Characterization of p190RhoGEF, a RhoA-specific guanine nucleotide exchange factor that interacts with microtubules.
Rho family GTPases control numerous cellular processes including cytoskeletal reorganization and transcriptional activation. Rho GTPases are activated by guanine nucleotide exchange factors (GEFs) which stimulate the exchange of bound GDP for GTP. We recently isolated a putative GEF, termed p190RhoGEF that binds to RhoA and, when overexpressed in neuronal cells, induces cell rounding and inhibits neurite outgrowth. Here we show that the isolated tandem Dbl homology/pleckstrin homology domain of p190RhoGEF activates RhoA in vitro, but not Rac1 or Cdc42, as determined by GDP release and protein binding assays. In contrast, full-length p190RhoGEF fails to activate RhoA in vitro. When overexpressed in intact cells, however, p190RhoGEF does activate RhoA with subsequent F-actin reorganization and serum response factor-mediated transcription. Immunofluorescence studies show that endogenous p190RhoGEF localizes to distinct RhoA-containing regions at the plasma membrane, to the cytosol and along microtubules. In vitro and in vivo binding experiments show that p190RhoGEF directly interacts with microtubules via its C-terminal region adjacent to the catalytic Dbl homology/pleckstrin homology domain. Our results indicate that p190RhoGEF is a specific activator of RhoA that requires as yet unknown binding partners to unmask its GDP/GTP exchange activity in vivo, and they suggest that p190RhoGEF may provide a link between microtubule dynamics and RhoA signaling.
van Horck
F P
FP
Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Ahmadian
M R
MR
Haeusler
L C
LC
Moolenaar
W H
WH
Kranenburg
O
O
D000199:Actins / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002451:Cell Compartmentation; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D026362:Serum Response Factor; D022502:Stress Fibers / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D014404:Tubulin / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D000199:Actins; D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D026362:Serum Response Factor; D014157:Transcription Factors; D014404:Tubulin; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein