The Ras target AF-6 has been shown to serve as one of the peripheral components of cell-cell adhesions, and is thought to participate in cell-cell adhesion regulation downstream of Ras. We here ...purified an AF-6-interacting protein with a molecular mass of ∼220 kD (p220) to investigate the function of AF-6 at cell-cell adhesions. The peptide sequences of p220 were identical to the amino acid sequences of mouse Fam. Fam is homologous to a deubiquitinating enzyme in Drosophila, the product of the fat facets gene. Recent genetic analyses indicate that the deubiquitinating activity of the fat facets product plays a critical role in controlling the cell fate. We found that Fam accumulated at the cell-cell contact sites of MDCKII cells, but not at free ends of plasma membranes. Fam was partially colocalized with AF-6 and interacted with AF-6 in vivo and in vitro. We also showed that AF-6 was ubiquitinated in intact cells, and that Fam prevented the ubiquitination of AF-6. These results indicate that AF-6 forms a complex with and serves as one of the substrates for Fam, and suggest that the degradation of peripheral components of cell-cell adhesions may be regulated by Fam.
Phospholipid composition of biological membranes differs between the cytoplasmic and exoplasmic leaflets. The type 4 P-type ATPases are phospholipid flippases that generate such membrane phospholipid ...asymmetry. Drs2p, a flippase in budding yeast, is involved in the endocytic recycling pathway. Drs2p is implicated in clathrin-coated vesicle formation, but the underlying mechanisms are not clearly understood. Here we show that the carboxyl-terminal cytoplasmic region of Drs2p directly binds to Rcy1p, an F-box protein that is also required for endocytic recycling. The Drs2p-binding region was mapped to the amino acids 574-778 region of Rcy1p and a mutant Rcy1p lacking this region was defective in endocytic recycling of a v-SNARE Snc1p. We isolated Drs2p point mutants that reduced the interaction with Rcy1p. The mutation sites were clustered within a small region (a.a. 1260-1268) of Drs2p. Although these point mutants did not exhibit clear phenotypes, combination of them resulted in cold-sensitive growth, defects in endocytic recycling of Snc1p and defective localization of Rcy1p to endosomal membranes like the drs2 null mutant. These results suggest that the interaction of Drs2p with Rcy1p plays an important role for Drs2p function in the endocytic recycling pathway.
A Novel GTPase-activating Protein for R-Ras (∗) Yamamoto, Takaharu; Matsui, Takeshi; Nakafuku, Masato ...
The Journal of biological chemistry,
12/1995, Volume:
270, Issue:
51
Journal Article
Peer reviewed
Open access
R-Ras, belonging to the Ras small GTP-binding protein superfamily, has been implicated in regulation of various cell functions such as gene expression, cell proliferation, and apoptotic cell death. ...In the present study, we purified an R-Ras-interacting protein with molecular mass of about 98 kDa (p98) from bovine brain cytosol by glutathione S-transferase (GST)-R-Ras affinity column chromatography. This protein bound to GTPγS (guanosine 5′-(3-O-thio)triphosphate, a nonhydrolyzable GTP analog)•R-Ras but not to GDP•R-Ras, GTPγS•R-Ras with a mutation in the effector domain (R-RasA64), GTPγS•Ha-Ras, or GTPγS•RalA. We obtained a cDNA encoding p98 on the basis of its partial amino acid sequences. The predicted protein consists of 834 amino acids whose calculated mass, 95,384 Da, is close to the apparent molecular mass of p98. The amino acid sequence shows a high degree of sequence similarity to the entire sequence of Gap1m, one of the GTPase-activating proteins (GAP) for Ha-Ras. A recombinant protein consisting of the GAP-related domain of p98 fused to maltose-binding protein stimulated GTPase activity of R-Ras, and showed a weak effect on that of Ha-Ras but not that of Rap1 or Rho. These results clearly indicate that p98 is a novel GAP for R-Ras. Thus, we designated this protein as R-Ras GAP.
In infantile leukemias and therapy-related leukemias, the MLL gene is frequently found to be disrupted and fused to various translocation partner genes, such as AF4/FEL, LTG9/AF9 and LTG19/ENL as a ...result of 11q23 translocations. We previously showed that the N-terminal portion common to various chimeric MLL products, as well as to MLL-LTG9 and MLL-LTG19, localizes in the nuclei, and therefore suggested that it might play an important role in leukemogenesis. In the present study, MLL-AF6 chimeric products found in the t(6;11)(q27;q23) translocation were analysed since AF6, a Ras-binding protein, exhibits a different subcellular localization from that of LTG9/AF9 and LTG19/ENL. Immunofluorescence staining data and cell fractionation analyses demonstrated that MLL-AF6 chimeric products localize in the nuclei despite the fact that AF6 itself localizes in the cytoplasm, confirming the importance of the nuclear localization of chimeric MLL products. The region in the N-terminal portion of MLL responsible for this nuclear localization was examined and found to be a region containing AT-hook motifs.
Schizosaccharomyces pombe Cap1 has been identified as the (adenylyl) cyclase-associated protein. Cap1 was able to bind Cap1 itself and actin. Cap1 localized at the growing tip, and this localization ...was dependent on the Cap1 P2 region. In a two-hybrid screening using cap1 as bait, we isolated csh3, which encodes a protein of 296 amino acids with an SH3 domain and a proline/glutamine-rich region. The binding of Csh3 and Cap1 was confirmed by in vivo pull down assays. Cooperative functions of Csh3 and Cap1 were observed. Deletion of both csh3 and cap1 resulted in heightened sensitivity to CaCl2, while disruption of either gene alone did not have any effect in this regard. In addition, over-expression of csh3 or cap1 alone did not affect cell growth, while over-expression of both genes resulted in growth retardation. Finally, while Csh3-GFP localized to the cytoplasm in wild-type cells, its localization was altered in cap1Δ cells, suggesting that the interaction between Csh3 and Cap1 controls the cellular localization of Csh3. These results demonstrate that Cap1 in Schizo. pombe is a multifunctional protein that functions through interaction with Cap1 itself and other proteins including adenylyl cyclase, actin and Csh3.
An SH3 protein in fission yeast
Septins are filament-forming proteins that function in cytokinesis in a wide variety of organisms. In budding yeast, the small GTPase Cdc42p triggers the recruitment of septins to the incipient ...budding site and the assembly of septins into a ring. We herein report that Bni1p and Cla4p, effectors of Cdc42p, are required for the assembly of the septin ring during the initiation of budding but not for its maintenance after the ring converts to a septin collar. In bni1Delta cla4-75-td mutant, septins were recruited to the incipient budding site. However, the septin ring was not assembled, and septins remained at the polarized growing sites. Bni1p, a formin family protein, is a member of the polarisome complex with Spa2p, Bud6p, and Pea2p. All spa2Delta cla4-75-td, bud6Delta cla4-75-td, and pea2Delta cla4-75-td mutants showed defects in septin ring assembly. Bni1p stimulates actin polymerization for the formation of actin cables. Point mutants of BNI1 that are specifically defective in actin cable formation also exhibited septin ring assembly defects in the absence of Cla4p. Consistently, treatment of cla4Delta mutant with the actin inhibitor latrunculin A inhibited septin ring assembly. Our results suggest that polarisome components and Cla4p are required for the initial assembly of the septin ring and that the actin cytoskeleton is involved in this process.
The small GTP binding protein Rho is implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid to form stress fibers and focal contacts. Here we have purified a ...Rho‐interacting protein with a molecular mass of approximately 164 kDa (p164) from bovine brain. This protein bound to GTPgammaS (a non‐hydrolyzable GTP analog).RhoA but not to GDP.RhoA or GTPgammaS.RhoA with a mutation in the effector domain (RhoAA37).p164 had a kinase activity which was specifically stimulated by GTPgammaS.RhoA. We obtained the cDNA encoding p164 on the basis of its partial amino acid sequences and named it Rho‐associated kinase (Rho‐kinase). Rho‐kinase has a catalytic domain in the N‐terminal portion, a coiled coil domain in the middle portion and a zinc finger‐like motif in the C‐terminal portion. The catalytic domain shares 72% sequence homology with that of myotonic dystrophy kinase and the coiled coil domain contains a Rho‐interacting interface. When COS7 cells were cotransfected with Rho‐kinase and activated RhoA, some Rho‐kinase was recruited to membranes. Thus it is likely that Rho‐kinase is a putative target serine/threonine kinase for Rho and serves as a mediator of the Rho‐dependent signaling pathway.
In the teaching of literature, it is important to know the process of interaction in which we as readers are producing our own "text" in the act of reading. We are reading, interpreting, and all the ...while revising the text. As long as reading is a sort of creating something new, we may even undermine the author's intention. Probably through such procedure we try to reconstruct "what we have lost" in order to find "where we should be." This seems to be the only way to justify the act of reading in what Senri Sugai calls the "essential dissociation between language and its referents."
The dynamic rearrangement of cell-cell junctions such as tight junctions and adherens junctions is a critical step in various cellular processes, including establishment of epithelial cell polarity ...and developmental patterning. Tight junctions are mediated by molecules such as occludin and its associated ZO-1 and ZO-2, and adherens junctions are mediated by adhesion molecules such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras results in the perturbation of cell-cell contacts. We previously identified the ALL-1 fusion partner from chromosome 6 (AF-6) as a Ras target. AF-6 has the PDZ domain, which is thought to localize AF-6 at the specialized sites of plasma membranes such as cell-cell contact sites. We investigated roles of Ras and AF-6 in the regulation of cell-cell contacts and found that AF-6 accumulated at the cell-cell contact sites of polarized MDCKII epithelial cells and had a distribution similar to that of ZO-1 but somewhat different from those of catenins. Immunoelectron microscopy revealed a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacted with ZO-1 in vitro. ZO-1 interacted with the Ras-binding domain of AF-6, and this interaction was inhibited by activated Ras. AF-6 accumulated with ZO-1 at the cell-cell contact sites in cells lacking tight junctions such as Rat1 fibroblasts and PC12 rat pheochromocytoma cells. The overexpression of activated Ras in Rat1 cells resulted in the perturbation of cell-cell contacts, followed by a decrease of the accumulation of AF-6 and ZO-1 at the cell surface. These results indicate that AF-6 serves as one of the peripheral components of tight junctions in epithelial cells and cell-cell adhesions in nonepithelial cells, and that AF-6 may participate in the regulation of cell-cell contacts, including tight junctions, via direct interaction with ZO-1 downstream of Ras.
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