FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2054838887> ?p ?o ?g. }

• W2054838887 endingPage "18400" @default.
• W2054838887 startingPage "18393" @default.
• W2054838887 abstract "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 vivodespite 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. 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 vivodespite 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. guanosine triphosphatase diffuse B-cell lymphoma the big sister of Dbl Dbl homology enzyme-linked immunosorbent assay guanine nucleotide exchange factor glutathioneS-transferase hemagglutinin 5)P2, inositol 4,5-bisphosphate the first cousin of LBC the second cousin of LBC N-methylanthraniloyl phosphate-buffered saline pleckstrin homology 5)P2, phosphatidylinositol 4,5-bisphosphate small unilamellar vesicles T-cell lymphoma invasion and metastasis 1 tris(hydroxymethyl)aminomethane wild-type green fluorescent protein enhanced GFP bovine serum albumin The Rho family GTPases1are an essential subset of the Ras superfamily of small molecular weight GTPases. Like Ras, Rho family GTPases cycle between GDP- and GTP-bound forms. When GDP-bound, Rho proteins are inactive and do not functionally couple to their downstream effectors. However, when GTP-bound, Rho GTPases elicit profound effects on the organization of the actin cytoskeleton, in addition to tightly regulating the activation state of transcription factors such as the serum response factor, c-Jun, and NF-κB (1Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2079) Google Scholar, 2Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5165) Google Scholar, 3Bishop A.L. Hall A. Biochem. J. 2000; 348: 241-255Crossref PubMed Scopus (1655) Google Scholar). GDP/GTP cycling within Rho proteins is primarily accomplished through the actions of two classes of regulatory proteins. GTPase activating proteins promote the inactive, GDP-bound form of Rho proteins by enhancing their intrinsic GTPase ability to convert bound GTP to GDP. In contrast, the actions of guanine nucleotide exchange factors (GEFs) upon Rho GTPases results in Rho activation by exchanging their bound GDP for GTP. Members of the Dbl family of oncoproteins act as GEFs exclusively for Rho GTPases (RhoGEFs) and, like constitutively activated members of Rho GTPases, can exhibit potent transformation potential within various cell types upon overexpression or constitutive activation (4Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (333) Google Scholar, 5Hoffman G.R. Cerione R.A. FEBS Lett. 2002; 513: 85-91Crossref PubMed Scopus (117) Google Scholar, 6Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (967) Google Scholar). Dbl family members invariably contain a Dbl homology (DH) domain in tandem with an adjacent, carboxyl-terminal pleckstrin homology (PH) domain. However, outside of this region, Dbl-related proteins share little sequence conservation and typically possess a variety of protein-signaling domains, presumably reflecting diversity in regulation and cellular function. The invariant positioning of PH domains immediately carboxyl-terminal to DH domains strongly implies a unique functional coupling. As demonstrated by several complementary studies, DH-associated PH domains are essential components of Dbl family activation of Rho family GTPases. Truncation of all or part of the PH domains of Dbl (7Zheng Y. Zangrilli D. Cerione R.A. Eva A. J. Biol. Chem. 1996; 271: 19017-19020Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), Dbs (8Whitehead I.P. Kirk H. Kay R. Oncogene. 1995; 10: 713-721PubMed Google Scholar), Lfc (9Whitehead I.P. Kirk H. Tognon C. Trigo-Gonzalez G. Kay R. J. Biol. Chem. 1995; 271: 18388-18395Abstract Full Text Full Text PDF Scopus (143) Google Scholar), and Lsc (10Whitehead I.P. Khosravi-Far R. Kirk H. Trigo-Gonzalez G. Der C.J. Kay R. J. Biol. Chem. 1996; 271: 18643-18650Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) results in the complete loss of cellular transformation. Replacing the PH domains of Lfc and Dbs with a membrane-targeting sequence restores at least partial transforming activity, indicating a role in membrane targeting for these PH domains, presumably through interaction with phosphoinositides, i.e.low affinity (micromolar KD), nonspecific ligands for the vast majority of PH domains (11Lemmon M.A. Ferguson K.M. Biochem. J. 2000; 350: 1-18Crossref PubMed Scopus (611) Google Scholar). In addition to possibly mediating the translocation of Dbl family proteins to cellular membranes, phosphoinositides may also potentiate or suppress the exchange activity of some Dbl family proteins in solution (12Han J. Luby-Phelps K. Das B. Shu X. Xia Y. Mosteller R.D. Krishna U.M. Falck J.R. White M.A. Broek D. Science. 1998; 279: 558-560Crossref PubMed Scopus (708) Google Scholar, 13Crompton A.M. Foley L.H. Wood A. Roscoe W. Stokoe D. McCormick F. Symons M. Bollag G. J. Biol. Chem. 2000; 275: 25751-25759Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 14Russo C. Gao Y. Mancini P. Vanni C. Porotto M. Falasca M. Torrisi M.R. Zheng Y. Eva A. J. Biol. Chem. 2001; 276: 19524-19531Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar); however, this does not appear to be a general mechanism of regulation (15Snyder J.T. Rossman K.L. Baumeister M.A. Pruitt W.M. Siderovski D.P. Der C.J. Lemmon M.A. Sondek J. J. Biol. Chem. 2001; 276: 45868-45875Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Moreover, recent evidence indicates that PH domains associated with DH domains can accelerate the catalytic exchange of nucleotides on Rho family GTPases independent of phosphoinositides. For example, a DH/PH fragment of Trio catalyzes nucleotide exchange within Rac ∼200-fold better than the isolated DH domain (16Liu X. Wang H. Eberstadt M. Schnuchel A. Olejniczak E.T. Meadows R.P. Schkeryantz J.M. Janowick D.A. Harlan J.E. Harris E.A. Staunton D.E. Fesik S.W. Cell. 1998; 95: 269-277Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). The PH domain of Dbs makes direct contacts to Cdc42, and these interactions are necessary for efficient guanine nucleotide exchangein vitro (17Rossman K.L. Worthylake D.K. Snyder J.T. Siderovski D.P. Campbell S.L. Sondek J. EMBO J. 2002; 21: 1315-1326Crossref PubMed Scopus (187) Google Scholar). In this study, we define functional roles for the PH domain of Dbs in directly mediating cellular transformation associated with downstream signaling events, especially the in vivo activation of GTPases. We found that mutations within the PH domain of Dbs, previously shown to be necessary for exchange in vitro, completely eliminated the ability of Dbs to transform NIH 3T3 cells. More interestingly, mutations within the PH domain that prevent binding of phosphoinositides, but not in vitronucleotide exchange by Dbs, also prevent Dbs from activating RhoAin vivo and do not allow the transformation of NIH 3T3 cells by a normally highly oncogenic form of Dbs. These effects were not caused by the mislocalization of Dbs, because none of the mutations affected subcellular targeting as assessed by cellular fractionations and indirect immunofluorescence. Therefore, the PH domain of Dbs must be functioning in some capacity other than a simple membrane anchor dependent upon binding phosphoinositides to alter the gross subcellular distribution of Dbs leading to GTPase activation. Indeed, the data are more consistent with phosphoinositide binding being required to direct orientation or conformations of the invariantly associated DH and PH domains with respect to cellular membranes and membrane-resident GTPases for regulated guanine nucleotide exchange. pAX142-dbs-HA6 and pCTV3H-dbs-HA6 contain cDNAs that encode fragments of murine Dbs fused to an HA epitope tag and include the Dbs DH/PH domain along with amino- and carboxyl-terminal flanking regions (residues 525–1097) (18Whitehead I.P. Lambert Q.T. Glaven J.A. Abe K. Rossman K.L. Mahon G.M. Trzaskos J.M. Kay R. Campbell S.L. Der C.J. Mol. Cell. Biol. 1999; 19: 7759-7770Crossref PubMed Google Scholar). pCTV3H-dbs-HA8 encodes an HA epitope-tagged