Multifunctional roles for the PH domain of Dbs in regulating Rho GTPase activation.
Dbl family members are guanine nucleotide exchange factors specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. Dbs, a Dbl family member specific for Cdc42 and RhoA, exhibits transforming activity when overexpressed in NIH 3T3 mouse fibroblasts. In this study, the PH domain of Dbs was mutated to impair selectively either guanine nucleotide exchange or phosphoinositide binding in vitro and resulting physiological alterations were assessed. As anticipated, substitution of residues within the PH domain of Dbs integral to the interface with GTPases reduced nucleotide exchange and eliminated the ability of Dbs to transform NIH 3T3 cells. More interestingly, substitutions within the PH domain that prevent interaction with phosphoinositides yet do not alter in vitro activation of GTPases also do not transform NIH 3T3 cell and fail to activate RhoA in vivo despite proper subcellular localization. Therefore, the PH domain of Dbs serves multiple roles in the activation of GTPases and cannot be viewed as a simple membrane-anchoring device. In particular, the data suggest that binding of phosphoinositides to the PH domain within the context of membrane surfaces may direct orientations or conformations of the linked DH and PH domains to regulate GTPases activation.
Rossman
Kent L
KL
Department of Pharmacology, The University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Cheng
Li
L
Mahon
Gwendolyn M
GM
Rojas
Rafael J
RJ
Snyder
Jason T
JT
Whitehead
Ian P
IP
Sondek
John
J
D016475:3T3 Cells; D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism* / Q000502:physiology*; D051379:Mice; D008958:Models, Molecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D064067:Rho Guanine Nucleotide Exchange Factors; D013050:Spectrometry, Fluorescence; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D018076:DNA, Complementary; D020662:Guanine Nucleotide Exchange Factors; C092132:Mcf2l protein, mouse; D064067:Rho Guanine Nucleotide Exchange Factors; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein