Modulation of Oncogenic DBL Activity by Phosphoinositol Phosphate Binding to Pleckstrin Homology Domain

Abstract

The Dbl family guanine nucleotide exchange factors (GEFs) contain a region of sequence similarity consisting of a catalytic Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain. PH domains are involved in the regulated targeting of signaling molecules to plasma membranes by protein-protein and/or protein-lipid interactions. Here we show that Dbl PH domain binding to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-triphosphate results in the inhibition of Dbl GEF activity on Rho family GTPase Cdc42. Phosphatidylinositol 4,5-bisphosphate binding to the PH domain significantly inhibits the Cdc42 interactive activity of the DH domain suggesting that the DH domain is subjected to the PH domain modulation under the influence of phosphoinositides (PIPs). We generated Dbl mutants unable to interact with PIPs. These mutants retained GEF activity on Cdc42 in the presence of PIPs and showed a markedly enhanced activating potential for both Cdc42 and RhoA in vivo while displaying decreased cellular transforming activity. Immunofluorescence analysis of NIH3T3 transfectants revealed that whereas the PH domain localizes to actin stress fibers and plasma membrane, the PH mutants are no longer detectable on the plasma membrane. These results suggest that modulation of PIPs in both the GEF catalytic activity and the targeting to plasma membrane determines the outcome of the biologic activity of Dbl.

Intersectin 1L Guanine Nucleotide Exchange Activity Is Regulated by Adjacent src Homology 3 Domains That Are Also Involved in Endocytosis

“…As has been seen for other DH domains (Liu et al, 1998), the exchange activity of the intersectin 1L DH domain alone is reduced relative to the exchange activity of the DHPH domains together ( Figure 3C and Hussain et al, 2001). Phosphatidylinositol phosphates have been shown to bind GEF associated PH domains and in some instances this binding regulates the DH domain exchange activity (Han et al, 1998;Nimnual et al, 1998;Crompton et al, 2000;Das et al, 2000;Russo et al, 2001;Snyder et al, 2001). When exchange assays are done with the intersectin 1L DH domain in the presence of phosphatidylinositol-(4,5)-P 2 (PIP 2 ) liposomes or soluble phosphatidylinositol phosphates, no effect is observed on the exchange activity (unpublished data), consistent with published results (Snyder et al, 2001).…”

“…The noncatalytic parts of the structurally complex GEFs link the exchange activity to cellular processes and inhibit the DH domain exchange activity (Zheng, 2001;Hoffman and Cerione, 2002). Cellular inputs, such as protein (Hart et al, 1998;Scita et al, 1999;Innocenti et al, 2002) and phospholipid binding to (Han et al, 1998;Nimnual et al, 1998;Crompton et al, 2000;Das et al, 2000;Russo et al, 2001), and phosphorylation of (Crespo et al, 1997;Han et al, 1997;Schuebel et al, 1998;Aghazadeh et al, 2000) these regulatory regions derepress exchange activity. Integration of cellular signals by Rho GEFs can focus GTPase activity to allow temporal and spatially localized activation of actin cytoskeletal rearrangements.…”

Homo- and hetero-oligomerization of PDZ-RhoGEF, LARG and p115RhoGEF by their C-terminal region regulates their in vivo Rho GEF activity and transforming potential

“…For example, the PI3K product phosphoinositol (3, 4, 5) trisphosphate binds to their PH domain and appears to alleviate an inhibitory effect of the PH domain on the DH domain (Han et al, 1998;Nimnual et al, 1998). By contrast, the GEF activity of Dbl towards Cdc42 was found to be inhibited by either phosphoinositol (4, 5) diphosphate or phospohoinositol (3, 4, 5) trisphosphate binding to the PH domain (Russo et al, 2001). These findings suggest that phosphatidylinositol phospholipids can regulate, either in a positive or negative fashion, the intramolecular interaction between the DH and PH domains.…”

Phosphorylation-independent membrane relocalization of ezrin following association with Dbl in vivo

“…The immunofluorescence staining for ezrin f.l or ezrin DC appeared localized to the cell plasma membrane, where costaining with Dbl PH domain could be observed (Figure 5d). Conversely, when NIH3T3 cells were transiently cotransfected with a PH domain mutant, unable to bind to PIPs and to localize to the plasma membrane (Russo et al, 2001), ezrin f.l. (Figure 5d) and ezrin DC (not shown) remained cytoplasmic.…”

“…To delineate the region of amino acids in the PH domain that may be involved in the interaction with ezrin, three Dbl mutant proteins, containing substitutions of basic amino acids in the PH domain, were employed as GST-tagged probes. The first mutant, (DH/PH-t), containing a triple substitution of Lys 712 to Ala, Lys 714 to Ala, and Arg 724 to Gly, unable to bind to PIPs and to localize to the plasma membrane has been previously described (Russo et al, 2001). The second mutant, carrying a double substitution of Arg 718 to Gly , and Lys 720 to Ala (DH/PH AL ), retains normal Figure 1 Dbl interacts in vitro with ezrin.…”