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内容記述 |
Cell adhesion and migration play essential roles in a wide variety of physiological and pathological aspects of the organization of multicellular organisms, such as embryogenesis, organogenesis, wound repair, inflammatory processes, and cancer invasion and metastasis. Adhesion and migration are primarily mediated by integrin binding to extracellular matrices (ECMs). Integrins recruit a chanacteristic set of cytoplasmic proteins, with scaffolding as well as signaling properties, at their cytoplasmic regions during this process. It is well documented in fibroblasts that integrin macroaggregates grow and shrink over time during cell migration, though the position of each macroaggregate remains fixed as the cell translocates. It is believed that there must be mechanisms that orchestrate the dynamics of protein recruitment and assembly at the cytoplasmic tails of integrins, but the molecular processes remain to be established. The precise subcellular locations where integrins initially assemble with their cytoplasmic binding proteins are also not known. <br /> The small GTP-binding proteins of the Rho family have been shown to play pivotal roles in regulating the dynamic properties of the actin-based cytoskeletal organization, which is also essential for cell migratory activity. For example, Rho A has been shown to be involved in the formation of actin stress fibers and local adhesion assembly in Swiss 3T3 cells. Furthermore, Rho A protein has been shown to participate in regulation of the phosphorylation status of myosin light chain, and thus regulate the contractility of the actomyosin network. Rho A can also activate phosphatidyl inositol 4- phosphate 5'-kinase to produce phosphatidyl inositol 4,5-bisphosphate, which interacts with gelsolin, profilin, and vinculin; and helps to regulate actin polymerization and cytoskeleton-membrane attachment. Rho A is moreover able to activate phospholipase D to produce phosphatidic acid, and to regulate actin polymerization. In spite of these extensive studies, however, the precise mechanism of how Rho A, as well as other Rho-family proteins, regulates focal adhesion assembly and its connection to actin fibers that ultimately leads to the regulation of cell migratory activity remains to be established. <br /> Recent studies by Norman et al., on the other hand, have shown that ADP-ribosylation factor 1 (ARF1), which belongs to another small GTP-binding protein family participates in paxillin recruitment to sites of focal contacts in Swiss 3T3. They also showed that ARF1 can potentiate the Rho A-stimulated stress fiber formation, and suggested that ARF 1 and Rho A activate complementary pathways that together lead to the formation of paxillin-rich focal adhesions at the ends of prominent actin stress fibers. <br /> ARF-family proteins have been implicated in the regulation of membrane and vesicle traffic in mammalian cells. Members of the family include six isoforms of ARF, and the ARF-like proteins. The six ARF isoforms are highly homologous to one another, and classified as class I, II or III based on sequence similarity. Class I includes ARF1, 2 and 3; class II, ARF4 and 5; and class III, ARF6. Among them, ARF1 has been most thoroughly studied. ARF1 has been shown to regulate membrane traffic at multiple sites within the cell. ARF1 colocalizes primarily with Golgi-associated proteins and acts at the Golgi; ARF1 also functions in ER-to-Golgi transport, trans-Golgi network, endosome-endosome fusion, protein secretion and fluid-phase endocytosis, as well as phospholipase D activation. The GTP bound form of ARF1 recruits protein coats, including the clathrin-associated adaptor proteins AP-1 and AP-3, and the nonclathrin coatomer, to membranes and initiates budding of the membrane vesicles. Subsequent hydrolysis of GTP to GDP by ARF1 may trigger disassembly of the coat from the vesicle, which is necessary for the vesicle to fuse to the target membranes. On the other hand, ARF6, the ARF which is most distantly related to ARF1, shows a rather wide distribution in the cytoplasm and localizes to an endosomal compartment and membrane ruffling regions. ARF6 primarily regulates endosomal trafficking as well as receptor-mediated endocytosis at the cell periphery, actin rearrangements beneath the plasma membrane, and cell spreading. Unlike other small GTP-binding family proteins such as Ras-family and Rho-family proteins, it is noteworthy that the intrinsic GTPase activity of ARF proteins is almost undetectable in vitro.<br /> Paxillin, one of the integrin-assembly proteins, is highly tyrosine phosphorylated upon integrin activation, and acts as an adaptor protein in integrin signaling. Paxillin can interact directly with several integrin-assembly proteins, including vinculin, talin, integrin b1, focal adhesion kinase, Pyk2, c-Src and Csk. The importance of paxillin in protein assembly and signaling has also been suggested by the lack of tyrosine phosphorylation in neutrophils isolated from a patient with a leukocyte adhesion deficiency, and its binding to Papilloma virus E6 proteins. Paxillin binding activity towards different types of E6 proteins correlates with degrees of disruption of the actin cytoskeletal architecture induced by infection with each type of Papilloma viruses. Human paxillin is composed of multiple isoforms (α,β and γ) with different biochemical properties and different patterns of expression. <br /> They have shown in fibroblasts that the cytoplasmic pool of paxillin primarily resides in the perinuclear region, a fraction of which seems to overlap with the Golgi apparatus. As will be described in this paper, there also appears to be a relatively large cytoplasmic pool in other types of cells, such as epithelial cells. They have, therefore, hypothesized that some intracellular active process, rather than a process of simple diffusion, may exist that helps to transport paxillin to sites of integrin macroaggregates at the plasma membrane. Paxillin is a soluble protein; thus they attempted to purify paxillin binding proteins that may be involved in localization of paxillin in the cytoplasm. <br /> The process of monocyte maturation in vitro provides a good model to explore the biochemical events involved in process of integrin activation. They have shown that human monocytes express all three isoforms of paxillin, and expression of all isoforms is augmented upon the cell maturation. Here, they report the isolation of a paxillin-binding protein, named PAG3 (Paxillin-associated protein with Arf GTPase-activating protein (GAP) activity, number 3), from mature U937 monocyte cells. PAG3 corresponds to KIAA0400 previously isolated by Ishikawa et al., 1997; and during their analysis, the same molecule was also identified as a Pyk2 binding protein and named Papα. PAG3/Pap α/KIAA0400 contains a zinc finger motif that is highly homologous to that of mammalian ARF1 GAP and yeast ARF GAP protein Gcs1. The zinc finger motif is essential for the ARF 1 GAP activity. Andreev et al. have shown that this protein exhibits a GAP activity against several isoforms of ARFs in vitro; and also demonstrated that this protein inhibits, ARF-dependent generation of post-Golgi vesicles and secretion of a truncated form of placental alkaline phosphatase. They show here that PAG3/Papα/KIAA0400 also binds to all three isoforms of human paxillin (α,β and γ), and is highly induced during monocyte maturation, during which integrins are activated and the cells become adherent and motile. They analyzed intracellular interactions among paxillin, PAG3 and ARFs. They also suggest that the GAP activity of PAG3 is involved in the recruitment of paxillin to focal contacts of adhesion plaques, and cell migratory activity. Finally, they discuss the relationship of ARF-mediated intracellular regulations to the subcellular localization of paxillin, and to cell migratory activities. |
要旨・審査要旨 / Abstract, Screening Result (463.9 kB)
