The role of RhoD in actin dynamics, receptor trafficking and cancer cell migration

The Ras subfamily of Rho GTPases plays an important role in myriads of cell processes, such as actin cytoskeleton dynamics, membrane trafficking and cell migration. Additionally, they also participate in diverse signal transduction pathways that regulate gene transcription, cell survival and cell growth. The ability of these proteins to function as a signaling molecule depends on their capacity to cycle between an active GTP-bound conformation and an inactive GDP-bound conformation. Till date, three Rho family members namely, RhoA, Rac1 and Cdc42 have been well studied as compared to other Rho family members.

This thesis highlights a less studied Rho GTPase, RhoD, which together with Rif constitutes a separate subgroup of the Rho GTPases. Ectopic expression of RhoD and Rif has a dramatic effect on the organization of the actin filament system observed as long flexible filopodia protrusions and the formation of short bundles of actin filaments. Moreover, RhoD has a role in regulating endosome dynamics and is a negative regulator of cell motility. This motivated us to find out more about the signaling pathways downstream of RhoD. We initiated our studies by identifying a number of novel binding partners for RhoD for instance, FILIP1, WHAMM, Rabankyrin-5 and the ZIP kinase. We observed that RhoD together with FILIP1 and WHAMM had a distinct role in actin dynamics, cell adhesion and cell migration as compared to the better studied members of the Rho subfamily. Furthermore, with the same RhoD binding partners, we elucidated another regulatory role of RhoD in Golgi homeostasis and ER-to-Golgi transport measured by VSV-G protein transport assay. Previous studied have identified Rabankyrin-5 as a Rab5 effector. Interestingly, these studies also reported that Rabankyrin-5 localizes to early endosomes and to macropinosomes of epithelial cells.

We found that Rabankyrin-5 participates in coordinating Rab5 and RhoD in endosome trafficking. We describe a novel mechanism by which RhoD, Rab5 and Rabankyrin-5 coordinate membrane trafficking events and endocytosis, for instance during the internalization of activated tyrosine kinase receptor, such as the PDGFRβ. Finally, we found that RhoD modulates focal adhesion dynamics and actin filament assembly through a novel effector, ZIP kinase. In summary, this thesis sheds light on the less studied Rho GTPase RhoD and provides novel insight into the mechanisms underlying its diverse cellular effects.

List of scientific papers

I. Gad AKB, Nehru V, Ruusala A, Aspenström P. RhoD regulates cytoskeletal dynamics via the actin nucleation-promoting factor WASp homologue associated with actin Golgi membranes and microtubules. Mol Biol Cell. 23 (24): 4807-19.
https://doi.org/10.1091/mbc.E12-07-0555

II. Nehru V, Yagi H, Sato M, Aspenström P. RhoD regulates ER to Golgi transport through its effectors Filamin A-binding protein, FILIP1, and WHAMM. [Manuscript]

III. Nehru V, Aspenström P. RhoD binds the Rab5 effector Rabankyrin-5 and has a role in trafficking of receptor tyrosine kinases. [Submitted]

IV. Nehru V, Almeida FN, Aspenström P. Interaction of RhoD and ZIP kinase modulates actin filament assembly and focal adhesion dynamics. Biochem Biophys Res Commun. 2013. [Accepted]
https://doi.org/10.1016/j.bbrc.2013.02.046