2/LPP-long) (2Petit M.M.R. Mols R. Schoenmakers E.F. Mandahl N. Van de Ven W.J. Genomics. 1996; 36: 118-129Crossref PubMed Scopus (186) Google Scholar). Our previous observations show that when GFP-tagged forms of these HMGA2/LPP fusion proteins are overexpressed in cells, both forms are expressed only in the nucleus (5Petit M.M. Fradelizi J. Golsteyn R.M. Ayoubi T.A. Menichi B. Louvard D. Van de Ven W.J. Friederich E. Mol. Biol. Cell. 2000; 11: 117-129Crossref PubMed Scopus (120) Google Scholar). However, in cells expressing very high levels of HMGA2/LPP-long, this protein is also present in the cytoplasm and in focal adhesions while in cells expressing similar levels of HMGA2/LPP-short, staining in focal adhesions is not observed (5Petit M.M. Fradelizi J. Golsteyn R.M. Ayoubi T.A. Menichi B. Louvard D. Van de Ven W.J. Friederich E. Mol. Biol. Cell. 2000; 11: 117-129Crossref PubMed Scopus (120) Google Scholar). These observations were the first indication that the LIM domains of LPP could play a role in targeting the LPP protein to focal adhesions. To investigate the role of the LIM domains in targeting the LPP protein to focal adhesions, we made a number of GFP fusion proteins containing full-length LPP molecules carrying mutations in one or two of its LIM domains (Fig. 1 B). The mutations in the LIM domains were made in such a way that these domains were completely destroyed: four of eight conserved zinc-binding cysteine and histidine residues were mutated to alanine (Fig. 1 C). We compared the intracellular distribution of these mutant LPP molecules to that of the wild-type protein also expressed as a GFP fusion protein (Fig. 1 B). The distribution of the GFP-tagged wild-type LPP protein is indistinguishable from that of the endogenous protein: GFP-LPP is highly concentrated in focal adhesions and, at steady state, only very low levels of the protein can be detected in the nucleus (Fig. 1, D and D′, and our previous observations, Ref. 5Petit M.M. Fradelizi J. Golsteyn R.M. Ayoubi T.A. Menichi B. Louvard D. Van de Ven W.J. Friederich E. Mol. Biol. Cell. 2000; 11: 117-129Crossref PubMed Scopus (120) Google Scholar). Mutations in any of the LPP LIM domains resulted in a reduction of the focal adhesion targeting capacity of the LPP protein (Fig. 1,E–G and E′–G′). The amount of reduction was different depending on which of the LIM domains was targeted by mutations. While mutations in the third LIM domain caused a minor reduction in focal adhesion targeting capacity (Fig. 1, Gand G′), mutations in the first LIM domain caused a more severe reduction (Fig. 1, E and E′), and mutations in the second LIM domain caused the most severe reduction (Fig. 1,F and F′). When two of the LPP LIM domains were targeted by mutations at the same time, a severe reduction in focal adhesion targeting capacity was observed in all possible cases (Fig. 1,H–J and H′–J′). Mutations in the second and third LIM domain (Fig. 1, I and I′), or in the first and the third LIM domain (Fig. 1, J and J′) reduced the level of LPP in focal adhesions in a similar way as when the second LIM domain was mutated. The most severe phenotype was observed when the first and second LIM domains were mutated at the same time. In this case, focal adhesion targeting of the LPP protein was almost completely abolished (Fig. 1, H and H′). In conclusion, our results suggest that the LIM domains of LPP play an important role in targeting the LPP protein to focal adhesions. LPP is a member of a family of proteins, which are all proline-rich in their N-terminal region and have three LIM domains in their C-terminal region. LPP family members include zyxin (10Beckerle M.C. J. Cell Biol. 1986; 103: 1679-1687Crossref PubMed Scopus (104) Google Scholar), TRIP6 (21Yi J. Beckerle M.C. Genomics. 1998; 49: 314-316Crossref PubMed Scopus (72) Google Scholar), ajuba (22Goyal R.K. Lin P. Kanungo J. Payne A.S. Muslin A.J. Longmore G.D. Mol. Cell. Biol. 1999; 19: 4379-4389Crossref PubMed Google Scholar), and LIMD1 (23Kiss H. Kedra D. Yang Y. Kost-Alimova M. Kiss C. O'Brien K.P. Fransson I. Klein G. Imreh S. Dumanski J.P. Hum Genet. 