MOLECULAR AND CELLULAR BIOLOGY, Apr. 2002, p. 2487–2497 Vol. 22, No. 8 0270-7306/02/$04.00ϩ0 DOI: 10.1128/MCB.22.8.2487–2497.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved. Critical but Distinct Roles for the Pleckstrin Homology and Cysteine-Rich Domains as Positive Modulators of Vav2 Signaling and Transformation Michelle A. Booden,1* Sharon L. Campbell,2 and Channing J. Der1 Department of Pharmacology1 and Department of Biochemistry and Biophysics,2 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Received 4 October 2001/Returned for modification 2 November 2001/Accepted 9 January 2002 Vav2, like all Dbl family proteins, possesses tandem Dbl homology (DH) and pleckstrin homology (PH) domains and functions as a guanine nucleotide exchange factor for Rho family GTPases. Whereas the PH domain is a critical positive regulator of DH domain function for a majority of Dbl family proteins, the PH domains of the related Vav and Vav3 proteins are dispensable for DH domain activity. Instead, Vav proteins contain a cysteine-rich domain (CRD) critical for DH domain function. We evaluated the contribution of the PH domain and the CRD to Vav2 guanine nucleotide exchange, signaling, and transforming activity. Unex- pectedly, we found that mutations of the PH domain impaired Vav2 signaling, transforming activity, and membrane association. However, these mutations do not influence exchange activity on Rac and only slightly affect exchange on RhoA and Cdc42. We also found that the CRD was critical for the exchange activity in vitro and contributed to Vav2 membrane localization. Finally, we found that phosphoinositol 3-kinase activation synergistically enhanced Vav2 transforming and signaling activity by stimulating exchange activity but not membrane association. In conclusion, the PH domain and CRD are mechanistically distinct, positive modu- lators of Vav2 DH domain function in vivo. Rho family proteins are members of the Ras superfamily of contrasting conclusions regarding the specific GTPases tar- small GTPases. Currently, 18 mammalian Rho family proteins geted by Vav (1, 12, 17, 23, 28, 35, 43). have been identified, with Rac1, Cdc42, and RhoA being the The invariant topography of DH and PH domains (DH/PH best characterized (37, 45). Rho family GTPases are guanine domain) found in all Dbl family proteins suggests a critical role nucleotide binding proteins that function as molecular switches for the PH domain in regulation of DH domain function. that cycle between active GTP-bound and inactive GDP-bound Extensive structure-function analyses of the DH/PH domains states. Dbl family proteins serve as guanine nucleotide ex- of various Dbl family proteins suggest that the PH domain may change factors (GEFs), which accelerate the intrinsic GDP/ serve two distinct functions to modulate DH domain activity GTP exchange activity of Rho GTPases to cause formation (8, 39). First, the PH domain may act as a positive modulator of the active GTP-bound protein (8, 39). The activated Rho of the intrinsic catalytic activity of the DH domain. For exam- GTPases then interact with a wide spectrum of downstream ple, a comparison of the catalytic activities of the DH and effector proteins to mediate cellular activities that include reg- DH/PH domains derived from several Dbl family proteins ulation of actin cytoskeletal organization, gene expression, and (e.g., Dbl, Trio, and Dbs) showed that the GEF activity exhib- cellular proliferation (3). A second class of regulatory proteins, ited by a PH-containing protein was up to 100-fold greater than Rho family-specific GTPase-activating proteins, stimulate in- that measured for the DH domain alone in vitro (24, 34, 44). trinsic GTPase activity to return these small GTPases to their Second, it may serve a membrane-targeting function and inactive GDP-bound state and to terminate downstream sig- regulate DH domain interaction with its membrane-bound naling (37). GTPase substrates. For example, the loss of function caused by Vav proteins (Vav, Vav2, and Vav3) are mammalian mem- mutation of the PH domains of Lfc and Dbs could be reversed bers of the Dbl family of proteins (7). All Vav proteins have by addition of a plasma membrane targeting sequence (40, 41). similar structural organizations. Like all Dbl family proteins, In contrast to what has been observed for many Dbl family Vav proteins possess a Dbl homology (DH) domain followed proteins, the PH domains of Vav proteins appear to serve as by a COOH-terminal pleckstrin homology (PH) domain (8, negative regulators of DH domain function and no role in 39). Previous studies indicate that the DH domain interacts membrane targeting has been described. Han and colleagues directly with Rho family GTPases to catalyze GDP release (8, determined that the PH domain of Vav serves as a negative 39). The Vav DH domains exhibit broad GTPase specificity regulator of DH domain GEF activity in vitro (18). This neg- and serve as GEFs for multiple Rho GTPases (RhoA, RhoG, ative regulatory function is promoted by phosphatidylinositol Rac1, and Cdc42), although different studies have reached 4,5-phosphate (PIP2), a substrate of phosphatidylinositol 3-ki- nase (PI3K), and is antagonized by the PI3K product, phos- phatidylinositol 3,4,5-phosphate (PIP ). Hence, PI3K activa- * Corresponding author. Mailing address: University of North Caro- 3 lina at Chapel Hill, CB 7295, Lineberger Comprehensive Cancer Cen- tion is proposed to facilitate the activation of Vav. Consistent ter, Chapel Hill, NC 27599. Phone: (919) 962-1057. Fax: (919) 966- with a negative regulatory function for the PH domain, Ma et 0162. E-mail: [email protected]. al. determined that a variant of Vav with its PH domain de- 2487 2488 BOODEN ET AL. MOL.CELL.BIOL. leted was activated constitutively in vivo (26). In evaluations of MATERIALS AND METHODS PH domain function in NH2-terminally truncated and consti- Molecular constructs. The pCGN-hygro mammalian expression vector regulates tutively activated versions of Vav proteins, mutation of the PH the expression of introduced cDNA sequences from a cytomegalovirus promoter, domains of Vav and Vav3 did not cause significant alteration in encodes hygromycin resistance, and introduces an NH2-terminal hemagglutinin (HA) epitope tag into the encoded protein (14). Human Vav2 truncation mutants GEF activity in vitro or growth and/or morphological trans- were generated by PCR-mediated approaches using wild-type full-length human- forming activity in vivo, suggesting that the PH domain is not Vav2 cDNA (pCMV5-Vav2; a generous gift from Dan Broek) as the template. A 5Ј a critical regulator of Vav DH domain function (18, 28). Since primer containing a BamHI site and sequences encoding eight Vav2 NH2-terminal Ј the role of the PH domain in Vav2 function has not been residues corresponding to residues 192 to 200 was used in conjunction with a 3 primer containing a BamHI restriction site and sequences encoding eight Vav2 addressed, it has not been previously established whether the COOH-terminal residues corresponding to residues 845 to 851 to generate a 2-kb PH domains of Vav family proteins exhibit similar or distinct fragment encoding the sequence from the DH domain to the end of the protein. functions. Three additional 3Ј primers containing eight Vav2 COOH-terminal residues corre- Vav proteins also contain additional protein-protein or sponding to residues 565 to 573, 510 to 518, or 368 to 377 were used to generate a 1.7-kb fragment encoding the DH and PH domains and the CRD (residues 192 to protein-lipid interaction domains that flank the DH/PH do- 573 [Vav2-DPC]), a 1.2-kb fragment encoding the DH and PH domains (Vav2-DP), mains. A calponin homology domain and an acidic amino and a 0.7-kb fragment encoding the DH domain (Vav2-D), respectively. All con- acid-rich domain positioned NH2 terminal to the DH and structs were digested with BamHI and ligated into the BamHI site of pCGN-hygro PH domains serve negative regulatory roles in Vav function in frame with the HA epitope tag at the NH2 terminus. cDNA sequences encoding missense mutations in the PH domain (K407A and W503L) or the CRD (K533A, (7). Tyrosine phosphorylation of the NH2-terminal region K538A, K563A, and V568E) of Vav2-DPC were introduced with the Quickchange relieves this negative regulatory function. Hence, mutant site-directed mutagenesis kit (Stratagene) in accordance with the manufacturer’s versions of all three Vav proteins with the NH2 termini protocol. All constructs were sequenced in their entirety to eliminate the possibility deleted are activated constitutively in a phosphorylation- of extra mutations. Cell culture and transformation assay. independent fashion and exhibit potent transforming activ- NIH 3T3 mouse fibroblasts were cul- tured in Dulbecco’s modified Eagle’s medium supplemented with 10% calf ity (2, 20). COOH terminal to the PH domain are a cysteine- serum. DNA transfections for transformation assays were performed by using the rich domain (CRD) and an Src homology 2 (SH2) domain calcium phosphate precipitation method as described previously (9). For each that is flanked by two SH3 domains. The role of the SH assay, the cognate empty vector was used as a control. The transfected cultures domains is to facilitate the phosphorylation of Vav (7). were maintained in culture media for 14 days, fixed, and stained with crystal violet (0.5%), and the number of foci of transformed cells was then quantitated. Consistent with this role, the SH domains are dispensable NIH 3T3 cells stably expressing the Vav2 mutants were established by transfec- for the transforming activities of the NH2-terminally trun- tion of pCGN-vav2 constructs and isolation of drug-resistant colonies after cated and transforming versions of Vav (35). In contrast, growth in medium supplemented with hygromycin (400 ␮g/␮l).