Cell Biology
Cytohesin-2/ARNO, through Its Interaction with Focal Adhesion Adaptor Protein Paxillin, Regulates Preadipocyte Migration via the Downstream Activation of Arf6*

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The formation of primitive adipose tissue is the initial process in adipose tissue development followed by the migration of preadipocytes into adipocyte clusters. Comparatively little is known about the molecular mechanism controlling preadipocyte migration. Here, we show that cytohesin-2, the guanine-nucleotide exchange factor for the Arf family GTP-binding proteins, regulates migration of mouse preadipocyte 3T3-L1 cells through Arf6. SecinH3, a specific inhibitor of the cytohesin family, markedly inhibits migration of 3T3-L1 cells. 3T3-L1 cells express cytohesin-2 and cytohesin-3, and knockdown of cytohesin-2 with its small interfering RNA effectively decreases cell migration. Cytohesin-2 preferentially acts upstream of Arf6 in this signaling pathway. Furthermore, we find that the focal adhesion protein paxillin forms a complex with cytohesin-2. Paxillin colocalizes with cytohesin-2 at the leading edges of migrating cells. This interaction is mediated by the LIM2 domain of paxillin and the isolated polybasic region of cytohesin-2. Importantly, migration is inhibited by expression of the constructs containing these regions. These results suggest that cytohesin-2, through a previously unexplored complex formation with paxillin, regulates preadipocyte migration and that paxillin plays a previously unknown role as a scaffold protein of Arf guanine-nucleotide exchange factor.

Adipocyte
Cell Migration
Cytoskeleton
Exchangers
Protein-Protein Interactions
Cytohesin
Paxillin

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*

This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to J. Y.) and from the Ministry of Health, Labor, and Welfare, by the Japan Health Science Foundation and the Ministry of Human Health and Welfare (to Y. M., J. Y., and A. T.) and by research grants from the Human Health Sciences Foundation (to J. Y.), the Naito Foundation (to J. Y.), the Suzuken Foundation (to J. Y.), and the Takeda Science Foundation (to J. Y.).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

1

Both authors contributed equally to this work.