1999; 105: 552-559Crossref PubMed Scopus (56) Google Scholar). While all family members are quite similar in their C-terminal LIM domains, there is only limited similarity in their proline-rich regions. The LPP protein contains regions similar to zyxin near the N terminus (the α-actinin binding site) (Fig. 2, A andB), TRIP6 in the center of the proline-rich region (Fig. 2,A and C), and a region similar to zyxin, TRIP6, and LIMD1 at the C terminus of the proline-rich region (Fig. 2,A and D). Recently, it was shown that when human or chicken zyxin lacking their α-actinin binding site are expressed as fusion proteins with GFP, targeting to focal adhesions is grossly impaired (14Nix D.A. Fradelizi J. Bockholt S. Menichi B. Louvard D. Friederich E. Beckerle M.C. J. Biol. Chem. 2001; 276: 34759-34767Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 24Reinhard M. Zumbrunn J. Jaquemar D. Kuhn M. Walter U. Trueb B. J. Biol. Chem. 1999; 274: 13410-13418Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 25Li B. Trueb B. J. Biol. Chem. 2001; 276: 33328-33335Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). We investigated whether this region has the same function in targeting LPP to focal adhesions as it has in zyxin. For this purpose, we made a construct expressing a GFP-LPP protein containing a deletion of its α-actinin binding site (GFP-LPPΔ41–57) (Fig. 2, A andB). However, no difference in focal adhesion targeting could be detected between the mutant LPP protein and the wild-type protein (Fig. 2, E and E′). We also investigated whether deletion of the TRIP6 similar region or the zyxin/TRIP6/LIMD1 similar region in LPP has an influence on the focal adhesion targeting capacity of the LPP protein. The function of the TRIP6 similar region is not known, either in LPP or in TRIP6. The zyxin/TRIP6/LIMD1 similar region contains a nuclear export signal (NES) in zyxin (26Nix D.A. Beckerle M.C. J. Cell Biol. 1997; 138: 1139-1147Crossref PubMed Scopus (201) Google Scholar). In LPP, the function of this region is not known. According to our previous results (5Petit M.M. Fradelizi J. Golsteyn R.M. Ayoubi T.A. Menichi B. Louvard D. Van de Ven W.J. Friederich E. Mol. Biol. Cell. 2000; 11: 117-129Crossref PubMed Scopus (120) Google Scholar) and Fig. 2, G andG′, it does not function as a NES in LPP. No difference in focal adhesion targeting could be detected between the wild-type protein and mutant GFP-LPP proteins containing a deletion of the TRIP6 similar region (GFP-LPPΔ205–230) (Fig. 2, A,C, F, and F′) or a deletion of the zyxin/TRIP6/LIMD1 similar region (GFP-LPPΔ387–408) (Fig. 2,A, D, G, and G′). In conclusion, none of the similar regions in the proline-rich region of LPP are important for the focal adhesion targeting of LPP. To obtain more insight into how the LIM domains of LPP function to target LPP to focal adhesions, we deleted the entire proline-rich region of the protein. In this way, we were able to investigate the focal adhesion targeting capacity of the LIM domains as a separate entity. We made a construct expressing a GFP fusion protein containing all three LIM domains of LPP (Fig. 3 A). In CV-1 cells, this GFP-LPP-(412–612) protein displayed robust targeting to focal adhesions (Fig. 3, B and B′). However, staining in focal adhesions was not as strong as the staining obtained with the full-length wild-type LPP protein. These observations indicate that targeting of the LIM domains to focal adhesions is not as powerful as for the full-length protein. This suggests that also the proline-rich region of LPP might have a function in targeting the protein to focal adhesions. To further analyze the focal adhesion targeting capacity of the LPP LIM domains, we investigated the targeting capacity of paired LIM domains and individual LIM domains of LPP. To do so, we made a number of constructs expressing GFP fusions containing LIM domains 1 and 2 (GFP-LPP-(412–531)), LIM domains 2 and 3 (GFP-LPP-(471–612)), LIM domain 1 (GFP-LPP-(412–473)), LIM domain 2 (GFP-LPP-(471–531)), or LIM domain 3 (GFP-LPP-(531–612)) (Fig. 3 A). Upon expression in CV-1 cells of these GFP-LPP fusion proteins, important observations could be made (Fig. 3, C–G and C′–G′). In contrast to the GFP-LPP protein containing all three LIM domains of LPP, which had strong focal adhesion targeting, paired LIM domains, either LIM 1 and 2, or LIM 2 and 3, as well as each individual LIM domain showed a drastic reduction in their focal adhesion targeting capacity. These results suggest that the three LIM domains of LPP cooperate to provide robust targeting of LPP to focal adhesions. The above-mentioned results on the focal adhesion targeting capacity of the LIM domains of LPP, as compared with the complete