- Email: [email protected]
[9] Guanine nucleotide exchange catalyzed by dbl oncogene product
[9]
EXPRESSIONOF Dbl AND DH DOMAIN
77
[9] G u a n i n e N u c l e o t i d e E x c h a n g e C a t a l y z e d by dbl Oncogene Product B y Y I ZHENG, MATTHEW J. H A R T , a n d RICHARD A . CERIONE
Introduction Guanine nucleotide exchange factors (GEFs) activate Ras family members of GTP-binding proteins by accelerating their rate of GDP release and therefore facilitating their GTP binding in cells) Unlike the case for Ras-GEFs, for which the in vivo activities can be examined by the extent of conversion of Ras-bound GDP to GTP by immunoprecipitation of Ras and thin-layer chromatography separation of the bound nucleotides (Gale et al. 2 and this series, Volume 255 [12]), the GEF activities for Rho-type GTPases are difficult to determine in this manner mostly because of the fast hydrolysis rates of members of this family (e.g., Rac and Cdc42Hs 3) and the presence of abundant R h o - G D I (GDP dissociation inhibitor) which renders most of the Rho-type proteins cytosolic and blocks the GEF action. 4 Thus, the in vitro reconstituted assay has been indispensable for detecting and quantitating the activities of potential Rho family GEF molecules. Oncogenic activation of dbl occurs as a result of an amino-terminal truncation of the proto-dbl product, resulting in a malignant transforming phenotype in NIH 3T3 cells (Ron et aL 5 and [38] in this volume). A region between residues 498 and 738 of proto-Dbl (designated the Dbl-homology domain or D H domain), retained by oncogenic Dbl, shares significant sequence similarity with the Saccharomyces cerevisiae cell division cycle protein Cdc24, 6 which acts together with the Rho-type GTP-binding protein Cdc42 to regulate bud site assembly in yeast: This clue has led to the discovery that Dbl acts as a potent GEF for Cdc42Hs. 8 The ability to purify the recombinant Dbl protein and to monitor the functional interactions i M. S. Boguski and F. McCormick, Nature 366, 643 (1993). 2 N. W. Gale, S. Kaplan, E. J. Lowenstein, J. Schlessinger, and D. Bar-Sagi, Nature 363, 88 (1993). 3 M. J. Hart, K. Shinjo, A. Hall, T. Evans, and R. A. Cerione, J. Biol. Chem. 266, 20840 (1991). 4 D. Leonard, M. J. Hart, J. V. Platko, A. Eva, W. Henzel, T. Evans, and R. A. Cerione, J. Biol. Chem. 267, 22860 (1992). 5 D. Ron, S. R. Tronick, S. A. Aaronson, and A. Eva, E M B O J. 7, 2465 (1988). 6 D. Ron, M. Zannini, M. Lewis, R. B. Wickner, L. T. Hunt, G. Graziani, S. R. Tronick, S. A. Aaronson, and A. Eva, N e w Biol. 3, 372 (1991). 7 D. G. Drubin, Cell 65, 1093 (1992). 8 M. J. Hart, A. Eva, T. Evans, S. A. Aaronson, and R. A. Cerione, Nature 354, 311 (1991).
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
78
GUANINE NUCLEOTIDE EXCHANGE
[9]
between Dbl and the Rho-type GTP-binding proteins by the in vitro GEF assay provides convincing biochemical evidence that the DH domain of the dbl oncogene product is sufficient in stimulating the GDP/GTP exchange for Rho-type proteins. 9 This chapter describes methods for the expression of Dbl and the DH domain in baculovirus expression systems and the in vitro assays to demonstrate that Dbl and its DH domain contain GEF activity for the Cdc42Hs and Rho GTP-binding proteins. Expression and Purification of dbl Oncogene Product in Insect S p o d o p t e r a f r u g i p e r d a Cells The entire coding sequence of the db! oncogene is removed from the mammalian expression vector p C l l ( p Z i p - n e o ) t° by BamHI digestion, and the released insert is ligated to the BamHI sites of the baculovirus transfer vector pAC373. The resulting construct pAC373dbl contains all but eight nucleotides of the Y-untranslated sequences of the baculovirus polyhedrin gene fused to the complete coding sequences of the db! oncogene, including the start ATG codon, pAC373dbl is cotransfected with the wild-type virus DNA into S. frugiperda (Sfg) insect cells to generate recombinant virus. The resulting virus is further purified according to the detailed procedure described by Piwnica-Worms. u In order to facilitate later purification steps, a version of Dbl joined at the N terminus to glutathione S-transferase (GST) is also expressed in Sf9 cells by fusion of cDNAs coding both GST and Dbl. The B a m H I insert, described earlier, that encodes oncogenic db! or the DH domain itself is first introduced into the BamHI site of the pGEX-2T vector to generate p G E X d b ! and pGEXDH. The cDNA encoding GST is digested between the X b a I - B a m H I sites and is then ligated together with the B a m H I - P s t I partial digestion fragments from p G E X d b ! or pGEXDH (which includes Dbl or the DH domain coding sequences) into the X b a I - P s t l sites of baculovirus transfer vector pVL1393. Recombinant viruses encoding GSTDbl and the G S T - D H domain are generated using the Baculogold kit from Pharmingen. Sf9 cells are grown to subconfluency and are infected with the respective recombinant baculovirus for 50-60 hr. Cells are then pelleted and resuspended in 5 ml of ice-cold buffer containing 20 mM TrIs-HC1, pH 8.0, 100 9 M. J. Hart, A. Eva, D. Zangrilli, S. A. Aaronson, T. Evans, R. A. Cerione, and Y. Zheng,
I. Biol. Chem. 269, 62 (1994). 10A. Eva, G. Vecchio,C. D. Rao, S. R. Tronick, and S. A. Aaronson,Proc. Natl. Acad. Sci. U.S.A. 85, 2061 (1988). 11H. Piwnica-Worms,in "Current Protocols in MolecularBiology,"(F. M. Ausubel et at, eds.), Wiley(Interscience),New York, Vol. 2, 16.8.1, 1994.
[9]
EXPRESSIONOV Dbl AND DH DOMAIN
79
mM NaC1, 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.2% Triton X-100, plus 0.5 mM phenylmethylsulfonyl fluoride, 10 tzg/ml leupeptin, and 10/zg/ml aprotinin. The resuspended insect cells are homogenized with a Dounce homogenizer for 10 min and then centrifuged for 15 min at 4 ° in a microfuge. The supernatants for the control as well as Dbl-expressing cell lysates can be used directly in the guanine nucleotide exchange experiments. Alternatively, supernatants from the cells expressing GST-Dbl and the G S T - D H domain are further incubated with glutathione-agarose for 2 hr at 4°. The glutathione-agarose beads are pelleted and washed four times in the cell suspension buffer. The GST fusion proteins are then eluted from the affinity beads in a buffer with 5 mM glutathione, 20 mM Tris-HC1, pH 8.0, 100 mM NaC1, 1 mM EDTA, and 0.5 mM DTT. The purified GSTDbl or G S T - D H domain can be dialyzed overnight at 4° to remove the free glutathione or used directly in a guanine nucleotide exchange assay.
G TP-Binding Assay To minimize the complications resulting from GTP hydrolysis during the time course of GTP binding, a nonhydrolyzable analog such as GTPyS is commonly used in place of GTP. Cdc42Hs or other GTP-binding proteins to be tested are first loaded with GDP in loading buffer supplemented with MgCI2. The guanine nucleotide exchange reaction is initiated by mixing the GDP-loaded G protein with Dbl or Dbl-containing lysates in the reaction buffer with [35S]GTPTS and is terminated at various time points by dilution of the reaction mixture in ice-cold termination buffer. The amount of [35S]GTPyS bound to the G protein is finally quantified by filtration of the terminated reactions through nitrocellulose filters. Loading buffer: 20 mM Tris-HCl, pH 8.0, 100 mM NaCI, 2 mM EDTA, 0.2 mM DTT, 100/xM AMP-PNP, and 10/xM GDP. Reaction buffer: 20 mM Tris-HC1, pH 8.0, 100 mM NaC1, 10 mM MgCI2, 100/xM AMP-PNP, 0.5 mg/ml bovine serum albumin, and 5/zM [35S]GTPTS (~11,000 cpm/pmol). Termination buffer: 20 mM Tris-HC1, pH 8.0, 100 mM NaC1, and 10 mM MgCI2. Figure 1 shows a time course of GTP binding to Cdc42Hs catalyzed by insect cell lysates in the presence or absence of Dbl. Approximately 2/zg of purified Cdc42Hs protein is first incubated in 60/zl loading buffer for 5 min at room temperature. MgC12 is then added to a final concentration of 5 mM and the loading incubation is continued for an additional 15 min. Aliquots (20/xl) of the GDP-loaded Cdc42Hs are mixed with 5/xl control cell lysates or lysates containing Dbl in the reaction buffer (total volume 100 tzl) to initiate the exchange reaction at room temperature. Aliquots
80
GUANINE NUCLEOTIDE EXCHANGE
[9]
4 o E e,
3
O. IO
2&
"O ,"
1
O ill
0 o
5
10
15
20
Time (min) FIG. 1. Effects of the Sf9 cell lysates containing Dbl on [35S]GTPyS binding to Cdc42Hs. Time course for the binding of [35S]GTPyS to Cdc42Hs. The Cdc42Hs was preloaded with GDP and then added to reaction incubations containing [35S]GTPyS together with aliquots from Sf9 control lysates ( 0 ) or from lysates expressing Dbl (A). From Hart e t al. 8 with permission.