Dbs DH domain (residues 525–833), whereas pCTV3P-dbs-HA7 encodes the Dbs DH domain fused to the plasma membrane-targeting sequence (GCMSCKCVLS) of H-Ras (Dbs DH plus CAAX). GST-C21 contains the Rho·GTP binding domain of Rhotekin (19Reid T. Furuyashiki T. Ishizaki T. Watanabe G. Watanabe N. Fujisawa K. Morii N. Madaule P. Narumiya S. J. Biol. Chem. 1996; 271: 13556-13560Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). pEGFP (Clontech) used for immunofluorescence studies contains GFP under the control of a cytomegalovirus promoter. The Dbs DH/PH domain (murine, residues 623–967), fused to a carboxyl-terminal hexa-histidine tag, was expressed inEscherichia coli from pET-28a (Novagen) (17Rossman K.L. Worthylake D.K. Snyder J.T. Siderovski D.P. Campbell S.L. Sondek J. EMBO J. 2002; 21: 1315-1326Crossref PubMed Scopus (187) Google Scholar). A pET-21a (Novagen) expression construct was used to bacterially express human placental Cdc42 (residues 1–188, C188S) (17Rossman K.L. Worthylake D.K. Snyder J.T. Siderovski D.P. Campbell S.L. Sondek J. EMBO J. 2002; 21: 1315-1326Crossref PubMed Scopus (187) Google Scholar). Human RhoA (residues 1–190, C190S), fused to an amino-terminal hexa-histidine tag, was expressed from pProEX-HT (Invitrogen). The PH domain of PLC-δ1 (residues 11–140), fused to an amino-terminal hexa-histidine tag, was also expressed from the pProEX-HT vector for bacterial expression. Site-directed substitutions placed within all Dbs DH/PH domains and Cdc42 expression constructs were prepared using the QuikChange site-directed mutagenesis kit (Stratagene) as per the manufacturer's instructions and were verified by automated sequencing. Protein expression in stably transfected NIH 3T3 cells was determined by Western blot analysis as described previously (9Whitehead I.P. Kirk H. Tognon C. Trigo-Gonzalez G. Kay R. J. Biol. Chem. 1995; 271: 18388-18395Abstract Full Text Full Text PDF Scopus (143) Google Scholar). Protein was visualized with Enhanced Chemiluminescence reagents (Amersham Biosciences). Protein expression and purification of Dbs DH/PH domains, Cdc42(C188S) and RhoA(C190S) from E. coli, were performed as described previously (17Rossman K.L. Worthylake D.K. Snyder J.T. Siderovski D.P. Campbell S.L. Sondek J. EMBO J. 2002; 21: 1315-1326Crossref PubMed Scopus (187) Google Scholar, 20Snyder J.T. Worthylake D.K. Rossman K.L. Betts L. Pruitt W.M. Siderovski D.P. Der C.J. Sondek J. Nat. Struct. Biol. 2002; 9: 468-475Crossref PubMed Scopus (188) Google Scholar). Fluorescence spectroscopic analysis of N-methylanthraniloyl (mant)-GTP incorporation into bacterially purified Cdc42 and RhoA was carried out using a PerkinElmer Life Sciences LS 50B spectrometer at 25 °C. Exchange reaction assay mixtures, containing 20 mm Tris, pH 7.5, 150 mm NaCl, 5 mm MgCl2, 1 mm dithiothreitol, and 100 μm mant-GTP (BIOMOL), and either 1 or 2 μm (as indicated) of either Cdc42 or RhoA protein, were prepared and allowed to equilibrate with continuous stirring. After equilibration, Dbs DH/PH domain proteins were added at 200 nm, and the rates of nucleotide loading (kobs) of Rho GTPases were determined by monitoring the decrease in Cdc42 or RhoA tryptophan fluorescence (λex = 295 nm, λem = 335 nm) in response to binding mant-GTP. The rates (kobs) of guanine nucleotide exchange were determined by fitting the data as single-exponential decays utilizing the program GraphPad Prism. Data were normalized to wild-type curves to yield the percent GDP released. NIH 3T3 cells were maintained in Dulbecco's modified Eagle medium (high glucose) supplemented with 10% bovine calf serum (NIH 3T3, JRH, Lenexa, KS). Primary focus formation assays were performed in NIH 3T3 cells exactly as described previously (21Clark G.J. Cox A.D. Graham S.M. Der C.J. Methods Enzymol. 1995; 255: 395-412Crossref PubMed Scopus (180) Google Scholar). Briefly, NIH 3T3 cells were transfected by calcium phosphate coprecipitation in conjunction with a glycerol shock. Focus formation was scored at 14 days. NIH 3T3 cells that stably express pCTV3H, pCTV3H-dbs-HA6, or the pCTV3H versions of Dbs mutants were generated by calcium phosphate coprecipitation followed by selection for 14 days in growth medium supplemented with hygromycin B (200 μg/ml). Multiple drug-resistant colonies (>100) were pooled together to establish stable cell lines. All assays for transformation were performed in triplicate. Potential lipid binding pockets on the surface of the Dbs PH domain were predicted using the SiteID option of SYBYL (Tripos Inc.). A coordinate file for the phosphoinositol ring of phosphatidylinositol 4,5-bisphosphate (Ins(4,5)P2) was created using SYBYL. All docking procedures utilized the DOCK suite of programs (version 4.0, I. D Kuntz, University of California at San Francisco) and were carried out in a manner previously reported (22Ewing T.J. Makino S. Skillman A.G. Kuntz I.D. J. Comput. Aided Mol. Des. 2001; 15: 411-428Crossref PubMed Scopus (1050) Google Scholar). Briefly, a molecular surface for each potential binding pocket was generated using the surface calculation algorithm MS (23Connolly M.L. J. Mol. Graph. 1993; 11: 139-141Crossref PubMed Scopus (546) Google Scholar) and used as an input for generating space-filling spheres using Sphgen. A scoring grid encompassing each pocket plus an additional radius of ∼8 Å was calculated using the grid. Parameters used for docking Ins(4,5)P2 into the Dbs PH domain were standard DOCK parameters for the single anchor search method using torsion drive and torsion minimization. Residues 846–852 in the disordered β1/β2 loop region were modeled in and minimized for some docking procedures. To validate our docking procedure, inositol 1,4,5-trisphosphate was successfully docked into the previously determined structure of the PH domain of PLC-δ bound to inositol 1,4,5-trisphosphate (RCSB accession number 1MAI). The ability of His6-tagged Dbs proteins to bind to phosphatidylinositol 4,5-bisphosphate was assessed by an enzyme linked immunosorbent assay (ELISA) utilizing a 96-well microtiter plate containing a various amounts of diC6-modified phosphatidylinositol 4,5-bisphosphate (Echelon Biosciences Inc.). Protein stock solutions were diluted to 10 μm in buffer C (20 mmTris, pH 7.5, 100 mm NaCl, and 5% glycerol) before adding 50 μl of each protein to the wells. 100 μl of an anti-His antibody (Santa Cruz Biotechnology) diluted 1:500 in buffer C was then incubated with each well, followed by 100 μl of an horseradish peroxidase-conjugated sheep anti-mouse IgG (Amersham Biosciences) diluted 1:1000 in buffer C. All incubations were performed for 1 h at 18 °C, and the wells were extensively washed with buffer C after each incubation step. Protein-lipid interactions were detected with the horseradish peroxidase substrate o-phenylenediamine (Sigma), and absorbances were measured at 490 nm. Dbs binding to small unilamellar vesicles (SUVs) containing phosphatidylinositol 4,5-bisphosphate was measured by surface plasmon resonance using a BIAcore 2000 instrument. SUVs (containing by molar fraction 80% dipalmitoyl phosphatidylcholine, 17% dipalmitoyl phosphatidylserine, 3% phosphatidylinositol 4,5-bisphosphate, and 0.1% N-biotinylated dipalmitoyl phosphatidylethanolamine) were prepared by sonicating lipids into 20 mm Hepes (pH 7.5) and 150 mm NaCl. SUVs were then captured on an SA5 chip (BIAcore) via a biotin-streptavidin interaction. Equal amounts of SUVs were immobilized on each respective flow cell, whereas an empty flow cell was maintained to control for nonspecific binding. 25 μl of various Dbs proteins in 20 mm Hepes (pH 7.5) and 150 mm NaCl were injected onto the flow cells at 50 μm and followed by 100 μl of buffer. Experiments were performed at 25 °C with a flow rate of 100 μl/min. Raw sensorgrams from each experiment were aligned, and the signal due to binding the empty flow cell was subtracted from each curve using the software BIAevaluation 3.0 (24Myszka D.G. Curr. Opin. Biotechnol. 1997; 8: 50-57Crossref PubMed Scopus (426) Google Scholar). The Rho binding domain of Rhotekin (GST-C21) (19Reid T. Furuyashiki T. Ishizaki T. Watanabe G. Watanabe N. Fujisawa K. Morii N. Madaule P. Narumiya S. J. Biol. Chem. 1996; 271: 13556-13560Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar) was expressed as a GST fusion in BL21(DE3) cells and immobilized by binding to glutathione-coupled Sepharose 4B beads (Amersham Biosciences). The immobilized GST-C21 was then used to precipitate GTP-bound RhoA from NIH 3T3 cell lysates. Cells were washed in cold phosphate-buffered saline and then lysed in 50 mmTris-HCl, pH 7.4, 2 mm MgCl2, 100 mm NaCl, 10% glycerol, 1% Nonidet P-40, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 μg/ml aprotinin, and 1 μg/ml phenylmethylsulfonyl fluoride. Cell lysates were then cleared by centrifugation at 10,000 × g for 5 min at 4 °C. Lysates used for affinity purification were normalized for endogenous Rho levels. Affinity purifications were carried out at 4 °C for 1 h, washed three times in an excess of lysis buffer, and then analyzed by Western blot. RhoA was detected by a monoclonal antibody (sc-418, Santa Cruz Biotechnology). NIH 3T3 cells were plated on coverslips and then transiently cotransfected with 1 μg of pEGFP (Clontech) and 3 μg of either pAX142 or pAX142-dbs-HA6 harboring the PH domains mutations by calcium phosphate precipitation. Cells were fixed with 3.7% formaldehyde (in PBS) for 10 min, permeabilized, and blocked in 0.1% Triton X-100, 3% BSA in PBS for 30 min. Coverslips were then incubated in a humidity chamber with an anti-HA mouse monoclonal antibody (BAbCO) for 1 h in 0.1% Triton X-100 with 0.1% BSA, washed in PBS, and then incubated with red-fluorescent Alexa Fluor 488-conjugated goat anti-mouse IgG (0.1% Triton X-100, 0.1% BSA, Molecular Probes) for 30 min in the dark. Coverslips were washed in PBS and mounted on glass slides using FluorSave reagent (Calbiochem). Cells were viewed with an Olympus IX50 inverted confocal microscope, and images were captured using the optronics digital charge-coupled device camera system. Mass populations of NIH 3T3 cells stably expressing Dbs-HA6 or Dbs-HA6 harboring a PH domain mutation were washed with ice-cold PBS and resuspended in cold HYPO buffer (10 mm Tris, pH 7.4, 1 mmMgCl2, 5 μg/ml leupeptin, 5 μg/ml aprotinin, 5 μg/ml pepstatin, 1 mm phenylmethylsulfonyl fluoride). Lysates were then homogenized, harvested with HYPO buffer supplemented with 0.15 m NaCl, and then centrifuged at 38,000 rpm for 30 min at 4 °C. The supernatant (cytosolic fraction) was removed, and the pellet (particulate fraction) was resuspended in HYPO buffer supplemented with 0.15 m NaCl. The protein concentrations of the total, cytosolic, and membrane fractions were determined with a bicinchoninic acid protein assay kit (Pierce). 30 μg of protein for each fraction was resolved by SDS-PAGE, transferred to Immobilon-P membranes (Millipore), and probed with anti-HA epitope antibody (BAbCO). The recently reported structure of a fragment of Dbs in complex with Cdc42 highlights a crucial role for the PH domain in promoting an extensive set of interactions between Dbs and Cdc42 necessary for efficient guanine nucleotide exchange in vitro(Fig. 1A) (17Rossman K.L. Worthylake D.K. Snyder J.T. Siderovski D.P. Campbell S.L. Sondek J. EMBO J. 2002; 21: 1315-1326Crossref PubMed Scopus (187) Google Scholar). In particular, Tyr-889 within β4 of the PH domain is critical for exchange because its substitution to phenylalanine dramatically decreases Dbs-catalyzed exchange of both Cdc42 and RhoA without affecting the overall arrangement of domains within the Dbs·Cdc42 complex (17Rossman K.L. Worthylake D.K. Snyder J.T. Siderovski D.P. Campbell S.L. Sondek J. EMBO J. 2002; 21: 1315-1326Crossref PubMed Scopus (187) Google Scholar). To clarify the structural role of the PH domain in contributing productively to the interface between Dbs and GTPases, a series of substitutions within the interface were assessed for functional importance. In particular, Arg-66 of Cdc42 interacts with multiple residues within the PH domain of Dbs, including Tyr-889. Yet substitution of Arg-66 to alanine does not affect nucleotide exchange (Fig. 1B, TableI, and see Ref. 17Rossman K.L. Worthylake D.K. Snyder J.T. Siderovski D.P. Campbell S.L. Sondek J. EMBO J. 2002; 21: 1315-1326Crossref PubMed Scopus (187) Google Scholar), suggesting that Tyr-889 likely stabilizes the electronic configuration of His-814 to promote interaction with Asp-65 of Cdc42. In support of the importance of Asp-65 to nucleotide exchange, amino acid substitutions placed at Asp-65 (D65A, D65E, and D65N) severely limit the ability of Dbs to activate Cdc42 (Fig. 1B and Table I). Of particular note, the isosteric substitution D65N in Cdc42 should not disrupt interactions with Asn-810 of the DH domain but will disrupt hydrogen bonding with His-814 of Dbs. Consistent with an “uncoupling” of Asp-65 from Tyr-889, Cdc42 (D65N) suffers a reduction of ∼7-fold in the activation by Dbs, on par with the effect of Dbs(Y889F) on wild-type Cdc42 (Table I).Table IRate constants of Dbs catalyzed guanine nucleotide exchange of mutant Cdc42 proteins2 μmCdc42Intrinsic ratePlus 0.2 μm DbsStimulationkobs s−1 × 10−3-foldWild type0.24 ± 0.0114.89 ± 0.0261D65A0.40 ± 0.001.05 ± 0.083D65E0.19 ± 0.002.34 ± 0.0012D65N0.28 ± 0.032.60 ± 0.149R66A0.34 ± 0.0219.56 ± 0.2657The rates (kobs) for GEF reactions were determined by fitting the data from Fig. 1B as single exponential decays. The -fold stimulation for each Dbs protein reflects the ratio of the kobs measured for the GEF-containing reaction to the unstimulated reaction containing no GEF. Open table in a new tab The rates (kobs) for GEF reactions were determined by fitting the data from Fig. 1B as single exponential decays. The -fold stimulation for each Dbs protein reflects the ratio of the kobs measured for the GEF-containing reaction to the unstimulated reaction containing no GEF. The in vitro exchange assays utilizing mutants of Dbs and Cdc42 strongly suggest that the PH domain of Dbs, acting in concert with the DH domain, is necessary to fully activate Rho family GTPases within cells. To further assess the physiological function of the PH domain of Dbs in activating Rho GTPases, we introduced a subset of the previously studied mutations into a transforming version of Dbs (Dbs-HA6) (8Whitehead I.P. Kirk H. Kay R. Oncogene. 1995; 10: 713-721PubMed Google Scholar) and measured the capacities of the mutated proteins to transform NIH 3T3 cells (Fig. 1C). Dramatically, whereas Dbs-HA6 possesses potent transformation activity, either H814A or Y889F completely eliminates the focus-forming activity of Dbs-HA6 in NIH 3T3 cells. Similarly, K885A, which is also within the PH domain, significantly reduces transformation by Dbs. Because transformation of Dbs depends upon activation of RhoA in NIH 3T3 cells (25Cheng L. Rossman K.L. Mahon G.M. Worthylake D.K. Korus M. Sondek J. Whitehead I.P. Mol. Cell. Biol. 2002; 22: 6895-6905Crossref PubMed Scopus (26) Google Scholar), these data suggest that the PH domain of Dbs is required for direct activation of Rho family proteins, irrespective of membrane targeting. These effects are not due to loss of affinity for phosphoinositides by Dbs, because Dbs(H814A), Dbs(Y889F), and Dbs(K885A) bind to phosphatidylinositol 4,5-bisphosphate similar to wild-type Dbs (Fig.2, B and C). Typically, the majority of pleckstrin homology domains bind phosphoinositide headgroups with low affinity and specificity. For instance, the PH domain of Dbs promiscuously binds several multiphosphorylated phosphoinositides with low affinity, including phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 4,5-bisphosphate (15Snyder J.T. Rossman K.L. Baumeister M.A. Pruitt W.M. Siderovski D.P. Der C.J. Lemmon M.A. Sondek J. J. Biol. Chem. 2001; 276: 45868-45875Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Primary sequence analysis and structural homology comparisons suggest Dbs binds phosphoinositides similarly to other PH domains largely through residues in and surrounding the β1/β2 and β3/β4 loops of the PH domain. Like most PH domains, the β1/β2 and β3/β4 loop region of Dbs exhibits a large envelope of positive electrostatic potential, compatible with binding the electronegative headgroups of phosphoinositides (17Rossman K.L. Worthylake D.K. Snyder J.T. Siderovski D.P. Campbell S.L. Sondek J. EMBO J. 2002; 21: 1315-1326Crossref PubMed Scopus (187) Google Scholar). To further define the lipid-binding pocket within the Dbs PH domain, we used a computational approach to dock various inositol phosphates onto the surface of the protein (Fig.2A). These analyses suggest that residues within the PH domain of Dbs that are predicted to contact phosphoinositides within this site, and/or contribute to the positive electrostatic potential include: Lys-849, Lys-851, Arg-855, and Lys-857 (within the β1/β2 loop); Arg-861 (at the base of β2); Lys-874 (within β3); and Lys-892 (within β4). To determine if regions comprising β1/β2 and β3/β4 of the PH domain of Dbs are responsible for mediating binding to phosphoinositides, we substituted the identified lysine and arginine residues with glutamate and assessed the effect of each mutation upon the ability of Dbs to bind phosphatidylinositol 4,5-bisphosphate. Initially, an enzyme-liked immunosorbent assay (ELISA) was used to assess binding to phosphatidylinositol 4,5-bisphosphate in the absence of secondary lipids (Fig. 2B). These results were supported by a more detailed analysis using surface plasmon resonance and small unilamellar vesicles containing a low molar fraction of phosphatidylinositol 4,5-bisphosphate within a background of negatively charged lipids (Fig. 2C). As we observed previously (15Snyder J.T. Rossman K.L. Baumeister M.A. Pruitt W.M. Siderovski D.P. Der C.J. Lemmon M.A. Sondek J. J. Biol. Chem. 2001; 276: 45868-45875Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), wild-type Dbs DH/PH domain binds significantly to phosphatidylinositol 4,5-bisphosphate, whereas the isolated DH domain of Dbs shows no binding. In comparison, Dbs proteins harboring either single or double glutamate substitutions within the putative phosphoinositide binding site of the PH domain do not significantly bind phosphatidylinositol 4,5-bisphosphate in either assay. In contrast, other mutations within the extended β3/β4 loop (K885A) or β4 loop (Y889F) of the PH domain, at sites distinct from the phosphoinositide-binding pocket, do not significantly affect binding of phosphoinositides. Therefore, as observed within the structures of other PH domains bound to inositol phosphates, the PH domain of Dbs binds lipids through portions of β1/β2 and β3/β4, and mutations within this pocket selectively abolish lipid binding. To assess if phosphoinositide binding to the PH domain is critical for Dbs transformation of NIH 3T3 cells, we placed each mutation into the Dbs-HA6 construct and compared transformation potentials in primary focus formation assays (Fig.3A). Strikingly, except for the low level of foci formation exhibited by Dbs(K874E), transformation was completely eliminated by all other mutations within the pocket of the PH domain responsible" @default.
• W2054838887 created "2016-06-24" @default.
• W2054838887 creator A5002265218 @default.
• W2054838887 creator A5025255901 @default.
• W2054838887 creator A5038883988 @default.
• W2054838887 creator A5039017412 @default.
• W2054838887 creator A5075451917 @default.
• W2054838887 creator A5083402067 @default.
• W2054838887 creator A5084453658 @default.
• W2054838887 date "2003-05-01" @default.
• W2054838887 modified "2023-10-11" @default.
• W2054838887 title "Multifunctional Roles for the PH Domain of Dbs in Regulating Rho GTPase Activation" @default.
• W2054838887 cites W1547066932 @default.
• W2054838887 cites W1583486328 @default.
• W2054838887 cites W1592950931 @default.
• W2054838887 cites W1887383255 @default.
• W2054838887 cites W1963941913 @default.
• W2054838887 cites W1988655374 @default.
• W2054838887 cites W1997784032 @default.
• W2054838887 cites W1999538252 @default.
• W2054838887 cites W2001234795 @default.
• W2054838887 cites W2016441216 @default.
• W2054838887 cites W2023323949 @default.
• W2054838887 cites W2024619839 @default.
• W2054838887 cites W2030616863 @default.
• W2054838887 cites W2031939553 @default.
• W2054838887 cites W2036887265 @default.
• W2054838887 cites W2039873071 @default.
• W2054838887 cites W2056180063 @default.
• W2054838887 cites W2058778918 @default.
• W2054838887 cites W2064284760 @default.
• W2054838887 cites W2066622533 @default.
• W2054838887 cites W2073686385 @default.
• W2054838887 cites W2073999242 @default.
• W2054838887 cites W2075497451 @default.
• W2054838887 cites W2077792958 @default.
• W2054838887 cites W2083847521 @default.
• W2054838887 cites W2085082922 @default.
• W2054838887 cites W2096791952 @default.
• W2054838887 cites W2101874950 @default.
• W2054838887 cites W2105746559 @default.
• W2054838887 cites W2118833233 @default.
• W2054838887 cites W2125905957 @default.
• W2054838887 cites W2134699117 @default.
• W2054838887 cites W2141206157 @default.
• W2054838887 cites W2151588652 @default.
• W2054838887 cites W2153637044 @default.
• W2054838887 cites W2154655439 @default.
• W2054838887 cites W2170225912 @default.
• W2054838887 cites W4239945442 @default.
• W2054838887 doi "https://doi.org/10.1074/jbc.m300127200" @default.
• W2054838887 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12637522" @default.
• W2054838887 hasPublicationYear "2003" @default.
• W2054838887 type Work @default.
• W2054838887 sameAs 2054838887 @default.
• W2054838887 citedByCount "84" @default.
• W2054838887 countsByYear W20548388872012 @default.
• W2054838887 countsByYear W20548388872013 @default.
• W2054838887 countsByYear W20548388872014 @default.
• W2054838887 countsByYear W20548388872015 @default.
• W2054838887 countsByYear W20548388872016 @default.
• W2054838887 countsByYear W20548388872017 @default.
• W2054838887 countsByYear W20548388872018 @default.
• W2054838887 countsByYear W20548388872019 @default.
• W2054838887 countsByYear W20548388872020 @default.
• W2054838887 countsByYear W20548388872021 @default.
• W2054838887 crossrefType "journal-article" @default.
• W2054838887 hasAuthorship W2054838887A5002265218 @default.
• W2054838887 hasAuthorship W2054838887A5025255901 @default.
• W2054838887 hasAuthorship W2054838887A5038883988 @default.
• W2054838887 hasAuthorship W2054838887A5039017412 @default.
• W2054838887 hasAuthorship W2054838887A5075451917 @default.
• W2054838887 hasAuthorship W2054838887A5083402067 @default.
• W2054838887 hasAuthorship W2054838887A5084453658 @default.
• W2054838887 hasBestOaLocation W20548388872 @default.
• W2054838887 hasConcept C12554922 @default.
• W2054838887 hasConcept C134306372 @default.
• W2054838887 hasConcept C185592680 @default.
• W2054838887 hasConcept C207332259 @default.
• W2054838887 hasConcept C2780298669 @default.
• W2054838887 hasConcept C33923547 @default.
• W2054838887 hasConcept C36503486 @default.
• W2054838887 hasConcept C62478195 @default.
• W2054838887 hasConcept C86803240 @default.
• W2054838887 hasConcept C95444343 @default.
• W2054838887 hasConceptScore W2054838887C12554922 @default.
• W2054838887 hasConceptScore W2054838887C134306372 @default.
• W2054838887 hasConceptScore W2054838887C185592680 @default.
• W2054838887 hasConceptScore W2054838887C207332259 @default.
• W2054838887 hasConceptScore W2054838887C2780298669 @default.
• W2054838887 hasConceptScore W2054838887C33923547 @default.
• W2054838887 hasConceptScore W2054838887C36503486 @default.
• W2054838887 hasConceptScore W2054838887C62478195 @default.
• W2054838887 hasConceptScore W2054838887C86803240 @default.
• W2054838887 hasConceptScore W2054838887C95444343 @default.
• W2054838887 hasIssue "20" @default.
• W2054838887 hasLocation W20548388871 @default.
• W2054838887 hasLocation W20548388872 @default.

• next