LPP protein, suggested that the proline-rich region of LPP also has a function in targeting the protein to focal adhesions. To confirm these results, we made a construct expressing a GFP-LPP protein containing only the proline-rich region, lacking all three LIM domains (GFP-LPP-(2–415)) (Fig. 4). CV-1 cells expressing this protein presented staining in focal adhesions indicating that the proline-rich region of LPP has focal adhesion targeting capacity (Fig. 5, A and A′). However, whereas the strength of focal adhesion targeting capacity of the LIM domains was comparable to that of the full-length protein, the targeting capacity of the proline-rich region, although clearly detectable, was found to be much weaker.Figure 5The LPP proline-rich region harbors targeting capacity for focal adhesions and stress fibers. CV-1 cells transiently transfected with constructs expressing GFP-LPP-(2–415) (A, A′), GFP-LPP-(62–415) (B,B′), GFP-LPP-(94–415) (C, C′), GFP-LPP-(94–258) (D, D′, E,E′), GFP-LPP-(94–258)Δ(205–230) (F,F′, G, G′), GFP-LPP-(179–415) (H, H′), GFP-LPP-(179–415)Δ(205–230) (I, I′). Cells were fixed and labeled for vinculin. Cells were visualized either for GFP (A–I) or vinculin (A′–I′).View Large Image Figure ViewerDownload (PPT) To narrow down the area in the proline-rich region responsible for its focal adhesion targeting capacity, we made several deletion constructs of the proline-rich region of LPP as depicted in Fig. 4. In this way, we were able to study the effect of deletions in the proline-rich region as a separate entity, isolated from the strong focal adhesion targeting effect of the LIM domains. We first deleted the α-actinin binding site in LPP (GFP-LPP-(62–415)), because α-actinin is known to be a component of focal adhesion sites and as such could provide a docking site for the proline-rich region of the LPP protein. However, deletion of the α-actinin binding site did not alter the targeting of the proline-rich region to focal adhesions (Fig. 5, B andB′). Deletion of the VASP binding sites in addition to the α-actinin binding site (GFP-LPP-(94–415)) had no effect on targeting to focal adhesions (Fig. 5, C and C′). Further deletion of the TRIP6 similar region in addition to the α-actinin and VASP binding sites (GFP-LPP-(94–415)Δ(205–230)) also had no effect (results not shown). These results indicate that the focal adhesion targeting capacity of the proline-rich region of LPP is located either between the VASP binding sites and the TRIP6 similar region or between the TRIP6 similar region and the C-terminal end of the proline-rich region. In order to discriminate between these two possibilities, we made constructs expressing GFP-LPP proteins containing either of these segments of the proline-rich region with the TRIP6 similar region (GFP-LPP-(94–258), GFP-LPP-(179–415)) or without the TRIP6 similar region (GFP-LPP-(94–258)Δ(205–230), GFP-LPP-(179–415)Δ(205–230)) (Fig. 4). GFP-LPP-(94–258) and GFP-LPP-(94–258)Δ(205–230) almost entirely lost the ability to target to focal adhesions (Fig. 5, D, D′,F, and F′). However, occasionally, targeting to stress fibers was observed (Fig. 5, E, E′,G, and G′). In this regard, we noticed that wild-type LPP protein is also occasionally observed along stress fibers. 3M. Petit, unpublished results. On the other hand, GFP-LPP-(179–415) and GFP-LPP-(179–415)Δ(205–230) did show targeting to focal adhesions in a way that was indistinguishable from the targeting capacity of the entire proline-rich region (Fig. 5,H, H′, I, and I′). These results indicate that the LPP proline-rich region harbors targeting capacity for focal adhesions and stress fibers and that these capacities are located between the TRIP6 similar region and the C-terminal end of the proline-rich region, and between the VASP binding sites and the TRIP6 similar region, respectively. Our results suggest that" @default.
• W2091210876 created "2016-06-24" @default.
• W2091210876 creator A5050912566 @default.
• W2091210876 creator A5057609788 @default.
• W2091210876 creator A5061490063 @default.
• W2091210876 date "2003-01-01" @default.
• W2091210876 modified "2023-10-02" @default.
• W2091210876 title "The Focal Adhesion and Nuclear Targeting Capacity of the LIM-containing Lipoma-preferred Partner (LPP) Protein" @default.
• W2091210876 cites W1546173464 @default.
• W2091210876 cites W1562998360 @default.
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