(15/xl) of samples are taken at various time points from the reaction mixture and added to 10 ml ice-cold termination buffer. The terminated reactions are filtered immediately through the BA85 nitrocellulose filters, followed by one wash with 5 ml ice-cold termination buffer. The filters are dissolved completely in scintillation fluid, and the radioactivity detained by the filter is measured by scintillation counting. A significant stimulation by Dbl lysates of the binding of [35S]GTPyS to Cdc42HS is observed within 5 min of the reaction, indicating that Dbl accelerates the rate of the GTPyS/GDP exchange on Cdc42Hs.
GDP Dissociation Assay The GDP dissociation assay is a direct measurement of the guanine nucleotide exchange activity of Dbl since the rate-limiting step in the nucleotide exchange reaction is GDP dissociation. The protocol for assaying Dblcatalyzed GDP dissociation is similar to that used to assay GTP binding except that - 1 0 / z M radioactive [3H]GDP (10 Ci/mmol) or [a-32p]GDP is used in the loading buffer replacing GDP and 1 mM GTP is used in the reaction buffer instead of [35S]GTPTS. It is important for GTP to be present during the exchange reaction, under conditions where [Cdc42Hs] > > [Dbl].
[9]
EXPRESSIONOV Dbl AND DH DOMAIN
81
The presence of GTP results in a Dbl-catalyzed exchange of the medium GTP for the [3H]GDP bound to Cdc42Hs and enables Dbl to act catalytically in stimulating GDP dissociation from multiple Cdc42Hs proteins (i.e., because each newly formed Cdc42Hs-GTP species dissociates from Dbl and thereby frees Dbl to bind to other Cdc42Hs-[3H]GDP complexes). Data are typically plotted as the percentage of radiolabeled GDP that remains bound to Cdc42Hs as a function of time. Figure 2 shows that the insect cell lysates expressing oncogenic Dbl markedly catalyze the dissociation of [3H]GDP from Cdc42Hs purified from human platelet membranes. The half-time for GDP dissociation in the presence of Dbl occurs within 5 min, and GDP dissociation is essentially complete in 20 min. In contrast, the control lysates cause <20% dissociation of radiolabeled GDP from Cdc42Hs even after 15 min. To show that purified Dbl and the D H domain are sufficient for GEF activity, both oncogenic Dbl and the D H domain are expressed in Sf9 insect ceils as GST fusion proteins and are then highly purified by glutathione-agarose chromatography (Fig. 3A). Figure 3B shows that the GST-Dbl and G S T - D H domain are each fully capable of stimulating the dissociation of GDP from the Cdc42Hs. Purified GST-Dbl acts catalytically to stimulate the dissociation of [3H]GDP, causing - 6 5 % of the total Cdc42Hs to lose labeled GDP a. (3 .i.
100
or; i,.,..i
"o r= o .,Q
80 60
q,,,,
o 40
o
E m
20
o ~
m
0
I
0
5
I
10
I
15
20
Time (rain) FIG. 2. Effects of the Sf9 cell lysates containing Dbl on [3H]GDP dissociation from Cdc42Hs. Time course for the dissociation of [3H]GDP from Cdc42Hs. Aliquots of Sf9 control cell lysates (O) or lysates expressing Dbl (/k) were added to the reaction incubations together with [3H]GDP-preloaded Cdc42Hs. From Hart et aL 8 with permission.
82
[9]
GUANINE NUCLEOTIDE EXCHANGE
A 498
735
925 1
2
3
•-,I- GST-Dbl Oncogenic Dbl 498
757
GST-DbI-H -.,,I- GST-CDC42
DbI-H iiiiTiiiii!~7~ i
B
i
1.001 o Q.
(:3 (D 0.75
o m
3
A 0.50
0 . 2 .=
I 4
I 8
I 12
I 16
I 20
T i m e (rain)
FIG. 3. Effects of purified GST-Dbl and G S T - D H on [3H]GDP dissociation from Cdc42Hs. (A) Left: Schematic diagram of proto-Dbl and a minimal unit containing the Dbl homology domain (Dbl-H). Right: S D S - P A G E (10% polyacrylamide) of the purified Cdc42Hs, G S T Dbl, and G S T - D H domain (GST-DbI-H) used in the experiments. (B) Measurements of the dissociation of [3H]GDP from the E. coli-expressed GST-Cdc42Hs protein in the presence of purified oncogenic Dbl or the DH domain. The time course for [3H]GDP dissociation from Cdc42Hs ( - 2 tzg) was measured in the presence of 2/zg of GST alone (O), 0.2/zg of Dbl ( • ) , or 1.5/zg of the D H domain (A). From Hart et aL 9 with permission.
[91
EXPRESSION OF Dbl AND D H DOMAIN
83
lOO
0
%o FIG. 4. Effects of the purified GST-Dbl on [3H]GDP dissociations from Ras and various Rho family GTPases. Two micrograms of each GTP-binding protein tested was preloaded with [3H]GDP and then mixed in reaction buffer with 1/xg of GST alone (solid bars) or with 0.1/xg GST-Dbl (cross-hatched bars) for 15 min before termination of reactions.
within the time course of the experiment, under conditions where the ratio of [Cdc42Hs] : [Dbl] is 20 : 1. Using the G D P dissociation assay, the specificity of D b l - G E F activity toward other related GTP-binding proteins was tested as shown in Fig. 4. In addition to Cdc42Hs, Dbl exhibits potent G E F activities toward R h o A , R h o B , RhoC, to a lesser extent R h o G , but shows little or no G E F activity on the Ras, R a c l , and TC10 proteins.
Discussion The " D b l family" of potential G E F s for Rho-type GTP-binding proteins have b e e n expanding rapidly since the first discovery of D b I - G E F activity. All m e m b e r s of this family share the c o m m o n structural features of an - 2 3 0 amino acid stretch that is designated the D H domain and a pleckstrin homology (PH) domain, which is located C-terminally adjacent to the D H domain and is thought to mediate p r o t e i n - p r o t e i n interactions in analogy to SH2 and SH3 domains. 12 M e m b e r s of the Dbl family appar12A. Musacchio, T. Gibson, P. Rice, J. Thompson, and M. Saraste, Trends Biochem. Sci. 18, 343 (1993).
84
GUANINE NUCLEOTIDEEXCHANGE
[9]
ently play roles in cell growth-related pathways and include Bcr, 13 R a s G R F , 14 mSos, 15 Cdc24,16 and the dbl, 9 ect2, a7 vav, 18 and lbc 19 oncogene products. Dbl, as well as the Ect2, Vav, and Lbc oncoproteins, are activated by protein truncation. We found that proto-Dbl, which exhibits - 6 0 - f o l d less transforming activity than Dbl in N I H 3T3 cells, exhibits as potent exchange activity for Cdc42Hs as the oncogenic form of Dbl (unpublished observation), implying that a third protein factor may be involved in the regulation of the activities of p r o t o - D b l in vivo. The efficient expression of Dbl-like proteins is critical for the detection of their G E F activity. A t t e m p t s to express Dbl and its D H domain in E. coli have resulted in nonfunctional proteins, most likely because of insolubility and i m p r o p e r folding. We also have had similar experience with the expression of Cdc24 in E. coli. The baculovirus expression system has been proven to be an efficient way to generate milligram quantities of purified Dbl and Cdc24 proteins. 9,16 Alternatively, stable expression in m a m m a l i a n cells has also been used to provide functional Dbl, although at a much lower level of expression. 9 On the contrary, the E. coli-expressed recombinant Cdc42Hs and Rho can interact with Dbl indistinguishably from the insect cell-expressed Cdc42Hs and Rho, 9 suggesting that posttranslational geranylgeranylations of the carboxyl-terminal cysteine of these G T P a s e s are not essential for functional interactions with Dbl. The D b l - C d c 4 2 H s interaction can also be observed directly by the complex formation assay (Miki et al.17 and [11] in this volume). This binding interaction is highly dependent on the nucleotide-bound state of Cdc42Hs, i.e., Dbl binds tightest to the guanine nucleotide-free form of Cdc42Hs, to a lesser extent to the G D P - b o u n d form of Cdc42Hs, and to a much less extent to the G T P - b o u n d form of Cdc42Hs. This property is consistent with the catalytic mechanism of a conventional G E F such that Dbl binds to the G D P - b o u n d f o r m of Cdc42Hs and stimulates G D P release and consequently facilitates G T P binding to the nucleotide-free Cdc42Hs.
13N. Heisterkamp, J. R. Stephenson, J. Groffen, P. F. Hansen, A. de Klein, C. R. Bartram, and G. Grasveld, Nature 306, 239 (1983). 14C. Shou, C. L. Farnsworth, B. G. Neel, and L. A. Feig, Nature 358, 351 (1992). 15S. E. Egan, B. W. Giddings, M. W. Brooks, L. Buday, A. M. Sizeland, and R. A. Weinberg, Nature 363, 45 (1993). 16y. Zheng, R. A. Cerione, and A. Bender, J. BioL Chem. 269, 2369 (1994). 17T. Miki, C. L. Smith, J. E. Long, A. Eva, and T. Fleming, Nature 362, 462 (1993). 18E. Gulbins, K. M. Coggeshall, G. Baier, S. Katzav, P. Burn, and A. Altman, Science 260, 822 (1993). 19D. Toksoz and D. A. Williams, Oncogene 9, 621 (1994).
EXPRESSIONOF Dbl AND DH DOMAIN
77
[9] G u a n i n e N u c l e o t i d e E x c h a n g e C a t a l y z e d by dbl Oncogene Product B y Y I ZHENG, MATTHEW J. H A R T , a n d RICHARD A . CERIONE
Introduction Guanine nucleotide exchange factors (GEFs) activate Ras family members of GTP-binding proteins by accelerating their rate of GDP release and therefore facilitating their GTP binding in cells) Unlike the case for Ras-GEFs, for which the in vivo activities can be examined by the extent of conversion of Ras-bound GDP to GTP by immunoprecipitation of Ras and thin-layer chromatography separation of the bound nucleotides (Gale et al. 2 and this series, Volume 255 [12]), the GEF activities for Rho-type GTPases are difficult to determine in this manner mostly because of the fast hydrolysis rates of members of this family (e.g., Rac and Cdc42Hs 3) and the presence of abundant R h o - G D I (GDP dissociation inhibitor) which renders most of the Rho-type proteins cytosolic and blocks the GEF action. 4 Thus, the in vitro reconstituted assay has been indispensable for detecting and quantitating the activities of potential Rho family GEF molecules. Oncogenic activation of dbl occurs as a result of an amino-terminal truncation of the proto-dbl product, resulting in a malignant transforming phenotype in NIH 3T3 cells (Ron et aL 5 and [38] in this volume). A region between residues 498 and 738 of proto-Dbl (designated the Dbl-homology domain or D H domain), retained by oncogenic Dbl, shares significant sequence similarity with the Saccharomyces cerevisiae cell division cycle protein Cdc24, 6 which acts together with the Rho-type GTP-binding protein Cdc42 to regulate bud site assembly in yeast: This clue has led to the discovery that Dbl acts as a potent GEF for Cdc42Hs. 8 The ability to purify the recombinant Dbl protein and to monitor the functional interactions i M. S. Boguski and F. McCormick, Nature 366, 643 (1993). 2 N. W. Gale, S. Kaplan, E. J. Lowenstein, J. Schlessinger, and D. Bar-Sagi, Nature 363, 88 (1993). 3 M. J. Hart, K. Shinjo, A. Hall, T. Evans, and R. A. Cerione, J. Biol. Chem. 266, 20840 (1991). 4 D. Leonard, M. J. Hart, J. V. Platko, A. Eva, W. Henzel, T. Evans, and R. A. Cerione, J. Biol. Chem. 267, 22860 (1992). 5 D. Ron, S. R. Tronick, S. A. Aaronson, and A. Eva, E M B O J. 7, 2465 (1988). 6 D. Ron, M. Zannini, M. Lewis, R. B. Wickner, L. T. Hunt, G. Graziani, S. R. Tronick, S. A. Aaronson, and A. Eva, N e w Biol. 3, 372 (1991). 7 D. G. Drubin, Cell 65, 1093 (1992). 8 M. J. Hart, A. Eva, T. Evans, S. A. Aaronson, and R. A. Cerione, Nature 354, 311 (1991).
METHODS IN ENZYMOLOGY, VOL. 256
Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
78
GUANINE NUCLEOTIDE EXCHANGE
[9]
between Dbl and the Rho-type GTP-binding proteins by the in vitro GEF assay provides convincing biochemical evidence that the DH domain of the dbl oncogene product is sufficient in stimulating the GDP/GTP exchange for Rho-type proteins. 9 This chapter describes methods for the expression of Dbl and the DH domain in baculovirus expression systems and the in vitro assays to demonstrate that Dbl and its DH domain contain GEF activity for the Cdc42Hs and Rho GTP-binding proteins. Expression and Purification of dbl Oncogene Product in Insect S p o d o p t e r a f r u g i p e r d a Cells The entire coding sequence of the db! oncogene is removed from the mammalian expression vector p C l l ( p Z i p - n e o ) t° by BamHI digestion, and the released insert is ligated to the BamHI sites of the baculovirus transfer vector pAC373. The resulting construct pAC373dbl contains all but eight nucleotides of the Y-untranslated sequences of the baculovirus polyhedrin gene fused to the complete coding sequences of the db! oncogene, including the start ATG codon, pAC373dbl is cotransfected with the wild-type virus DNA into S. frugiperda (Sfg) insect cells to generate recombinant virus. The resulting virus is further purified according to the detailed procedure described by Piwnica-Worms. u In order to facilitate later purification steps, a version of Dbl joined at the N terminus to glutathione S-transferase (GST) is also expressed in Sf9 cells by fusion of cDNAs coding both GST and Dbl. The B a m H I insert, described earlier, that encodes oncogenic db! or the DH domain itself is first introduced into the BamHI site of the pGEX-2T vector to generate p G E X d b ! and pGEXDH. The cDNA encoding GST is digested between the X b a I - B a m H I sites and is then ligated together with the B a m H I - P s t I partial digestion fragments from p G E X d b ! or pGEXDH (which includes Dbl or the DH domain coding sequences) into the X b a I - P s t l sites of baculovirus transfer vector pVL1393. Recombinant viruses encoding GSTDbl and the G S T - D H domain are generated using the Baculogold kit from Pharmingen. Sf9 cells are grown to subconfluency and are infected with the respective recombinant baculovirus for 50-60 hr. Cells are then pelleted and resuspended in 5 ml of ice-cold buffer containing 20 mM TrIs-HC1, pH 8.0, 100 9 M. J. Hart, A. Eva, D. Zangrilli, S. A. Aaronson, T. Evans, R. A. Cerione, and Y. Zheng,
I. Biol. Chem. 269, 62 (1994). 10A. Eva, G. Vecchio,C. D. Rao, S. R. Tronick, and S. A. Aaronson,Proc. Natl. Acad. Sci. U.S.A. 85, 2061 (1988). 11H. Piwnica-Worms,in "Current Protocols in MolecularBiology,"(F. M. Ausubel et at, eds.), Wiley(Interscience),New York, Vol. 2, 16.8.1, 1994.
[9]
EXPRESSIONOV Dbl AND DH DOMAIN
79
mM NaC1, 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.2% Triton X-100, plus 0.5 mM phenylmethylsulfonyl fluoride, 10 tzg/ml leupeptin, and 10/zg/ml aprotinin. The resuspended insect cells are homogenized with a Dounce homogenizer for 10 min and then centrifuged for 15 min at 4 ° in a microfuge. The supernatants for the control as well as Dbl-expressing cell lysates can be used directly in the guanine nucleotide exchange experiments. Alternatively, supernatants from the cells expressing GST-Dbl and the G S T - D H domain are further incubated with glutathione-agarose for 2 hr at 4°. The glutathione-agarose beads are pelleted and washed four times in the cell suspension buffer. The GST fusion proteins are then eluted from the affinity beads in a buffer with 5 mM glutathione, 20 mM Tris-HC1, pH 8.0, 100 mM NaC1, 1 mM EDTA, and 0.5 mM DTT. The purified GSTDbl or G S T - D H domain can be dialyzed overnight at 4° to remove the free glutathione or used directly in a guanine nucleotide exchange assay.
G TP-Binding Assay To minimize the complications resulting from GTP hydrolysis during the time course of GTP binding, a nonhydrolyzable analog such as GTPyS is commonly used in place of GTP. Cdc42Hs or other GTP-binding proteins to be tested are first loaded with GDP in loading buffer supplemented with MgCI2. The guanine nucleotide exchange reaction is initiated by mixing the GDP-loaded G protein with Dbl or Dbl-containing lysates in the reaction buffer with [35S]GTPTS and is terminated at various time points by dilution of the reaction mixture in ice-cold termination buffer. The amount of [35S]GTPyS bound to the G protein is finally quantified by filtration of the terminated reactions through nitrocellulose filters. Loading buffer: 20 mM Tris-HCl, pH 8.0, 100 mM NaCI, 2 mM EDTA, 0.2 mM DTT, 100/xM AMP-PNP, and 10/xM GDP. Reaction buffer: 20 mM Tris-HC1, pH 8.0, 100 mM NaC1, 10 mM MgCI2, 100/xM AMP-PNP, 0.5 mg/ml bovine serum albumin, and 5/zM [35S]GTPTS (~11,000 cpm/pmol). Termination buffer: 20 mM Tris-HC1, pH 8.0, 100 mM NaC1, and 10 mM MgCI2. Figure 1 shows a time course of GTP binding to Cdc42Hs catalyzed by insect cell lysates in the presence or absence of Dbl. Approximately 2/zg of purified Cdc42Hs protein is first incubated in 60/zl loading buffer for 5 min at room temperature. MgC12 is then added to a final concentration of 5 mM and the loading incubation is continued for an additional 15 min. Aliquots (20/xl) of the GDP-loaded Cdc42Hs are mixed with 5/xl control cell lysates or lysates containing Dbl in the reaction buffer (total volume 100 tzl) to initiate the exchange reaction at room temperature. Aliquots
80
GUANINE NUCLEOTIDE EXCHANGE
[9]
4 o E e,
3
O. IO
2&
"O ,"
1
O ill
0 o
5
10
15
20
Time (min) FIG. 1. Effects of the Sf9 cell lysates containing Dbl on [35S]GTPyS binding to Cdc42Hs. Time course for the binding of [35S]GTPyS to Cdc42Hs. The Cdc42Hs was preloaded with GDP and then added to reaction incubations containing [35S]GTPyS together with aliquots from Sf9 control lysates ( 0 ) or from lysates expressing Dbl (A). From Hart e t al. 8 with permission.
(15/xl) of samples are taken at various time points from the reaction mixture and added to 10 ml ice-cold termination buffer. The terminated reactions are filtered immediately through the BA85 nitrocellulose filters, followed by one wash with 5 ml ice-cold termination buffer. The filters are dissolved completely in scintillation fluid, and the radioactivity detained by the filter is measured by scintillation counting. A significant stimulation by Dbl lysates of the binding of [35S]GTPyS to Cdc42HS is observed within 5 min of the reaction, indicating that Dbl accelerates the rate of the GTPyS/GDP exchange on Cdc42Hs.
GDP Dissociation Assay The GDP dissociation assay is a direct measurement of the guanine nucleotide exchange activity of Dbl since the rate-limiting step in the nucleotide exchange reaction is GDP dissociation. The protocol for assaying Dblcatalyzed GDP dissociation is similar to that used to assay GTP binding except that - 1 0 / z M radioactive [3H]GDP (10 Ci/mmol) or [a-32p]GDP is used in the loading buffer replacing GDP and 1 mM GTP is used in the reaction buffer instead of [35S]GTPTS. It is important for GTP to be present during the exchange reaction, under conditions where [Cdc42Hs] > > [Dbl].
[9]
EXPRESSIONOV Dbl AND DH DOMAIN
81
The presence of GTP results in a Dbl-catalyzed exchange of the medium GTP for the [3H]GDP bound to Cdc42Hs and enables Dbl to act catalytically in stimulating GDP dissociation from multiple Cdc42Hs proteins (i.e., because each newly formed Cdc42Hs-GTP species dissociates from Dbl and thereby frees Dbl to bind to other Cdc42Hs-[3H]GDP complexes). Data are typically plotted as the percentage of radiolabeled GDP that remains bound to Cdc42Hs as a function of time. Figure 2 shows that the insect cell lysates expressing oncogenic Dbl markedly catalyze the dissociation of [3H]GDP from Cdc42Hs purified from human platelet membranes. The half-time for GDP dissociation in the presence of Dbl occurs within 5 min, and GDP dissociation is essentially complete in 20 min. In contrast, the control lysates cause <20% dissociation of radiolabeled GDP from Cdc42Hs even after 15 min. To show that purified Dbl and the D H domain are sufficient for GEF activity, both oncogenic Dbl and the D H domain are expressed in Sf9 insect ceils as GST fusion proteins and are then highly purified by glutathione-agarose chromatography (Fig. 3A). Figure 3B shows that the GST-Dbl and G S T - D H domain are each fully capable of stimulating the dissociation of GDP from the Cdc42Hs. Purified GST-Dbl acts catalytically to stimulate the dissociation of [3H]GDP, causing - 6 5 % of the total Cdc42Hs to lose labeled GDP a. (3 .i.
100
or; i,.,..i
"o r= o .,Q
80 60
q,,,,
o 40
o
E m
20
o ~
m
0
I
0
5
I
10
I
15
20
Time (rain) FIG. 2. Effects of the Sf9 cell lysates containing Dbl on [3H]GDP dissociation from Cdc42Hs. Time course for the dissociation of [3H]GDP from Cdc42Hs. Aliquots of Sf9 control cell lysates (O) or lysates expressing Dbl (/k) were added to the reaction incubations together with [3H]GDP-preloaded Cdc42Hs. From Hart et aL 8 with permission.
82
[9]
GUANINE NUCLEOTIDE EXCHANGE
A 498
735
925 1
2
3
•-,I- GST-Dbl Oncogenic Dbl 498
757
GST-DbI-H -.,,I- GST-CDC42
DbI-H iiiiTiiiii!~7~ i
B
i
1.001 o Q.
(:3 (D 0.75
o m
3
A 0.50
0 . 2 .=
I 4
I 8
I 12
I 16
I 20
T i m e (rain)
FIG. 3. Effects of purified GST-Dbl and G S T - D H on [3H]GDP dissociation from Cdc42Hs. (A) Left: Schematic diagram of proto-Dbl and a minimal unit containing the Dbl homology domain (Dbl-H). Right: S D S - P A G E (10% polyacrylamide) of the purified Cdc42Hs, G S T Dbl, and G S T - D H domain (GST-DbI-H) used in the experiments. (B) Measurements of the dissociation of [3H]GDP from the E. coli-expressed GST-Cdc42Hs protein in the presence of purified oncogenic Dbl or the DH domain. The time course for [3H]GDP dissociation from Cdc42Hs ( - 2 tzg) was measured in the presence of 2/zg of GST alone (O), 0.2/zg of Dbl ( • ) , or 1.5/zg of the D H domain (A). From Hart et aL 9 with permission.
[91
EXPRESSION OF Dbl AND D H DOMAIN
83
lOO
0
%o FIG. 4. Effects of the purified GST-Dbl on [3H]GDP dissociations from Ras and various Rho family GTPases. Two micrograms of each GTP-binding protein tested was preloaded with [3H]GDP and then mixed in reaction buffer with 1/xg of GST alone (solid bars) or with 0.1/xg GST-Dbl (cross-hatched bars) for 15 min before termination of reactions.
within the time course of the experiment, under conditions where the ratio of [Cdc42Hs] : [Dbl] is 20 : 1. Using the G D P dissociation assay, the specificity of D b l - G E F activity toward other related GTP-binding proteins was tested as shown in Fig. 4. In addition to Cdc42Hs, Dbl exhibits potent G E F activities toward R h o A , R h o B , RhoC, to a lesser extent R h o G , but shows little or no G E F activity on the Ras, R a c l , and TC10 proteins.
Discussion The " D b l family" of potential G E F s for Rho-type GTP-binding proteins have b e e n expanding rapidly since the first discovery of D b I - G E F activity. All m e m b e r s of this family share the c o m m o n structural features of an - 2 3 0 amino acid stretch that is designated the D H domain and a pleckstrin homology (PH) domain, which is located C-terminally adjacent to the D H domain and is thought to mediate p r o t e i n - p r o t e i n interactions in analogy to SH2 and SH3 domains. 12 M e m b e r s of the Dbl family appar12A. Musacchio, T. Gibson, P. Rice, J. Thompson, and M. Saraste, Trends Biochem. Sci. 18, 343 (1993).
84
GUANINE NUCLEOTIDEEXCHANGE
[9]
ently play roles in cell growth-related pathways and include Bcr, 13 R a s G R F , 14 mSos, 15 Cdc24,16 and the dbl, 9 ect2, a7 vav, 18 and lbc 19 oncogene products. Dbl, as well as the Ect2, Vav, and Lbc oncoproteins, are activated by protein truncation. We found that proto-Dbl, which exhibits - 6 0 - f o l d less transforming activity than Dbl in N I H 3T3 cells, exhibits as potent exchange activity for Cdc42Hs as the oncogenic form of Dbl (unpublished observation), implying that a third protein factor may be involved in the regulation of the activities of p r o t o - D b l in vivo. The efficient expression of Dbl-like proteins is critical for the detection of their G E F activity. A t t e m p t s to express Dbl and its D H domain in E. coli have resulted in nonfunctional proteins, most likely because of insolubility and i m p r o p e r folding. We also have had similar experience with the expression of Cdc24 in E. coli. The baculovirus expression system has been proven to be an efficient way to generate milligram quantities of purified Dbl and Cdc24 proteins. 9,16 Alternatively, stable expression in m a m m a l i a n cells has also been used to provide functional Dbl, although at a much lower level of expression. 9 On the contrary, the E. coli-expressed recombinant Cdc42Hs and Rho can interact with Dbl indistinguishably from the insect cell-expressed Cdc42Hs and Rho, 9 suggesting that posttranslational geranylgeranylations of the carboxyl-terminal cysteine of these G T P a s e s are not essential for functional interactions with Dbl. The D b l - C d c 4 2 H s interaction can also be observed directly by the complex formation assay (Miki et al.17 and [11] in this volume). This binding interaction is highly dependent on the nucleotide-bound state of Cdc42Hs, i.e., Dbl binds tightest to the guanine nucleotide-free form of Cdc42Hs, to a lesser extent to the G D P - b o u n d form of Cdc42Hs, and to a much less extent to the G T P - b o u n d form of Cdc42Hs. This property is consistent with the catalytic mechanism of a conventional G E F such that Dbl binds to the G D P - b o u n d f o r m of Cdc42Hs and stimulates G D P release and consequently facilitates G T P binding to the nucleotide-free Cdc42Hs.
13N. Heisterkamp, J. R. Stephenson, J. Groffen, P. F. Hansen, A. de Klein, C. R. Bartram, and G. Grasveld, Nature 306, 239 (1983). 14C. Shou, C. L. Farnsworth, B. G. Neel, and L. A. Feig, Nature 358, 351 (1992). 15S. E. Egan, B. W. Giddings, M. W. Brooks, L. Buday, A. M. Sizeland, and R. A. Weinberg, Nature 363, 45 (1993). 16y. Zheng, R. A. Cerione, and A. Bender, J. BioL Chem. 269, 2369 (1994). 17T. Miki, C. L. Smith, J. E. Long, A. Eva, and T. Fleming, Nature 362, 462 (1993). 18E. Gulbins, K. M. Coggeshall, G. Baier, S. Katzav, P. Burn, and A. Altman, Science 260, 822 (1993). 19D. Toksoz and D. A. Williams, Oncogene 9, 621 (1994).