ABSTRACT
Afadin is an actin-filament-binding protein that binds to nectin, an immunoglobulin-like cell-cell adhesion molecule, and plays an important role in the formation of adherens junctions. Here, we show that afadin, which did not bind to nectin and was localized at the leading edge of moving cells, has another role: enhancement of the directional, but not random, cell movement. When NIH3T3 cells were stimulated with platelet-derived growth factor (PDGF), afadin colocalized with PDGF receptor, alphavbeta3 integrin and nectin-like molecule-5 at the leading edge and facilitated the formation of leading-edge structures and directional cell movement in the direction of PDGF stimulation. However, these phenotypes were markedly perturbed by knockdown of afadin, and were dependent on the binding of afadin to active Rap1. Binding of Rap1 to afadin was necessary for the recruitment of afadin and the tyrosine phosphatase SHP-2 to the leading edge. SHP-2 was previously reported to tightly regulate the activation of PDGF receptor and its downstream signaling pathway for the formation of the leading edge. These results indicate that afadin has a novel role in PDGF-induced directional cell movement, presumably in cooperation with active Rap1 and SHP-2.
Subject(s)
Cell Movement/drug effects , Microfilament Proteins/physiology , Platelet-Derived Growth Factor/pharmacology , Animals , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Blotting, Western , Cattle , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Immunoprecipitation , Mice , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , NIH 3T3 Cells , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , rap1 GTP-Binding Proteins/genetics , rap1 GTP-Binding Proteins/metabolismABSTRACT
The nectin-afadin complex is involved in the formation of cell-cell junctions, such as adherens junctions (AJs) and tight junctions (TJs). Nectins are Ca(2+)-independent immunoglobulin-like cell-cell adhesion molecules, whereas afadin is an intracellular nectin-binding protein that connects nectins to the cadherin-catenin system at AJs and to the claudin-zona-occludens (ZO) protein system at TJs. Afadin(-/-) mice show embryonic lethality, resulting from impaired migration and improper differentiation of cells due to disorganization of cell-cell junctions during gastrulation. However, it remains to be elucidated whether disruption of afadin affects apoptosis. In the present study, we first found that embryoid bodies derived from afadin-knockout embryonic stem (ES) cells contained many more apoptotic cells than those derived from wild-type ES cells. We also revealed that apoptosis induced by serum starvation or Fas-ligand stimulation was increased in cultured NIH3T3 cells when afadin or nectin-3 was knocked down. The nectin-afadin complex was involved in the platelet-derived growth factor (PDGF)-induced activation of phosphatidylinositol 3-kinase (PI3K)-Akt signaling for cell survival. This complex was associated with PDGF receptor on the plasma membrane at cell-cell adhesion sites. Thus, the nectin-afadin complex is involved in PDGF-induced cell survival, at least through the PI3K-Akt signaling pathway.
Subject(s)
Cell Adhesion Molecules/metabolism , Cell Survival/physiology , Microfilament Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Platelet-Derived Growth Factor/metabolism , Animals , Apoptosis/drug effects , Apoptosis/genetics , Cell Adhesion Molecules/antagonists & inhibitors , Cell Adhesion Molecules/genetics , Cell Survival/drug effects , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Enzyme Activation , Fas Ligand Protein/pharmacology , Humans , Intercellular Junctions/metabolism , Intercellular Junctions/ultrastructure , Mice , Mice, Knockout , Microfilament Proteins/genetics , NIH 3T3 Cells , Nectins , RNA, Small Interfering , Signal Transduction , Up-RegulationABSTRACT
In epithelial cells, tight junctions (TJs) and adherens junctions (AJs) form junctional complexes. At AJs, cadherins and nectins are the major cell-cell adhesion molecules. Nectins first form cell-cell adhesions and then recruit cadherins to the nectin-based cell-cell adhesion sites to form AJs in coordination with the activation of integrin alpha(v)beta(3), followed by the formation of TJs. We previously demonstrated that when MDCK cells precultured at a low Ca(2+) concentration were treated with the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA), incomplete AJs and a TJ-like structure were achieved. However, it remains unknown how PKC is activated and how it regulates the formation of cell-cell junctions. When MDCK cells precultured at a low Ca(2+) concentration were treated with TPA, incomplete AJs were formed without the activation of integrin alpha(v)beta(3). Treatment of cells with TPA also enhanced the phosphorylation of FAK, which transmits the outside-in signal of integrin and plays a role in the nectin-induced formation of AJs. In addition, inhibition of PKC suppressed the formation of AJs. These results indicate that the activation of PKC functions downstream of integrin alpha(v)beta(3) and upstream of FAK, and is important for the nectin-induced formation of AJs.
