Regulation by Afadin of Cyclical Activation and Inactivation of Rap1, Rac1, and RhoA Small G Proteins at Leading Edges of Moving NIH3T3 Cells

Abstract

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.

Suppression of SIPA-1 expression may reduce bladder cancer invasion and metastasis via the downregulation of E-cadherin and ZO-1

“…Previous studies have suggested that SIPA-1 is involved in certain types of cancer activity, including mutation (23,24), apoptosis (12,25), proliferation (4,15), invasion (15,16) and metastasis (13,26,27). In the present study, it was observed that SIPA-1 additionally regulates bladder cancer cell metastasis and invasion (Figs.…”

“…1). The molecular mechanisms underlying the SIPA-1-mediated regulation of E-cadherin remain unclear, and it has been hypothesized that the protein afadin serves a pivotal function in the dynamic cyclical activation and inactivation of Rap1 via the coordinated regulation of SIPA-1 (24,30). ZO-1 encodes a protein located on the cytoplasmic membrane surface of intercellular tight junctions.…”

The effects of modifying RhoA and Rac1 activities on heterotypic contact inhibition of locomotion

“…All three proteins are involved in actin polymerization and have been shown to be localized in the cell leading edge during protrusion formation [18][19][20][21][22]. We found both increasing (V12Rac1) and lowering (N17Rac1) activity resulted in defective heterotypic CIL.…”

Role of Afadin in Vascular Endothelial Growth Factor– and Sphingosine 1-Phosphate–Induced Angiogenesis

“…Indeed, we recently reported that the interaction of Rap1 with afadin increases the activity of Rap1 in NIH3T3 cells and that this interaction prevents the SPA-1-induced inactivation of Rap1. 19 It was reported that both the transinteraction of nectins and the trans-interaction of VEcadherin induces the activation of Rap1. 38,39 Therefore, afadin potentially regulates the activity of Rap1 by two mechanisms: (1) prevention of the inactivation of Rap1 by direct binding to Rap1 and/or RapGAP; and (2) enhancement of the formation of the nectin-and/or VE-cadherin-dependent cell-cell adhesion, although the latter mechanism might be involved during and/or after the formation of cell-cell adhesion.…”

“…16, 17 We have demonstrated that afadin regulates cell adhesion and polarity in epithelial cells and neurons, and cell movement of fibroblasts. 14,18,19 The roles of afadin in tubulogenesis and angiogenesis are unknown, but the current literature implies afadin may be a promising candidate protein linking Rap1 to angiogenesis. In this study, we therefore attempt to clarify the roles of Rap1 and afadin in the VEGF-and sphingosine 1-phosphate (S1P)-induced angiogenesis.…”