Role of Substrates and Products of PI 3-kinase in Regulating Activation of Rac-Related Guanosine Triphosphatases by Vav

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Science  23 Jan 1998:
Vol. 279, Issue 5350, pp. 558-560
DOI: 10.1126/science.279.5350.558


Mitogen stimulation of cytoskeletal changes and c-jun amino-terminal kinases is mediated by Rac small guanine nucleotide-binding proteins. Vav, a guanosine diphosphate (GDP)–guanosine triphosphate (GTP) exchange factor for Rac that stimulates the exchange of bound GDP for GTP, bound to and was directly controlled by substrates and products of phosphoinositide (PI) 3-kinase. The PI 3-kinase substrate phosphatidylinositol-4,5-bisphosphate inhibited activation of Vav by the tyrosine kinase Lck, whereas the product phosphatidylinositol-3,4,5-trisphosphate enhanced phosphorylation and activation of Vav by Lck. Control of Vav in response to mitogens by the products of PI 3-kinase suggests a mechanism for Ras-dependent activation of Rac.

Mitogens induce Rac-mediated changes in the actin cytoskeleton as well as Rac-mediated regulation of gene expression. Rac-related proteins are activated by a family of guanine nucleotide exchange factors (GEFs) related to the Dbl oncogene product that catalyze the exchange of bound GDP for GTP on Rac-related guanosine triphosphatases (GTPases) (1,2). The functional properties of the Dbl homology domain of the Vav oncogene product are regulated by Lck-dependent tyrosine phosphorylation (3, 4). However, this observation alone does not explain the apparent interaction of Vav with the Ras signaling pathway (5, 6). T cell receptor activation results in activation of Vav, Ras, and PI 3-kinase (7). Each of these events results in activation of Rac-related GTPases (3, 4, 8). However, the mechanism by which Ras, PI 3-kinase, and Vav might interact to mediate activation of Rac is unknown. Vav, like all known Dbl-related molecules, has a pleckstrin homology (PH) domain on the COOH-terminal side of the GEF domain (1). Because some PH domains bind substrates and products of PI 3-kinase (9-11), we tested whether phosphoinositides bound to the PH domain of Vav and affected Vav GEF activity for Rac-related GTPases.

We examined the ability of Vav to stimulate guanine nucleotide exchange on Rac in the presence of substrates or products of PI 3-kinase. We used water-soluble analogs of phosphatidylinositides that are biologically active (12, 13). A His-tagged Vav fusion protein was incubated with the Lck tyrosine kinase to allow the kinase reaction to go to completion. Lck was removed by successive washings of the immobilized Vav protein. Phosphorylated Vav, but not unphosphorylated Vav, stimulated the release of [3H]GDP from Rac (Fig. 1A) (3). The presence of a water-soluble PI 3-kinase substrate, C8 phosphatidylinositol-4,5-bisphosphate [C8PtdIns(4,5)P2], in the GEF reaction resulted in a 90% inhibition of Vav GEF activity. The natural PtdIns(4,5)P2, when incorporated into micelles, also inhibited Vav-GEF activity to a similar extent (14). In contrast, the presence of PI 3-kinase products, C8 phosphatidylinositol-3,4-bisphosphate [C8PtdIns(3,4)P2] or C8 phosphatidylinositol-3,4,5-trisphosphate [C8 PtdIns(3,4,5)P3], in the GEF reaction resulted in a twofold stimulation of Vav GEF activity (Fig. 1A). We examined Vav GEF activity in the presence of the inhibitor C8PtdIns(4,5)P2 (10 μM) together with either C8PtdIns(3,4)P2 (10 μM) or C8PtdIns(3,4,5)P3 (10 μM). Stimulation of Vav GEF activity was still observed, indicating that the products of PI 3-kinase bind Vav with a higher affinity than does C8PtdIns(4,5)P2. Similar effects on Vav GEF activity were obtained with the Rac-related GTPases Cdc42 and RhoA as substrates (14) (Fig. 1A). Thus, activation of PI 3-kinase results in conversion of an inhibitor of Vav-GEF function to an activator of Vav.

Figure 1

Interaction of phosphoinositides with the PH domain of Vav and modulation of Vav GEF activity. (A) Regulation of Vav GEF activity in vitro. Lck-phosphorylated Vav was incubated with Rac, Cdc42, or RhoA and C8PtdIns(4,5)- P2 (10 μM), C8PtdIns(3,4)P2 (10 μM), or C8PtdIns(3,4,5)P3 (10 μM) (12). Guanine nucleotide release assays were done as described (3). (B) Dose-dependent binding of 125I- labeled C8PtdIns(4,5)P2 to Vav. His-tagged Vav(L) protein (3) immobilized on Ni-agarose was incubated with the indicated concentration of Bolton-Hunter reagent–labeled C8PtdIns(4,5)P2-NH2 (25). Material associated with His-Vav(L) immobilized on Ni-agarose was collected, and the amount of 125I-phosphoinositide was counted with a gamma counter. (C) Failure of C8PtdIns(4,5)P2 to displace C8PtdIns(3,4,5)P3from Vav. C8PtdIns(3,4,5)P3-NH2 was labeled with 125I as above. His-Vav(L) protein immobilized on Ni-agarose was incubated with125I-C8PtdIns(3,4,5)P3 alone or in the presence of 50 μM C8PtdIns(4,5)P2 or 50 μM C8PtdIns(3,4,5)P3. The amount of125I-C8PtdIns(3,4,5)P3 associated with Vav was determined as described above. (D) PH domain mutants of Vav are defective in binding C8PtdIns(3,4,5)P3. Wild-type or mutant His-Vav protein (10 pmol) (26) was incubated with a saturating amount of 125I-C8PtdIns(3,4,5)P3, and the amount of C8PtdIns(3,4,5)P3 binding to Vav was determined as above.

To examine whether Vav interacts directly with PtdIns(4,5)P2 and PtdIns(3,4,5)-P3, we incubated 10 pmol of recombinant His-tagged Vav immobilized on Ni-agarose beads with various concentrations of 125I-labeled C8PtdIns(4,5)P2(1 to 50 μM). After 2 hours, we removed material that was not bound to Vav. 125I-C8PtdIns(4,5)P2 bound Vav with an apparent dissociation constant K d of 3 to 4 μM (Fig. 1B). Vav also bound125I-C8PtdIns(3,4,5)P3, and this binding was blocked by the presence of excess unlabeled C8PtdIns(3,4,5)P3 (50 μM final concentration) (Fig.1C). Unlabeled C8PtdIns(4,5)P2 was less efficient at blocking the interaction of Vav with125I-C8PtdIns(3,4,5)P3. The PH domain of another Dbl family member (Sos1) also has a fivefold selectivity for binding PtdIns- (3,4,5)P3 over PtdIns(4,5)P2(15).

The PH domain of Vav likely mediates the effects of phosphoinositides because numerous other PH domains bind phosphoinositides (16). The structures of two distinct PH domains complexed with PtdIns reveal that positively charged amino acids in a loop between β sheet 1 and β sheet 2 have direct interaction with PtdIns (9-11). Alignment of the sequence of the PH domain of Vav with these structures revealed that the PH domain of Vav has positively charged amino acids in positions roughly corresponding to those known to directly interact with phosphoinositides. We created three mutant proteins containing a single or double amino acid substitutions at these residues [Vav(PH-b), Arg422 → Gly422; Vav(PH-e), Lys404 → Ala404; and Vav(PH-m), Lys404 → Ala404, Arg422 → Gly422]. His-tagged fusions of wild-type Vav and each of the PH domain mutants of Vav were incubated with saturating amounts of125I-C8PtdIns(3,4,5)P3, and material not bound to Vav proteins was removed by successive washings. The mutant Vav(PH-e) bound less than 20% of the amount of125I-C8PtdIns(3,4,5)P3 that bound to wild-type Vav (Fig. 1D). The mutants Vav(PH-b) and Vav(PH-m) bound less than one-third of the amount of125I-C8PtdIns(3,4,5)P3 that bound to wild-type Vav. Each of the PH domain mutants was examined for GEF activity with [3H]GDP-Cdc42 as a substrate. In the absence of phosphoinositides, each of the mutant proteins stimulated [3H]GDP release to an extent similar to that of wild-type Vav, but the presence of phosphoinositides had no effect on GEF activity of the PH domain mutant proteins (17). Thus, the PH domain of Vav apparently mediates the effects of phosphoinositides on Vav GEF activity.

Tyr174 of Vav is thought to be the site of phosphorylation by Lck that regulates Vav function (18). A Vav mutant protein with a Tyr (Y) to Phe (F) substitution at position 174 (Y174F) was not phosphorylated by Lck in vitro (Fig. 2A). We investigated whether phosphoinositide binding affected Lck-dependent activation of Vav. A His-tagged Vav protein was incubated with recombinant Lck protein in the presence of [32P]ATP and C8PtdIns(4,5)P2, C8PtdIns-(3,4,5)P3, or no phosphoinositide for 0 to 30 min, and the products were separated by SDS-polyacrylamide gel electrophoresis (PAGE) to visualize phosphorylation of Vav. Phosphorylation of Vav in the presence of C8PtdIns(3,4,5)P3 was detectable after 5 min of incubation and maximal after 10 min (Fig. 2B). Phosphorylation of Vav was enhanced in the presence of C8PtdIns- (3,4,5)P3 compared with that in the presence of C8PtdIns(4,5)P2 or in the absence of phosphoinositides. C8PtdIns(3,4)P2 also enhanced the phosphorylation of Vav by Lck (17). Phosphorylation of Vav by Lck results in an increase in GEF activity (3). Thus, products of PI 3-kinase, in addition to their direct effect on activation of Vav GEF activity, also indirectly activate Vav GEF function by enhancing Lck-dependent phosphorylation of Vav.

Figure 2

Stimulation of Lck-dependent phosphorylation of Vav in vitro by C8PtdIns(3,4,5)P3. (A) Phosphorylation of His-tagged Vav(L) protein, but not of a Vav Tyr174 → Phe174 (Y174F) mutant, by Lck. The indicated Vav protein was incubated with baculovirus-produced Lck (27) in the presnce of [32P]ATP for 30 min and separated by SDS-PAGE. Phosphorylation of Vav was visualized by autoradiography. (B) Phosphorylation of Vav(L) in the absence of phosphoinositide or in the presence of C8PtdIns(4,5)P2 (50 μM) or C8PtdIns-(3,4,5)P3 (50 μM). Portions of the reactions were collected at the indicated time and prepared for SDS-PAGE and autoradiography.

We propose a model in which the PH domain of Vav, when complexed to PtdIns (4,5)P2, inhibits Vav GEF activity. The activation of PI 3-kinase could then serve to eliminate a Vav inhibitor and simultaneously produce activators of Vav GEF activity. Vav is a member of a large family of molecules containing a Dbl homology domain and a PH domain that could be similarly regulated (1). Ras activation of Rac is mediated by PI 3-kinase (19). RasV12C40, a Ras effector mutant that retains the ability to bind to and activate PI 3-kinase but not other Ras effectors (19, 20, 21), cooperated with wild-type Vav to induce membrane ruffling in REF-52 fibroblasts (Fig.3), supporting our suggestion that Ras activation of Rac is mediated by a Dbl-related molecule. Furthermore, regulation through the Dbl homology and PH domains of Sos, a bifunctional GEF for both Ras and Rac, is apparently similar to the regulation that we have proposed for Vav (22). The regulation of Vav by both PI 3-kinase and a protein kinase is similar to the dual regulation of the PH domain containing protein kinase B (PKB/AKT) (23). Perhaps such dual regulation is a general feature of signaling molecules with PH domains.

Figure 3

Cooperation of RasV12C40 and Vav in the induction of membrane ruffling. Quiescent REF-52 cells were microinjected with plasmids expressing (A) RasV12C40 (5 μg/ml), (B) RasV12C40 (5 μg/ml) and a myc-Vav fusion (50 μg/ml), or (C) a myc-Vav fusion (50 μg/ml) alone (28). Six hours after injection, cells were fixed and stained with fluorescein-labeled phalloidin to visualize the actin cytoskeleton (right) and with an antibody to Ras (those cells injected with Ras plasmid) or an antibody to Myc (those cells injected with Vav plasmid alone) (21). Injection of RasV12C40 alone (left) (5 μg/ml) (A) resulted in expression below the amount required to induce membrane ruffling (21). The results shown are representative of at least four independent experiments.

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