Characterization of functional domains of mDia1, a link between the small GTPase Rho and the actin cytoskeleton.
The widely expressed diaphanous proteins, a subclass of formins, comprise links between the Rho GTPases and the actin-based cytoskeleton. They contain several functional domains that are thought to be responsible for interaction with different ligands: the FH1 domain for binding the actin-associated protein profilin; the RBD for targeting activated Rho; and the C-terminal CIID module for autoregulation of the overall diaphanous activity. Using deletion constructs of the murine mDia1, we have analyzed the functional properties of these three domains separately in in vitro assays and in transiently and stably transfected cell lines. We show that the proline-rich FH1 domain effectively binds to profilins in vitro as well as in cells, that the RBD complexes with the CIID in a species-restricted manner and that overexpression of RBD causes spontaneous ruffling and loss of stress fibers, together with loss of directional motility. Supertransfection of cells stably expressing the RBD with dominant negative Rac effectively suppresses ruffling. Our data contribute to the understanding of the function of these domains in linking the actin cytoskeleton with the Rho-signaling cascade. Furthermore, they suggest that inactivation of Rho by exogenous RBD causes upregulation of Rac activity in the transfected cells.
Krebs
A
A
Cell Biology, Zoological Institute, Technical University of Braunschweig, D-38092 Braunschweig, Germany.
Rothkegel
M
M
Klar
M
M
Jockusch
B M
BM
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002465:Cell Movement / Q000502:physiology*; D022081:Cell Surface Extensions / Q000378:metabolism; D003285:Contractile Proteins; D006224:Cricetinae; D003599:Cytoskeleton / Q000378:metabolism*; D000080865:Formins; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D051304:Profilins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D014162:Transfection; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D000199:Actins; D002352:Carrier Proteins; D003285:Contractile Proteins; C491464:Diap1 protein, mouse; D000080865:Formins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008840:Microfilament Proteins; C495738:PFN1 protein, human; C495739:Pfn1 protein, mouse; D051304:Profilins; D049452:Green Fluorescent Proteins; D020741:rho GTP-Binding Proteins