Regulation by afadin of cyclical activation and inactivation of Rap1, Rac1, and RhoA small G proteins at leading edges of moving NIH3T3 cells.
Cyclical activation and inactivation of Rho family small G proteins, such as Rho, Rac, and Cdc42, are needed for moving cells to form leading edge structures in response to chemoattractants. However, the mechanisms underlying the dynamic regulation of their activities are not fully understood. We recently showed that another small G protein, Rap1, plays a crucial role in the platelet-derived growth factor (PDGF)-induced formation of leading edge structures and activation of Rac1 in NIH3T3 cells. We showed here that knockdown of afadin, an actin-binding protein, in NIH3T3 cells resulted in a failure to develop leading edge structures in association with an impairment of the activation of Rap1 and Rac1 and inactivation of RhoA in response to PDGF. Overexpression of a constitutively active mutant of Rap1 (Rap1-CA) and knockdown of SPA-1, a Rap1 GTPase-activating protein that was negatively regulated by afadin by virtue of binding to it, in afadin-knockdown NIH3T3 cells restored the formation of leading edge structures and the reduction of the PDGF-induced activation of Rac1 and inactivation of RhoA, suggesting that the inactivation of Rap1 by SPA-1 is responsible for inhibition of the formation of leading edge structures. The effect of Rap1-CA on the restoration of the formation of leading edge structures and RhoA inactivation was diminished by additional knockdown of ARAP1, a Rap-activated Rho GAP, which localized at the leading edges of moving NIH3T3 cells. These results indicate that afadin regulates the cyclical activation and inactivation of Rap1, Rac1, and RhoA through SPA-1 and ARAP1.
Miyata
Muneaki
M
Division of Molecular and Cellular Biology, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Japan.
Rikitake
Yoshiyuki
Y
Takahashi
Motonori
M
Nagamatsu
Yuichi
Y
Yamauchi
Yusuke
Y
Ogita
Hisakazu
H
Hirata
Ken-ichi
K
Takai
Yoshimi
Y
D000818:Animals; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D004789:Enzyme Activation; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D041681:NIH 3T3 Cells; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein; D020827:rap1 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein
D020690:GTPase-Activating Proteins; D008840:Microfilament Proteins; D009479:Neuropeptides; D009687:Nuclear Proteins; D010982:Platelet-Derived Growth Factor; C484602:Rac1 protein, mouse; C091108:Sipa1 protein, mouse; C108835:afadin; C526380:RhoA protein, mouse; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D020827:rap1 GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein