FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2064750103> ?p ?o ?g. }

• W2064750103 endingPage "16851" @default.
• W2064750103 startingPage "16844" @default.
• W2064750103 abstract "Fish is a scaffolding protein and Src substrate. It contains an amino-terminal Phox homology (PX) domain and five Src homology 3 (SH3) domains, as well as multiple motifs for binding both SH2 and SH3 domain-containing proteins. We have determined that the PX domain of Fish binds 3-phosphorylated phosphatidylinositols (including phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate). Consistent with this, a fusion protein of green fluorescent protein and the Fish PX domain localized to punctate structures similar to endosomes in normal fibroblasts. However, the full-length Fish protein was largely cytoplasmic, suggesting that its PX domain may not be able to make intermolecular interactions in unstimulated cells. In Src-transformed cells, we observed a dramatic re-localization of some Fish molecules to actin-rich structures called podosomes; the PX domain was both necessary and sufficient to effect this translocation. We used a phage display screen with the fifth SH3 domain of Fish and isolated ADAM19 as a binding partner. Subsequent analyses in mammalian cells demonstrated that Fish interacts with several members of the ADAMs family, including ADAMs 12, 15, and 19. In Src-transformed cells, ADAM12 co-localized with Fish in podosomes. Because members of the ADAMs family have been implicated in growth factor processing, as well as cell adhesion and motility, Fish could be acting as an adaptor molecule that allows Src to impinge on these processes. Fish is a scaffolding protein and Src substrate. It contains an amino-terminal Phox homology (PX) domain and five Src homology 3 (SH3) domains, as well as multiple motifs for binding both SH2 and SH3 domain-containing proteins. We have determined that the PX domain of Fish binds 3-phosphorylated phosphatidylinositols (including phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate). Consistent with this, a fusion protein of green fluorescent protein and the Fish PX domain localized to punctate structures similar to endosomes in normal fibroblasts. However, the full-length Fish protein was largely cytoplasmic, suggesting that its PX domain may not be able to make intermolecular interactions in unstimulated cells. In Src-transformed cells, we observed a dramatic re-localization of some Fish molecules to actin-rich structures called podosomes; the PX domain was both necessary and sufficient to effect this translocation. We used a phage display screen with the fifth SH3 domain of Fish and isolated ADAM19 as a binding partner. Subsequent analyses in mammalian cells demonstrated that Fish interacts with several members of the ADAMs family, including ADAMs 12, 15, and 19. In Src-transformed cells, ADAM12 co-localized with Fish in podosomes. Because members of the ADAMs family have been implicated in growth factor processing, as well as cell adhesion and motility, Fish could be acting as an adaptor molecule that allows Src to impinge on these processes. phox homology green fluorescent protein glutathioneS-transferase phosphatidylinositol 3-phosphate 4)P2, phosphatidylinositol 3,4-bisphosphate epidermal growth factor Src homology phosphate-buffered saline tetramethylrhodamine iso- thiocyanate Fish was originally isolated in a screen to identify Src substrates (1Lock P. Abram C.L. Gibson T. Courtneidge S.A. EMBO J. 1998; 17: 4346-4357Crossref PubMed Scopus (148) Google Scholar). It has a PX1domain at its amino terminus and five SH3 domains, as well as multiple polyproline-rich motifs that could mediate association with SH3 domains, several possible phosphorylation sites for both serine/threonine and tyrosine kinases, and potentially four alternatively spliced forms. The presence of these domains and motifs in Fish suggests that it might act as a scaffold or docking molecule for both proteins and lipids.PX domains are independently folding modules of ∼120 amino acids, with an overall hydrophobic character but few totally conserved amino acids. They are frequently found in combination with protein interaction domains such as SH3 domains and exist in a diverse array of proteins with wide ranging functions (2Ponting C.P. Protein Sci. 1996; 5: 2353-2357Crossref PubMed Scopus (265) Google Scholar). For example, the p40phox and p47phox subunits of the NADPH oxidase system of phagocytes contain PX domains. The enzymes CISK (cytokine-independent survivalkinase) and phospholipase D have PX domains, as do several proteins that function in vesicular sorting (for example the sorting nexins), and proteins involved in cytoskeletal organization (including the yeast bud emergence proteins). The binding capabilities of several PX domains have been reported recently. All PX domains tested bind to phosphorylated phosphatidylinositol lipids. The most common binding partner is PtdIns3P, but some PX domains will bind PtdIns(3,4)P2 and other substituted phosphatidylinositol molecules (3Sato T.K. Overduin M. Emr S.D. Science. 2001; 294: 1881-1885Crossref PubMed Scopus (202) Google Scholar). When PX domains were first identified, it was also noted that many of them contained a PXXP motif, suggesting that they might be able to bind SH3 domains (2Ponting C.P. Protein Sci. 1996; 5: 2353-2357Crossref PubMed Scopus (265) Google Scholar). Indeed, structural analysis of the PX domain of p47phox by NMR showed that its PXXP motif is on the surface of the domain and is able to bind to the second SH3 domain of p47phox (4Hiroaki H. Ago T. Ito T. Sumimoto H. Kohda D. Nat. Struct. Biol. 2001; 8: 526-530Crossref PubMed Scopus (149) Google Scholar). These data suggested that SH3 binding might impact the ability of a PX domain simultaneously to bind PtdIns3P. In the related protein p40phox, which also has a PXXP motif in its PX domain, lipid and SH3 binding to the PX domain appears to be neither cooperative nor antagonistic (5Bravo J. Karathanassis D. Pacold C.M. Pacold M.E. Ellson C.D. Anderson K.E. Butler P.J. Lavenir I. Perisic O. Hawkins P.T. Stephens L. Williams R.L. Mol. Cell. 2001; 8: 829-839Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). However, it remains possible that in other proteins, lipid and SH3 domain binding might influence each other. The PX domain of Fish has both a PXXP motif and the conserved residues required for phospholipid binding.We recently reported that Fish is a Src substrate both in vitro and in vivo in Src-transformed cells (1Lock P. Abram C.L. Gibson T. Courtneidge S.A. EMBO J. 1998; 17: 4346-4357Crossref PubMed Scopus (148) Google Scholar). Fish is also tyrosine-phosphorylated in a Src-dependent manner in normal cells treated with concentrations of cytochalasin D that result in rearrangement of the cortical actin cytoskeleton. Furthermore, tyrosine phosphorylation of Fish, albeit with slow kinetics, was detected in Rat1 cells in response to treatment with growth factors such as platelet-derived growth factor, lysophosphatidic acid, and bradykinin that are known to promote changes in the actin cytoskeleton (1Lock P. Abram C.L. Gibson T. Courtneidge S.A. EMBO J. 1998; 17: 4346-4357Crossref PubMed Scopus (148) Google Scholar). These data suggest that Fish may impact, or be impacted by, cytoskeletal regulation.The cytoskeleton in Src-transformed cells is grossly abnormal. Very few actin filaments are detected. Instead, much of the F-actin has a ring-like appearance in the cortex of the cells and is found in structures that have been called podosomes (6David-Pfeuty T. Singer S.J. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 6687-6691Crossref PubMed Scopus (214) Google Scholar, 7Tarone G. Cirillo D. Giancotti F.G. Comoglio P.M. Marchisio P.C. Exp. Cell Res. 1985; 159: 141-157Crossref PubMed Scopus (339) Google Scholar). Each podosome is a fine, cylindrical, actin-rich structure on the ventral surface of the cell. In Src-transformed fibroblasts, these podosomes cluster to form rings or semi-circles that are called rosettes. Although it was thought that these structures might simply represent remnants of focal adhesions, more recent research has suggested that podosomes are involved in driving locomotion and invasion of Src-transformed cells (8Chen W.T. J. Exp. Zool. 1989; 251: 167-185Crossref PubMed Scopus (267) Google Scholar). Podosomes contain a number of cytoskeleton-associated proteins, including N-WASP, cortactin, paxillin, and p190RhoGAP(9Mizutani K. Miki H. He H. Maruta H. Takenawa T. Cancer Res. 2002; 62: 669-674PubMed Google Scholar, 10Bowden E.T. Barth M. Thomas D. Glazer R.I. Mueller S.C. Oncogene. 1999; 18: 4440-4449Crossref PubMed Scopus (308) Google Scholar, 11Nakahara H. Mueller S.C. Nomizu M. Yamada Y. Yeh Y. Chen W.T. J. Biol. Chem. 1998; 273: 9-12Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Src-transformed cells are not the only cells that contain podosomes; they have also been reported in invasive human breast cancer and melanoma cells (10Bowden E.T. Barth M. Thomas D. Glazer R.I. Mueller S.C. Oncogene. 1999; 18: 4440-4449Crossref PubMed Scopus (308) Google Scholar, 12Monsky W.L. Lin C.Y. Aoyama A. Kelly T. Akiyama S.K. Mueller S.C. Chen W.T. Cancer Res. 1994; 54: 5702-5710PubMed Google Scholar), raising the possibility that these structures might also be involved in metastatic properties of human tumor cells. Osteoclasts and macrophages also contain structures called podosomes (13Teti A. Marchisio P.C. Zallone A.Z. Am. J. Physiol. 1991; 261: C1-C7Crossref PubMed Google Scholar). In this case, one large actin ring is formed, from which protrusions emerge that are involved in bone remodeling. It is not yet clear whether the podosomes of osteoclasts and of transformed cells contain the same components.The metzincin family of metalloproteases contains not just the matrix metalloproteases (some of which co-localize with podosomes) but also ADAMs family proteases (14Seals D.F. Courtneidge S.A. Genes Dev. 2003; 17: 7-30Crossref PubMed Scopus (881) Google Scholar, 15Primakoff P. Myles D.G. Trends Genet. 2000; 16: 83-87Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 16Black R.A. White J.M. Curr. Opin. Cell Biol. 1998; 10: 654-659Crossref PubMed Scopus (428) Google Scholar, 17Schlondorff J. Blobel C.P. J. Cell Sci. 1999; 112: 3603-3617Crossref PubMed Google Scholar). In addition to a metalloprotease domain, ADAMs proteins have disintegrin, cysteine-rich, and EGF-like domains that are involved in cell adhesion, a membrane spanning sequence, and a cytoplasmic tail. In many members of the ADAMs family, this cytoplasmic tail contains multiple PXXP motifs that can mediate the interaction with SH3 domain-containing proteins. Members of the ADAMs family function as sheddases (by cleaving active growth factors and cytokines from their inactive precursors), as well as mediating cell and matrix interactions.To isolate proteins that bound to the SH3 domains of Fish, we chose to use a phage display screen. Phage display has been used extensively for the generation of monoclonal antibodies and for screening peptide libraries. Recent advances in technology have allowed larger insert sequences to be expressed on the phage surface, thus facilitating the rapid screening of cDNA libraries against target proteins or peptides (18Zozulya S. Lioubin M. Hill R.J. Abram C. Gishizky M.L. Nat. Biotechnol. 1999; 17: 1193-1198Crossref PubMed Scopus (72) Google Scholar, 19Crameri R. Suter M. Gene. 1993; 137: 69-75Crossref PubMed Scopus (175) Google Scholar, 20Crameri R. Jaussi R. Menz G. Blaser K. Eur. J. Biochem. 1994; 226: 53-58Crossref PubMed Scopus (146) Google Scholar). Phage display does have limitations, such as issues with the production of the target protein in bacterial cells. However, one advantage over other methods is that once reagents are generated, the process is very rapid, with screening taking just a few days. Furthermore, although the two-hybrid system often results in the detection of large numbers of low affinity interactors, phage display screening allows the identification of only the highest affinity interacting molecules.To determine the role of Fish in signaling pathways downstream of Src, we have begun to look for binding partners of Fish. Given Fish's array of lipid and protein binding domains, we used both lipid binding assays and phage display screens in our analyses. Here we describe the identification of molecules that interact with the PX domain and the fifth SH3 domain of Fish.DISCUSSIONWe have demonstrated that the PX domain of Fish binds predominantly to PtdIns3P and also to PtdIns(3,4)P2, on filters spotted with the lipids. Many PX domains appear to bind PtdIns3P (3Sato T.K. Overduin M. Emr S.D. Science. 2001; 294: 1881-1885Crossref PubMed Scopus (202) Google Scholar, 30Yu J.W. Lemmon M.A. J. Biol. Chem. 2001; 276: 44179-44184Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 31Ellson C.D. Andrews S. Stephens L.R. Hawkins P.T. J. Cell Sci. 2002; 115: 1099-1105Crossref PubMed Google Scholar), but binding to PtdIns(3,4)P2 is more unusual. Only the PX domain of p47phox, which is most closely related to the Fish PX domain, has also been shown to bind PtdIns(3,4)P2 (23Kanai F. Liu H. Field S.J. Akbary H. Matsuo T. Brown G.E. Cantley L.C. Yaffe M.B. Nat. Cell Biol. 2001; 3: 675-678Crossref PubMed Scopus (494) Google Scholar). Although the filter binding assays of the type we used here may not reveal the full specificity or complexity of binding of a given PX domain, the data we have obtained are consistent with our subcellular localization analyses. For example, the isolated PX domain of Fish, when expressed as a GFP fusion protein, was distributed in the cytoplasm in a punctate fashion (that may reflect an endosomal localization) in normal fibroblasts. In contrast to the isolated PX domain, the full-length Fish protein did not show a punctate staining pattern. Rather, it appeared to be more diffusely cytoplasmic. These data suggest that, in the context of the full-length protein, the PX domain of Fish might not be able to make intermolecular contacts. A similar situation has been observed with the related protein, p47phox. In unstimulated neutrophils, p47phox is cytoplasmic, and its PX domain makes an intramolecular contact with its second SH3 domain. Stimulation of the neutrophil results in p47phox adopting an open conformation and associating with membranes containing PtdIns(3,4)P2 (4Hiroaki H. Ago T. Ito T. Sumimoto H. Kohda D. Nat. Struct. Biol. 2001; 8: 526-530Crossref PubMed Scopus (149) Google Scholar, 32Ago T. Nunoi H. Ito T. Sumimoto H. J. Biol. Chem. 1999; 274: 33644-33653Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 33Karathanassis D. Stahelin R.V. Bravo J. Perisic O. Pacold C.M. Cho W. Williams R.L. EMBO J. 2002; 21: 5057-5068Crossref PubMed Scopus (256) Google Scholar). A similar mechanism may exist for Fish. Indeed, we have preliminary evidence that the PX domain of Fish makes contact with the third SH3 domain and that this intramolecular interaction is released by Src phosphorylation. 2D. Salinsky, C. L. Abram, and S. A. Courtneidge, unpublished observations. We observed a striking re-distribution of Fish in Src-transformed cells, with much of the protein found co-localized with actin in structures called podosomes. The PX domain of Fish was both necessary and sufficient to localize to podosomes. Interestingly, in the same cell type, a GFP-FYVE domain fusion protein was localized to the endosomal compartment via interaction with PtdIns3P. These data suggest that, in these Src-transformed cells, the Fish PX domain binds lipids other than PtdIns3P and that this may account for the Fish localization observed. The most likely candidate lipid is PtdIns(3,4)P2, which was able to bind to the PX domain in vitro and which can be produced from phosphatidylinositol 3,4,5-trisphosphate by the action of a 5-phosphatase (34Erneux C. Govaerts C. Communi D. Pesesse X. Biochim. Biophys. Acta. 1998; 1436: 185-199Crossref PubMed Scopus (126) Google Scholar), or by the action of PtdIns3P 4-kinases on PtdIns3P (35Banfic H. Tang X. Batty I.H. Downes C.P. Chen C. Rittenhouse S.E. J. Biol. Chem. 1998; 273: 13-16Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Alternatively, it is also possible that the PX domain of Fish is targeted to podosomes via interaction with a protein. We are currently determining whether podosomes contain PtdIns(3,4)P2 and whether the PX domain must bind either lipid and/or protein to associate with podosomes.Podosomes are interesting structures, found normally in invasive cells such as osteoclasts and macrophages (13Teti A. Marchisio P.C. Zallone A.Z. Am. J. Physiol. 1991; 261: C1-C7Crossref PubMed Google Scholar) but also present in Src-transformed cells and invasive breast carcinoma and melanoma cell lines (6David-Pfeuty T. Singer S.J. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 6687-6691Crossref PubMed Scopus (214) Google Scholar, 7Tarone G. Cirillo D. Giancotti F.G. Comoglio P.M. Marchisio P.C. Exp. Cell Res. 1985; 159: 141-157Crossref PubMed Scopus (339) Google Scholar, 8Chen W.T. J. Exp. Zool. 1989; 251: 167-185Crossref PubMed Scopus (267) Google Scholar, 10Bowden E.T. Barth M. Thomas D. Glazer R.I. Mueller S.C. Oncogene. 1999; 18: 4440-4449Crossref PubMed Scopus (308) Google Scholar, 12Monsky W.L. Lin C.Y. Aoyama A. Kelly T. Akiyama S.K. Mueller S.C. Chen W.T. Cancer Res. 1994; 54: 5702-5710PubMed Google Scholar, 36Mueller S.C. Ghersi G. Akiyama S.K. Sang Q.X. Howard L. Pineiro-Sanchez M. Nakahara H. Yeh Y. Chen W.T. J. Biol. Chem. 1999; 274: 24947-24952Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). They are actin-rich protrusions of ∼0.4 ॖm in diameter that extend from underneath the cell body into the extracellular matrix. Podosomes are very dynamic structures, with a half-life in the order of minutes compared with focal adhesions, which are stable for several hours (8Chen W.T. J. Exp. Zool. 1989; 251: 167-185Crossref PubMed Scopus (267) Google Scholar). Podosomes contain several actin-binding proteins (see the Introduction), many of which are also Src substrates. In addition, and of interest in regard to the ability of podosomes to degrade the extracellular matrix, the podosomes of Src-transformed cells also contain the matrix metalloprotease MT1-MMP (also known as MMP14), which processes the gelatinases MMP2 and MMP9 to their active forms (37Nakahara H. Howard L. Thompson E.W. Sato H. Seiki M. Yeh Y. Chen W.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7959-7964Crossref PubMed Scopus (360) Google Scholar). Other podosome-associated proteins include औ1 integrins (38Mueller S.C. Chen W.T. J. Cell Sci. 1991; 99: 213-225Crossref PubMed Google Scholar), the tyrosine kinase Pyk2 (in osteoclasts) (39Pfaff M. Jurdic P. J. Cell Sci. 2001; 114: 2775-2786Crossref PubMed Google Scholar,40Sanjay A. Houghton A. Neff L. DiDomenico E. Bardelay C. Antoine E. Levy J. Gailit J. Bowtell D. Horne W.C. Baron R. J. Cell Biol. 2001; 152: 181-195Crossref PubMed Scopus (337) Google Scholar), and Src itself (8Chen W.T. J. Exp. Zool. 1989; 251: 167-185Crossref PubMed Scopus (267) Google Scholar, 41Mueller S.C. Yeh Y. Chen W.T. J. Cell Biol. 1992; 119: 1309-1325Crossref PubMed Scopus (120) Google Scholar). 3L. Maurer and S. A. Courtneidge, unpublished observations. We now show that, in Src-transformed cells, podosomes contain both Fish and ADAM12.We have demonstrated that the fifth SH3 domain of the Src substrate, Fish, binds to the cytoplasmic tail of several ADAMs family metalloproteases, particularly ADAMs 12, 15, and 19. Members of the ADAMs family are characterized by the presence of protease and disintegrin domains in the extracellular region, in addition to a pro-domain, a cysteine-rich region, and an EGF-like domain, followed by a transmembrane domain and a cytoplasmic tail of variable length. ADAMs have functions in many different cell processes, including myoblast fusion, fertilization, cell fate determination, and growth factor and cytokine processing (14Seals D.F. Courtneidge S.A. Genes Dev. 2003; 17: 7-30Crossref PubMed Scopus (881) Google Scholar, 15Primakoff P. Myles D.G. Trends Genet. 2000; 16: 83-87Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 16Black R.A. White J.M. Curr. Opin. Cell Biol. 1998; 10: 654-659Crossref PubMed Scopus (428) Google Scholar, 17Schlondorff J. Blobel C.P. J. Cell Sci. 1999; 112: 3603-3617Crossref PubMed Google Scholar). On a biochemical level, the ADAMs have three distinctive properties. First, the disintegrin domain (probably in conjunction with the cysteine-rich and EGF-like domains) has the ability to associate with integrin receptors, particularly those containing औ1 subunits, and therefore can modulate cell/cell interactions (14Seals D.F. Courtneidge S.A. Genes Dev. 2003; 17: 7-30Crossref PubMed Scopus (881) Google Scholar, 42Evans J.P. Bioessays. 2001; 23: 628-639Crossref PubMed Scopus (134) Google Scholar). Second, many of the ADAMs are active proteases that act as sheddases, that is they act to release active growth factors and cytokines from cell surfaces. Third, many of the ADAMs have extended cytoplasmic tails that mediate interactions with several proteins, both with and without SH3 domains.In this study we identified ADAMs 12, 15, and 19, but not ADAM9, as binding partners for Fish. Each of these ADAMs contains multiple PXXP motifs in their cytoplasmic tails (14Seals D.F. Courtneidge S.A. Genes Dev. 2003; 17: 7-30Crossref PubMed Scopus (881) Google Scholar), and indeed, some binding partners for these ADAMs have already been reported. These include the association of the SH3 domain-containing proteins Grb2, phosphatidylinositol 3-kinase, and Src with ADAM12 (43Kang Q. Cao Y. Zolkiewska A. J. Biol. Chem. 2001; 276: 24466-24472Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 44Suzuki A. Kadota N. Hara T. Nakagami Y. Izumi T. Takenawa T. Sabe H. Endo T. Oncogene. 2000; 19: 5842-5850Crossref PubMed Scopus (51) Google Scholar) and endophilin 1, Grb2, SH3PX1, and Src family kinases with ADAM15 (45Howard L. Nelson K.K. Maciewicz R.A. Blobel C.P. J. Biol. Chem. 1999; 274: 31693-31699Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar,46Poghosyan Z. Robbins S.M. Houslay M.D. Webster A. Murphy G. Edwards D.R. J. Biol. Chem. 2002; 277: 4999-5007Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The cytoplasmic domains of ADAMs 12, 15, and 19 contain polyproline-rich motifs that likely represent the binding site for the Fish SH3 domain. Whether the association of Fish with various ADAMs family members is regulated by Src or Fish acts as an adaptor to allow Src to regulate the ADAMs family remains to be determined.It has been suggested that the cytoplasmic tails of the ADAMs may connect intracellular signals to the extracellular activity of these proteins. For example, overexpression of the cytoplasmic tail of ADAM12 inhibits myoblast fusion (47Galliano M.F. Huet C. Frygelius J. Polgren A. Wewer U.M. Engvall E. J. Biol. Chem. 2000; 275: 13933-13939Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), and similar overexpression of the ADAM9 tail inhibits 12-O-tetradecanoylphorbol-13-acetate-induced heparin-binding EGF shedding (48Izumi Y. Hirata M. Hasuwa H. Iwamoto R. Umata T. Miyado K. Tamai Y. Kurisaki T. Sehara-Fujisawa A. Ohno S. Mekada E. EMBO J. 1998; 17: 7260-7272Crossref PubMed Scopus (474) Google Scholar). Presumably this inhibition occurs because the isolated cytoplasmic tails sequester proteins that normally bind to the full-length protein and are required for ADAMs function. Our future analyses will therefore involve testing the effect of Fish binding on ADAMs function in both normal and Src-transformed cells. Fish was originally isolated in a screen to identify Src substrates (1Lock P. Abram C.L. Gibson T. Courtneidge S.A. EMBO J. 1998; 17: 4346-4357Crossref PubMed Scopus (148) Google Scholar). It has a PX1domain at its amino terminus and five SH3 domains, as well as multiple polyproline-rich motifs that could mediate association with SH3 domains, several possible phosphorylation sites for both serine/threonine and tyrosine kinases, and potentially four alternatively spliced forms. The presence of these domains and motifs in Fish suggests that it might act as a scaffold or docking molecule for both proteins and lipids. PX domains are independently folding modules of ∼120 amino acids, with an overall hydrophobic character but few totally conserved amino acids. They are frequently found in combination with protein interaction domains such as SH3 domains and exist in a diverse array of proteins with wide ranging functions (2Ponting C.P. Protein Sci. 1996; 5: 2353-2357Crossref PubMed Scopus (265) Google Scholar). For example, the p40phox and p47phox subunits of the NADPH oxidase system of phagocytes contain PX domains. The enzymes CISK (cytokine-independent survivalkinase) and phospholipase D have PX domains, as do several proteins that function in vesicular sorting (for example the sorting nexins), and proteins involved in cytoskeletal organization (including the yeast bud emergence proteins). The binding capabilities of several PX domains have been reported recently. All PX domains tested bind to phosphorylated phosphatidylinositol lipids. The most common binding partner is PtdIns3P, but some PX domains will bind PtdIns(3,4)P2 and other substituted phosphatidylinositol molecules (3Sato T.K. Overduin M. Emr S.D. Science. 2001; 294: 1881-1885Crossref PubMed Scopus (202) Google Scholar). When PX domains were first identified, it was also noted that many of them contained a PXXP motif, suggesting that they might be able to bind SH3 domains (2Ponting C.P. Protein Sci. 1996; 5: 2353-2357Crossref PubMed Scopus (265) Google Scholar). Indeed, structural analysis of the PX domain of p47phox by NMR showed that its PXXP motif is on the surface of the domain and is able to bind to the second SH3 domain of p47phox (4Hiroaki H. Ago T. Ito T. Sumimoto H. Kohda D. Nat. Struct. Biol. 2001; 8: 526-530Crossref PubMed Scopus (149) Google Scholar). These data suggested that SH3 binding might impact the ability of a PX domain simultaneously to bind PtdIns3P. In the related protein p40phox, which also has a PXXP motif in its PX domain, lipid and SH3 binding to the PX domain appears to be neither cooperative nor antagonistic (5Bravo J. Karathanassis D. Pacold C.M. Pacold M.E. Ellson C.D. Anderson K.E. Butler P.J. Lavenir I. Perisic O. Hawkins P.T. Stephens L. Williams R.L. Mol. Cell. 2001; 8: 829-839Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). However, it remains possible that in other proteins, lipid and SH3 domain binding might influence each other. The PX domain of Fish has both a PXXP motif and the conserved residues required for phospholipid binding. We recently reported that Fish is a Src substrate both in vitro and in vivo in Src-transformed cells (1Lock P. Abram C.L. Gibson T. Courtneidge S.A. EMBO J. 1998; 17: 4346-4357Crossref PubMed Scopus (148) Google Scholar). Fish is also tyrosine-phosphorylated in a Src-dependent manner in normal cells treated with concentrations of cytochalasin D that result in rearrangement of the cortical actin cytoskeleton. Furthermore, tyrosine phosphorylation of Fish, albeit with slow kinetics, was detected in Rat1 cells in response to treatment with growth factors such as platelet-derived growth factor, lysophosphatidic acid, and bradykinin that are known to promote changes in the actin cytoskeleton (1Lock P. Abram C.L. Gibson T. Courtneidge S.A. EMBO J. 1998; 17: 4346-4357Crossref PubMed Scopus (148) Google Scholar). These data suggest that Fish may impact, or be impacted by, cytoskeletal regulation. The cytoskeleton in Src-transformed cells is grossly abnormal. Very few actin filaments are detected. Instead, much of the F-actin has a ring-like appearance in the cortex of the cells and is found in structures that have been called podosomes (6David-Pfeuty T. Singer S.J. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 6687-6691Crossref PubMed Scopus (214) Google Scholar, 7Tarone G. Cirillo D. Giancotti F.G. Comoglio P.M. Marchisio P.C. Exp. Cell Res. 1985; 159: 141-157Crossref PubMed Scopus (339) Google Scholar). Each podosome is a fine, cylindrical, actin-rich structure on the ventral surface of the cell. In Src-transformed fibroblasts, these podosomes cluster to form rings or semi-circles that are called rosettes. Although it was thought that these structures might simply represent remnants of focal adhesions, more recent research has suggested that podosomes are involved in driving locomotion and invasion of Src-transformed cells (8Chen W.T. J. Exp. Zool. 1989; 251: 167-185Crossref PubMed Scopus (267) Google Scholar). Podosomes contain a number of cytoskeleton-associated proteins, including N-WASP, cortactin, paxillin, and p190RhoGAP(9Mizutani K. Miki H. He H. Maruta H. Takenawa T. Cancer Res. 2002; 62: 669-674PubMed Google Scholar, 10Bowden E.T. Barth M. Thomas D. Glazer R.I. Mueller S.C. Oncogene. 1999; 18: 4440-4449Crossref PubMed Scopus (308) Google Scholar, 11Nakahara H. Mueller S.C. Nomizu M. Yamada Y. Yeh Y. Chen W.T. J. Biol. Chem. 1998; 273: 9-12Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Src-transformed cells are not the only cells that contain podosomes; they have also been reported in invasive human breast cancer and melanoma cells (10Bowden E.T. Barth M. Thomas D. Glazer R.I. Mueller S.C. Oncogene. 1999; 18: 4440-4449Crossref PubMed Scopus (308) Google Scholar, 12Monsky W.L. Lin C.Y. Aoyama A. Kelly T. Akiyama S.K. Mueller S.C. Chen W.T. Cancer Res. 1994; 54: 5702-5710PubMed Google Scholar), raising the possibility that these structures might also be involved in metastatic properties of human tumor cells. Osteoclasts and macrophages also contain structures called podosomes (13Teti A. Mar" @default.
• W2064750103 created "2016-06-24" @default.
• W2064750103 creator A5015330857 @default.
• W2064750103 creator A5017746510 @default.
• W2064750103 creator A5019630008 @default.
• W2064750103 creator A5041057500 @default.
• W2064750103 creator A5059664245 @default.
• W2064750103 creator A5085916827 @default.
• W2064750103 creator A5087221706 @default.
• W2064750103 date "2003-05-01" @default.
• W2064750103 modified "2023-09-27" @default.
• W2064750103 title "The Adaptor Protein Fish Associates with Members of the ADAMs Family and Localizes to Podosomes of Src-transformed Cells" @default.
• W2064750103 cites W1250812676 @default.
• W2064750103 cites W1496054948 @default.
• W2064750103 cites W1517631297 @default.
• W2064750103 cites W1526208457 @default.
• W2064750103 cites W1581364974 @default.
• W2064750103 cites W1929801845 @default.
• W2064750103 cites W1933881136 @default.
• W2064750103 cites W1964616832 @default.
• W2064750103 cites W1972062729 @default.
• W2064750103 cites W1974729368 @default.
• W2064750103 cites W1981148940 @default.
• W2064750103 cites W1982036031 @default.
• W2064750103 cites W1984707983 @default.
• W2064750103 cites W1986273917 @default.
• W2064750103 cites W1989954297 @default.
• W2064750103 cites W1990180927 @default.
• W2064750103 cites W1999942847 @default.
• W2064750103 cites W2001485278 @default.
• W2064750103 cites W2002980966 @default.
• W2064750103 cites W2003113302 @default.
• W2064750103 cites W2005759392 @default.
• W2064750103 cites W2007503777 @default.
• W2064750103 cites W2008084782 @default.
• W2064750103 cites W2024999779 @default.
• W2064750103 cites W2030553086 @default.
• W2064750103 cites W2040888996 @default.
• W2064750103 cites W2051337966 @default.
• W2064750103 cites W2055370880 @default.
• W2064750103 cites W2060224232 @default.
• W2064750103 cites W2061921618 @default.
• W2064750103 cites W2063988619 @default.
• W2064750103 cites W2067000579 @default.
• W2064750103 cites W2077475973 @default.
• W2064750103 cites W2082379606 @default.
• W2064750103 cites W2083514445 @default.
• W2064750103 cites W2085370249 @default.
• W2064750103 cites W2118893166 @default.
• W2064750103 cites W2126514623 @default.
• W2064750103 cites W2139688694 @default.
• W2064750103 cites W2145837099 @default.
• W2064750103 cites W2156846932 @default.
• W2064750103 cites W2160094174 @default.
• W2064750103 cites W2286626550 @default.
• W2064750103 cites W2341892311 @default.
• W2064750103 cites W2344829769 @default.
• W2064750103 cites W4231183555 @default.
• W2064750103 doi "https://doi.org/10.1074/jbc.m300267200" @default.
• W2064750103 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12615925" @default.
• W2064750103 hasPublicationYear "2003" @default.
• W2064750103 type Work @default.
• W2064750103 sameAs 2064750103 @default.
• W2064750103 citedByCount "229" @default.
• W2064750103 countsByYear W20647501032012 @default.
• W2064750103 countsByYear W20647501032013 @default.
• W2064750103 countsByYear W20647501032014 @default.
• W2064750103 countsByYear W20647501032015 @default.
• W2064750103 countsByYear W20647501032016 @default.
• W2064750103 countsByYear W20647501032017 @default.
• W2064750103 countsByYear W20647501032018 @default.
• W2064750103 countsByYear W20647501032019 @default.
• W2064750103 countsByYear W20647501032020 @default.
• W2064750103 countsByYear W20647501032021 @default.
• W2064750103 countsByYear W20647501032022 @default.
• W2064750103 countsByYear W20647501032023 @default.
• W2064750103 crossrefType "journal-article" @default.
• W2064750103 hasAuthorship W2064750103A5015330857 @default.
• W2064750103 hasAuthorship W2064750103A5017746510 @default.
• W2064750103 hasAuthorship W2064750103A5019630008 @default.
• W2064750103 hasAuthorship W2064750103A5041057500 @default.
• W2064750103 hasAuthorship W2064750103A5059664245 @default.
• W2064750103 hasAuthorship W2064750103A5085916827 @default.
• W2064750103 hasAuthorship W2064750103A5087221706 @default.
• W2064750103 hasBestOaLocation W20647501031 @default.
• W2064750103 hasConcept C108636557 @default.
• W2064750103 hasConcept C141073059 @default.
• W2064750103 hasConcept C142669718 @default.
• W2064750103 hasConcept C1491633281 @default.
• W2064750103 hasConcept C183786373 @default.
• W2064750103 hasConcept C185592680 @default.
• W2064750103 hasConcept C2909208804 @default.
• W2064750103 hasConcept C505870484 @default.
• W2064750103 hasConcept C54355233 @default.
• W2064750103 hasConcept C62478195 @default.
• W2064750103 hasConcept C86803240 @default.
• W2064750103 hasConcept C95444343 @default.
• W2064750103 hasConceptScore W2064750103C108636557 @default.

• next