Rac1 Recruits the Adapter Protein CMS/CD2AP to Cell-Cell Contacts

Trynette J Van Duijn,Eloise C Anthony,Paul J Hensbergen,André M Deelder,Peter L Hordijk

doi:10.1074/jbc.m109.099481

Trynette J Van Duijn, Eloise C Anthony + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m109.099481

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Abstract

Rac1 is a member of the Rho family of small GTPases, which regulate cell adhesion and migration through their control of the actin cytoskeleton. Rho-GTPases are structurally very similar, with the exception of a hypervariable domain in the C terminus. Using peptide-based pulldown assays in combination with mass spectrometry, we previously showed that the hypervariable domain in Rac1 mediates specific protein-protein interactions. Most recently, we found that the Rac1 C terminus associates to the ubiquitously expressed adapter protein CMS/CD2AP. CD2AP is critical for the formation and maintenance of a specialized cell-cell contact between kidney podocyte foot processes, the slit diaphragm. Here, CD2AP links the cell adhesion protein nephrin to the actin cytoskeleton. In addition, CMS/CD2AP binds actin-regulating proteins, such as CAPZ and cortactin, and has been implicated in the internalization of growth factor receptors. We found that CD2AP specifically interacts with the C-terminal domain of Rac1 but not with that of other Rho family members. Efficient interaction between Rac1 and CD2AP requires both the proline-rich domain and the poly-basic region in the Rac1 C terminus, and at least two of the three N-terminal SH3 domains of CD2AP. CD2AP co-localizes with Rac1 to membrane ruffles, and small interfering RNA-based experiments showed that CD2AP links Rac1 to CAPZ and cortactin. Finally, expression of constitutive active Rac1 recruits CD2AP to cell-cell contacts in epithelial cells, where we found CD2AP to participate in the control of the epithelial barrier function. These data identify CD2AP as a novel Rac1-associated adapter protein that participates in the regulation of epithelial cell-cell contact.

Highlights

The small GTPases of the Rho family regulate many important cellular processes including cell proliferation, migration, and vesicle trafficking
We found that the Rac1 C terminus associates to the ubiquitously expressed adapter protein CMS/ CD2AP
We found that CD2AP interacts with the C-terminal domain of Rac1 but not with that of other Rho family members

Summary

To whom correspondence should be addressed

GTPase-activating proteins on the other hand, stimulate the intrinsic GTPase activity, i.e. the hydrolysis of GTP to GDP, which results in inactivation of the GTPase [2]. We found the Rac GEF ␤-PIX that binds to the Rac C terminus to recruit Rac to focal adhesions and the peripheral membrane, where Rac activity induces the formation of lamellipodia and membrane ruffles [4, 5]. Rac Recruits Adapter Protein CMS/CD2AP to Cell-Cell Contacts barrier [19]. CD2AP is required for the formation and maintenance of this junction, by linking the cell-cell adhesion protein nephrin to the cytoskeleton [20, 21]. We found that the SH3 domains of CD2AP are required for its selective association to the hypervariable C terminus of Rac. We have identified the adapter protein CD2AP as a novel Rac1-associated protein, which is involved in the control of epithelial cell-cell contact

EXPERIMENTAL PROCEDURES

Findings

DISCUSSION