Quantitative analysis of the effect of phosphoinositide interactions on the function of Dbl family proteins.
Normally, Rho GTPases are activated by the removal of bound GDP and the concomitant loading of GTP catalyzed by members of the Dbl family of guanine nucleotide exchange factors (GEFs). This family of GEFs invariantly contain a Dbl homology (DH) domain adjacent to a pleckstrin homology (PH) domain, and while the DH domain usually is sufficient to catalyze nucleotide exchange, possible roles for the conserved PH domain remain ambiguous. Here we demonstrate that the conserved PH domains of three distinct Dbl family proteins, intersectin, Dbs, and Tiam1, selectively bind lipid vesicles only when phosphoinositides are present. While the PH domains of intersectin and Dbs promiscuously bind several multiphosphorylated phosphoinositides, Tiam1 selectively interacts with phosphatidylinositol 3-phosphate (K(D) approximately 5-10 microm). In addition, and in contrast to recent reports, catalysis of nucleotide exchange on nonprenylated Rac1 provided by various extended portions of Tiam1 is not influenced by (a) soluble phosphoinositide head groups, (b) dibutyl versions of phosphoinositides, or (c) lipid vesicles containing phosphoinositides. Likewise, GEF activity afforded by DH/PH fragments of intersectin and Dbs are also not altered by phosphoinositide interactions. These results strongly suggest that unless all relevant components are localized to a lipid membrane surface, Dbl family GEFs generally are not intrinsically modulated by binding phosphoinositides.
Snyder
J T
JT
Department of Biochemistry and Biophysics, Program in Molecular and Cellular Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Rossman
K L
KL
Baumeister
M A
MA
Pruitt
W M
WM
Siderovski
D P
DP
Der
C J
CJ
Lemmon
M A
MA
Sondek
J
J
D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D015151:Immunoblotting; D010716:Phosphatidylinositols / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D064067:Rho Guanine Nucleotide Exchange Factors; D020349:Surface Plasmon Resonance; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1
D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; D020662:Guanine Nucleotide Exchange Factors; C092132:Mcf2l protein, mouse; D010716:Phosphatidylinositols; D011506:Proteins; D064067:Rho Guanine Nucleotide Exchange Factors; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; C513260:Tiam1 protein, mouse; C115919:intersectin 1