Critical role of the pleckstrin homology domain in Dbs signaling and growth regulation.
Dbl family proteins act as guanine nucleotide exchange factors and positive regulators of Rho GTPase function by stimulating formation of the active, GTP-bound state. All Dbl family Rho guanine nucleotide exchange factors possess an invariant tandem domain structure consisting of a Dbl homology (DH) catalytic domain followed by a pleckstrin homology (PH) regulatory domain. We determined previously that the PH domain of Dbs was critical for the intrinsic catalytic activity of the DH domain in vitro and for Dbs transformation in vivo. In this study, we evaluated the role of phosphoinositide binding to the PH domain in regulating the DH domain function of Dbs in vitro and in vivo. We determined that mutation of basic amino acids located within the beta1-beta2 and beta3-beta4 loops of the PH domain resulted in impaired phospholipid binding in vitro, yet full guanine nucleotide exchange activity in vitro was retained for RhoA and Cdc42. Surprisingly, these mutants were compromised in their ability to activate Rho GTPases in vivo and to cause transformation of NIH 3T3 cells. However, Dbs subcellular localization was impaired by these PH domain mutations, supporting a role for phospholipid interactions in facilitating membrane association. Despite the importance of phospholipid binding for Dbs function in vivo, we found that Dbs signaling and transforming activity was not stimulated by phosphatidylinositol 3-kinase activation. We suggest that the PH domain of Dbs facilitates two distinct roles in the regulation of DH domain function, one critical for GTPase association and activation in vitro and one critical for phosphoinositide binding and GTPase interaction in vivo, that together promote Dbs association with membranes.
Fuentes
Ernesto J
EJ
Department of Biochemistry and Biophysics, University of North Carolina, Lineberger Comprehensive Cancer Center, Chapel Hill, North Carolina 27599, USA.
Karnoub
Antoine E
AE
Booden
Michelle A
MA
Der
Channing J
CJ
Campbell
Sharon L
SL
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001798:Blood Proteins / Q000235:genetics*; D020134:Catalytic Domain; D002455:Cell Division / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics* / Q000378:metabolism*; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010716:Phosphatidylinositols / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics*; D017434:Protein Structure, Tertiary; D064067:Rho Guanine Nucleotide Exchange Factors; D015398:Signal Transduction / Q000502:physiology*; D022502:Stress Fibers / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D001798:Blood Proteins; D020662:Guanine Nucleotide Exchange Factors; C092132:Mcf2l protein, mouse; D010716:Phosphatidylinositols; D010750:Phosphoproteins; D064067:Rho Guanine Nucleotide Exchange Factors; C039077:platelet protein P47; D020558:GTP Phosphohydrolases; D020764:cdc42 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein