Localization of nectin-free afadin at the leading edge and its involvement in directional cell movement induced by platelet-derived growth factor.

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.

Miyata M ，Ogita H ，Komura H ，Nakata S ，Okamoto R ，Ozaki M ，Majima T ，Matsuzawa N ，Kawano S ，Minami A ，Waseda M ，Fujita N ，Mizutani K ，Rikitake Y ，Takai Y ... -  《-》

Sequential activation of Rap1 and Rac1 small G proteins by PDGF locally at leading edges of NIH3T3 cells.

Takahashi M ，Rikitake Y ，Nagamatsu Y ，Hara T ，Ikeda W ，Hirata K ，Takai Y ... -  《-》

Involvement of the nectin-afadin complex in PDGF-induced cell survival.

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.

Kanzaki N ，Ogita H ，Komura H ，Ozaki M ，Sakamoto Y ，Majima T ，Ijuin T ，Takenawa T ，Takai Y ... -  《JOURNAL OF CELL SCIENCE》

Role of afadin in vascular endothelial growth factor- and sphingosine 1-phosphate-induced angiogenesis.

Angiogenesis contributes to physiological and pathological conditions, including atherosclerosis. The Rap1 small G protein regulates vascular integrity and angiogenesis. However, little is known about the effectors of Rap1 involved in angiogenesis. It is not known whether afadin, an adherens junction protein that connects immunoglobulin-like adhesion molecule nectins to the actin cytoskeleton and binds activated Rap1, plays a role in angiogenesis. We investigated the role of endothelial afadin in angiogenesis and attempted to clarify the underlying molecular mechanism. Treatment of human umbilical vein endothelial cells (HUVECs) with vascular endothelial growth factor (VEGF) and sphingosine 1-phosphate (S1P) induced the activation of Rap1. Activated Rap1 regulated intracellular localization of afadin. Knockdown of Rap1 or afadin by small interfering RNA inhibited the VEGF- and S1P-induced capillary-like network formation, migration, and proliferation, and increased the serum deprivation-induced apoptosis of HUVECs. Knockdown of Rap1 or afadin decreased the accumulation of adherens and tight junction proteins to the cell-cell contact sites. Rap1 regulated the interaction between afadin and phosphatidylinositol 3-kinase (PI3K), recruitment of the afadin-PI3K complex to the leading edge, and the activation of Akt, indicating the involvement of Rap1 and afadin in the PI3K-Akt signaling pathway. Binding of afadin to Rap1 regulated the activity of Rap1 in a positive-feedback manner. In vivo, conditional deletion of afadin in mouse vascular endothelium using a Cre-loxP system impaired the VEGF- and S1P-induced angiogenesis. These results demonstrate a novel molecular mechanism by which Rap1 and afadin regulate the VEGF- and S1P-induced angiogenesis.

Tawa H ，Rikitake Y ，Takahashi M ，Amano H ，Miyata M ，Satomi-Kobayashi S ，Kinugasa M ，Nagamatsu Y ，Majima T ，Ogita H ，Miyoshi J ，Hirata K ，Takai Y ... -  《-》

Interaction and localization of Necl-5 and PDGF receptor beta at the leading edges of moving NIH3T3 cells: Implications for directional cell movement.

Amano H ，Ikeda W ，Kawano S ，Kajita M ，Tamaru Y ，Inoue N ，Minami Y ，Yamada A ，Takai Y ... -  《-》