Subject(s)
Adherens Junctions/metabolism , Integrin alphaVbeta3/metabolism , Protein Kinase C/metabolism , Adherens Junctions/drug effects , Adherens Junctions/ultrastructure , Animals , CSK Tyrosine-Protein Kinase , Calcium/pharmacology , Cell Adhesion Molecules/metabolism , Cell Line , Dogs , Enzyme Activation , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Models, Biological , Nectins , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction/drug effects , Tetradecanoylphorbol Acetate/pharmacology , Transfection , src-Family KinasesABSTRACT
Cell-matrix and cell-cell junctions cross-talk together, and these two junctions cooperatively regulate cell movement, proliferation, adhesion, and polarization. However, the mechanism of this cross-talk remains unknown. An immunoglobulin-like cell-cell adhesion molecule nectin first trans-interacts with each other to form cell-cell adhesion and induces activation of Rap1, Cdc42, and Rac small G proteins through c-Src. Trans-interacting nectin then recruits another cell-cell adhesion molecule cadherin to the nectin-based cell-cell adhesion sites and forms adherens junctions (AJs). Here, we show that integrin alpha(v)beta3 functionally and physically associates with nectin. Integrin alpha(v)beta3 colocalized with nectin at the nectin-based cell-cell adhesion sites. The association of integrin alpha(v)beta3 with nectin was direct and was mediated through their extracellular regions. This interaction was necessary for the nectin-induced signaling. Focal adhesion kinase, which relays the integrin-initiated outside-in signals to the intracellular signaling molecules, was also involved in the nectin-induced signaling. During the formation of AJs, the high affinity form of integrin alpha(v)beta3 co-localized with nectin at the primordial cell-cell contact sites, and then after the establishment of AJs, this high affinity form of integrin alpha(v)beta3 was converted to the low affinity form, which continued to co-localize with nectin. Thus, integrin alpha(v)beta3 and nectin play pivotal roles in the cross-talk between cell-matrix and cell-cell junctions and the formation of cadherin-based AJs.
Subject(s)
Cell Adhesion Molecules/chemistry , Integrin alphaVbeta3/chemistry , Intercellular Junctions/metabolism , Animals , Cell Adhesion , Cell Adhesion Molecules/metabolism , Cell Communication , Dogs , Genetic Vectors , Immunoglobulins/chemistry , Mice , NIH 3T3 Cells , Nectins , Protein Binding , Signal Transduction , Wound HealingABSTRACT
The enantioselectivity of a Burkholderia cepacia lipase toward secondary alcohols could be both increased and decreased rationally by introducing only a single mutation on the basis of the mechanism proposed previously.
Subject(s)
Lipase/genetics , Lipase/metabolism , Mutagenesis, Site-Directed , Alcohols/chemistry , Burkholderia cepacia/enzymology , DNA, Bacterial/genetics , Escherichia coli/genetics , Models, Molecular , Reverse Transcriptase Polymerase Chain Reaction , StereoisomerismABSTRACT
A highly sensitive method for the determination of bisphenol-A in water with semi-micro column high-performance liquid chromatography using 2-methoxy-4-(2-phthalimidinyl)phenylsulfonyl chloride as a fluorescent labeling reagent has been developed. The labeling reaction was carried out at 70 degrees C for 20 min in borate buffer (pH 9.5). The derivative eluted at 11.6 min on a reversed-phase column with methanol-water (78:22, v/v) at a flow-rate of 0.2 ml/min. The fluorescence was monitored at 308 nm for excitation and 410 nm for emission. The detection limit (S/N = 3) was 10 fmol per injection. The labeling yield was about 95%.