[31] Activation of Raf-1 by Ras in intact cells

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activated MEKK. The procedures for performing coupled M E K K assays, including synthesis and purification of recombinant wild-type MEK-1 and catalytically inactive MAPK, have been described. 9 Alternatively, a mutant form of kinase-inactive MEK-1 (MEK-1 T286A/T292A/T386A), which lacks the sites phosphorylated by M A P K but contains those sites necessary for MEK-1 activation that are phosphorylated by M E K K or Raf, may be used as a substrate for M E K K assays. 1° Acknowledgments This work was supported by National Institutes of Health Grants DK 37871, GM 30324, CA 58187, and GM 15843 (to C. A. Lange-Carter). NOTE ADDED IN PROOF. Subsequent to the writing of this manuscript, we have fotmd thai the originally cloned MEKK1 eDNA is a partial clone. We have isolated additional MEKK1 eDNA sequences extending 5' to our current open reading frame (Fig, 1) and have identified a MEKK1 immunoreactive species of 180kD in cell lysates and MEKK immunoprecipitates. The 98kD M E K K immunoreactive band (Fig. 2) is most likely derived from a larger M E K K species.

~oA. M. Gardner, R. R. Vaillancourt. C. A. Lange-Carter, and G. L. Johnson. Mol. Biol. Cell. 5, 193 (1994).

[31] Activation of R a f - 1 b y R a s i n I n t a c t C e l l s

By D E B O R A H

K. MORRISON

Introduction The Ras and Raf-1 protooncogene products are key proteins involved in the transmission of many proliferative and developmental signals. Raf-I and Ras serve as intermediates in these signaling pathways by connecting upstream tyrosine kinases with downstream serine/threonine kinases, such as mitogen-activated protein kinase ( M A P K or E R K ) and M A P K kinase (MKK, also known as MEK.) l Ras is a membrane-localized guanine nucleotide-binding protein that is biologically active in the GTPbound state, 2 whereas Raf-1 is a serine/threonine kinase located primarily in the cytosol. 3 Genetic and biochemical studies demonstrate that Raf-1 1T. M. Roberts, Nature (London) 360, 534 (1992). 2 H. R. Bourne, D. A. Sanders, and F. McCormick, Nature (London) 349, 117 (1991). 3 D. K. Morrison, Cancer Cells 2, 377 (1990).

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functions d o w n s t r e a m of R a s in m a n y signaling p a t h w a y s / ' 4 T h e following lines of e v i d e n c e f u r t h e r suggest that R a s m a y p l a y a role in the a c t i v a t i o n of Raf-1. First, the a c t i v a t i o n of Raf-1 in m a n y cases is d e p e n d e n t on the activity of Ras. 5~' S e c o n d , c o e x p r e s s i o n of Raf-1 with v - R a s in the b a c u l o v i rus e x p r e s s i o n system e n h a n c e s the k i n a s e activity of Raf-l.7 Finally, Raf1 can i n t e r a c t d i r e c t l y with G T P - b o u n d forms o f R a s in vitro a n d in vivo. s lO A r g - 8 9 of Raf-1 is a r e s i d u e r e q u i r e d for the R a s - R a f - 1 i n t e r a c t i o n . ~ M u t a t i o n of this R a f - I r e s i d u e disrupts the a s s o c i a t i o n with R a s in vitro a n d in the y e a s t t w o - h y b r i d system a n d p r e v e n t s the R a s - m e d i a t e d e n z y m a t i c a c t i v a t i o n of Raf-1 in the b a c u l o v i r u s e x p r e s s i o n system (Fig. 1). T h e b i n d ing of Raf-1 a n d Ras d o e s not itself s t i m u l a t e the kinase activity of Raf-1, but a p p e a r s to function by t a r g e t i n g R a f - I to the p l a s m a m e m b r a n e . 12~3 T h e e v e n t s that occur at the m e m b r a n e to activate Raf-1 a r e u n k n o w n ; h o w e v e r , t r y o s i n e kinases, s e r i n e / t h r e o n i n e k i n a s e s such as p r o t e i n k i n a s e C, a n d p o s s i b l y lipid ligands m a y c o n t r i b u t e to R a f - I activation. ~4'~5 F o r e x a m p l e , as shown in Fig. 1, the m a x i m a l s t i m u l a t i o n of Raf-1 activity in the b a c u l o v i r u s system r e q u i r e s c o e x p r e s s i o n of b o t h R a s a n d an a c t i v a t e d t y r o s i n e k i n a s e ] H o w e v e r , t y r o s i n e k i n a s e s can induce Raf-1 activity in the a b s e n c e of a f u n c t i o n a l R a s p r o t e i n 7 o r R a s b i n d i n g ] l s u g g e s t i n g that Raf-1 can b e a c t i v a t e d by R a s - i n d e p e n d e n t p a t h w a y s as well. T h e r e f o r e , while the R a s / R a f - 1 a s s o c i a t i o n is i m p o r t a n t for certain aspects of Raf-1 function, the exact m e c h a n i s m ( s ) a n d p r o t e i n s or factors d i r e c t l y i n v o l v e d in the activation of Raf-1 still r e m a i n s to be e l u c i d a t e d . R a t i o n a l e for U s i n g B a c u l o v i r u s E x p r e s s i o n S y s t e m F u r t h e r studies e x a m i n i n g the role of Ras in Raf-I activation r e q u i r e a s y s t e m that is easily m a n i p u l a t e d a n d in which b i o l o g i c a l l y active R a s 4 S. A. Moodic and A. Wolfman. Trends Genet. 10, 44 (1994). 5 B. Dickson, F. Sprenger, D. Morrison, and E. Hafen, Nature (London) 360, 600 (1992). ~'K. W. Wood, C. Sarnecki, T. M. Roberts, and J. Blenis, Cell (CarnbrMge, Mass.) 68, 1t)41 (1992). 7N. G. Williams, T. M. Roberts, and P. Li. Proc. Natl. Acad. Sci. U.S.A. 89, 2922 (1992). s A. B. Vojtek. S. M. Hollenberg, and J. A. Cooper, Cell (Cambridge, Mass.) 74, 205 (1993). ') B. Hallberg, S. I. Rayter, and J. Downward, J. Biot Chem. 269, 3913 (1994). m R. E. Finney, S. M. Robbins, and J. M. Bishop, Curr. BioL 3, 805 (1993). it j. R. Fabian, A. B. Vojtek, J. A. Cooper, and D. K. Morrison, Pro(:. Natl. Acad. Sci. U.S.A. 91, 5982 (1994). ~-~D. Stokoe, S. G. Macdonald, K. Cadwallader, M. Symons. and J. F. Hancock, Science 264, 1463 (1994). i3 S. J. Leevers, H. F. Paterson, and C. J. Marshall. Nature (Londott) 369, 411 (1994). 14j. R. Fabian, 1. Daar, and D. K. Morrison, Mol. Cell. Biol. 13, 7133 (1993). is W. Kolch, G. Heidecker, G. Kochs. R. Hummel, H. Vahidi, H. Mischak, G. Finkenzeller. D. Marme, and U. R. Rapp, Nature (London) 364, 249 (1993).

[31]

ACTIVATION OF Raf-1 BY Ras IN INTACTCEL[.S

A

KD/Raf-1

WT/Raf-1

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+ 200

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+

+

303

Raf-1R89L o II

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+

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+

+

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MKK1 45-

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Fu(;. 1. Analysis of the kinase activity of Raf-1 proteins expressed in Sf9 insect cells. (A) Wild-type Raf-1 (WT/Raf-1), kinase-defcctive Raf-I (KD/Raf-I), and Raf-1 containing an Arg-to-Leu mutation at amino acid residue 89 (Raf-1R'wL) were expressed in Sf9 cells in the absence (Alone) or presence of v-Ras (+Ras), activated Src (+Src), or v-Ras and Src (+ Ras/Src). Raf-I proteins were immunoprecipitated from infected cell lysates and in vitro kinase assays were performed as described in text. Assays were terminated by the addition of gel loading buffer, the samples were resolved on a 7.5% SDS-polyacrylamide gel, and the phosphoproteins were visualized by autoradiography. Molecular size markers (in kilodaltons) are shown on the left. (B) lmmunoprecipitated Raf-I was detected by immunoblotting analysis using antibodies to Raf-I.

and Raf-I proteins can be overexpressed. In this chapter we describe the use of the baculovirus expression system to study the contribution of Ras to Raf-1 activity in intact cells. The baculovirus expression system has been chosen for this analysis for the following reasons. First, this system allows for high levels of recombinant protein expression that facilitate protein purification. Second, this is a eukaryotic expression system that, unlike expression in bacteria, is conducive to the proper folding, disulfide bond formation, phosphorylation, and other posttranslational modifications re-

304

CELL EXPRESSIONAND ANALYSISin Vitro

[31]

quired for obtaining biologically active proteins. These features are particularly important in terms of the Raf-1 protein, because certain serine residues must be constitutively phosphorylated in order for the protein to be a functionl kinase.16 Third, Sf9 (Spodoptera frugiperda, fall armyworm ovary) cells can be simultaneously infected with multiple viruses encoding one or more components of a signaling pathway. In addition, the levels of expression of these proteins can be easily varied. Finally, this system allows the examination of mutant Ras and Raf-1 proteins in the absence of endogenous wild-type proteins. While the baculovirus expression system has proved to be valuable for the analysis of Raf-1 activity, the protocols for cell lysis, immunoprecipitation, and in vitro Raf-1 kinase assays that are presented in this chapter can be applied to studies examining the activation of Raf-I in various other cell systems, including growth factor-treated mammalian cells and Xenopus oocytes.

G e n e r a t i o n of R e c o m b i n a n t B a c u l o v i r u s e s The baculovirus system employing the Autographa californica nuclear polyhedrosis virus (AcNPV) has been widely used for the expression of recombinant, biologically active proteins in Sf9 cells. Protocols and methodology required for setting up this expression system have been thoroughly described in the baculovirus laboratory manual by Summers and Smith. 17 Here, the procedures for generating recombinant baculoviruses are briefly described, with an emphasis placed on those aspects of our techniques that differ from the standard protocols. To obtain recombinant viruses expressing the Ras and Raf-1 proteins, c D N A fragments containing the entire coding sequences for v-Ras and Raf-1 are first isolated and inserted into a baculovirus transfer vector. The transfer vector places the gene of interest under the control of the AcNPV polyhedrin gene promoter and in the context of flanking viral sequences. pVL1393 (Invitrogen, San Diego, CA) and pAcC4 (R. Clark, Chiron, Emeryville, CA) are two transfer vectors that we routinely use for expressing the recombinant gene as a nonfused protein. After a recombinant transfer plasmid (containing the gene of interest) has been constructed, it is cotransfected into Sf9 cells together with a modified version of baculovirus genomic D N A (BaculoGold baculovirus DNA; PharMigen, San Diego, CA). In vivo, the gene of interest is transferred to the viral genome by homologous recombination and recombinant viruses are produced by the transfected 1~D. K. Morrison, G. Heidecker, U. R. Rapp, and T. D. Copeland. J. Biol. Chem. 268, 17309 (1993). 17M. D. Summers and G. E. Smith, Tex. Agric. Exp. Stn. [Bull] B-1555 (t987).

[311

ACTIVATIONOF Raf-1 BY Ras IN INTACTCELLS

305

cells. Using BaculoGold baculovirus DNA, which is defective for the production of wild-type viruses, all of the viruses generated should be recombinant. However, because - 1 0 % of the recombinant viruses may not express the foreign gene, the viruses produced by the transfected Sf9 cells are isolated by plaque purification and monitored for recombinant protein expression by immunoblot analysis. Once recombinant viruses expressing Ras and Raf-1 have been identified, high-titer stocks [>108 plaque-forming units (PFU)/ml] are prepared. Infection of Sf9 Cells To obtain maximal expression of recombinant proteins with the baculovirus system, it is essential that infection experiments be performed using Sf9 cells that are completely viable and in log-phase growth. Routinely, Sf9 cells are grown at 27 ° in Grace's medium (GIBCO, Grand Island, NY) supplemented with Yeastolate (3.3 g/liter), Lactoalbumin hydrolysate (3.3 g/liter), and (10% v/v) fetal calf serum. Cell stocks are grown as suspension cultures and are maintained at a density of 1-4 × 106. For small-scale infections, 2 × 106 Sf9 cells are seeded onto 60-mm tissue culture plates in a total volume of 3 ml of complete medium and allowed to attach to the plate surface for 1 hr. After the cells have adhered, the medium is removed and 1 ml of virus inoculum is added. The virus inoculum consists of high-titered, purified virus stocks ( - 1 × 10 s PFU/ml) expressing the desired recombinant proteins (in this case, v-Ras and Raf-1) diluted in fresh medium. For these assays, in which maximal protein expression and the coexpression of multiple proteins in cells are desired, a high multiplicity of infection is required. Routinely, we infect with 5-10 PFU of each virus per Sf9 cell. However, the amount of virus may need to be adjusted in order to maintain equivalent expression levels of multiple proteins in cells simultaneously infected with more than one virus. Once the virus inoculum is added, the Sf9 cells are incubated at 27 ° for 1 hr. Following the infection period, an additional 3 ml of complete medium is added and the cells are incubated at 28 ° for 40-48 hr. For the expression of recombinant proteins other than v-Ras and R a M , time course experiments should be performed to determine the time of optimal protein production. P r e p a r a t i o n of Cell Lysates Infected Sf9 cells are dislodged from the tissue culture dish by pipetting, transferred to a 15-ml conical tube, and centrifuged at 1000 rpm for 5 min at 4 °. The resulting cell pellets are gently resuspended and washed twice with cold (4 °) phosphate-buffered saline (PBS, 1 ml/2 × 106 cells). The

306

CELLEXPRESSIONAND ANALYSISin Vitro

[31]

washed cell pellets are then resuspended in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer containing 20 mM Tris (pH 8.0), 137 mM NaC1, 10% (v/v) glycerol, 1% (v/v) Nonidet P-40 (NP-40), 0.1% (w/v) sodium dodecyl sulfate (SDS), 0.5% (w/v) sodium deoxycholate, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM aprotinin, 20 /,M leupeptin, and 5 mM sodium vanadate (600/,1 of lysis buffer per 2 × 106 cells) and incubated on ice for 15 min. The celt lysates are transferred to a 1.5-ml microfuge tube and centrifuged at 16,000 g for 10 min at 4° to remove insoluble cellular debris. Aliquots (20-40/,1) of the clarified lysates are removed and assayed for recombinant protein production by immunoblot analysis. The remaining clarified lysates are then either used immediately for immunoprecipitation assays or are quick frozen in a dry iceethanol bath and stored at 80°. Immunoblotting Cell lysates prepared as described above are resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto 0.2-~m pore size nitrocellulose membranes (Schleicher & Schuell, Keene, NH). Residual binding sites on the membrane are blocked by incubating the filter with 2% (w/v) bovine serum albumin (BSA, fraction V; Sigma, St. Louis, MO) in TBS [10 mM Tris (pH 8.0), 150 mM NaC1] for 1 hr at room temperature. The filter is then washed three times (5 min/ wash) with TBST [TBS containing 0.2% (v/v) Tween 20] and incubated with primary antibody diluted in TBST overnight at 4° on a rocking platform. Following incubation with the primary antibody, the filters are washed three times with TBST (5 min/wash) and probed for 1 hr with a horseradish peroxidase-conjugated secondary antibody (Boehringer Mannheim Biochemicals, Indianapolis, IN) diluted 1 : 20,000 in TBST. The filters are again washed three times with TBST (5 min/wash) and immune reactions are detected by enhanced chemiluminescence using ECL reagents from Amersham (Arlington Heights, IL). Immunoprecipitation Assays The anti-Raf-1 antibody that has been most commonly used for immunoprecipitation assays is a peptide antibody generated against the last 12 Cterminal amino acid residues of Raf-1. An affinity-purified rabbit antibody of this type is commercially available through Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Transduction Laboratories (Lexington, KY) has generated an anti-Raf-1 mouse monoclonal antibody using a 24-kDa protein fragment corresponding to amino acid residues 162-378 of human Raf-1

[3 1]

ACTIVATION OF Raf-1 BY Ras ly IWrACTCELLS

307

as an i m m u n o g e n . O u r l a b o r a t o r y n o w exclusively uses this m o n o c l o n a l a n t i b o d y to i m m u n o p r e c i p i t a t e Raf-1. W e find that this a n t i b o d y is preferable to o t h e r Raf-1 a n t i b o d i e s that we have tested for i m m u n o p r e c i p i t a t i n g kinase-active Raf-1 with high affinity. Figure 2 shows a c o m p a r i s o n of the anti-Raf-1 m o n o c l o n a l a n t i b o d y ( T r a n s d u c t i o n L a b o r a t o r i e s ) a n d the antiRaf-1 C - t e r m i n a l p e p t i d e a n t i b o d y (Santa Cruz Biotechnology, Inc.) in i m m u n o p r e c i p i t a t i o n e x p e r i m e n t s m e a s u r i n g Raf-1 activity from u n t r e a t e d a n d p l a t e l e t - d e r i v e d growth factor ( P D G F ) - t r e a t e d N I H 3T3 cells. T o i m m u n o p r e c i p i t a t e Raf-1 from cell lysates, the Raf-1 a n t i b o d y is first p r e b o u n d to p r o t e i n A - S e p h a r o s e b e a d s ( P h a r m a c i a , U p p s a l a , Sweden). In

o~ Raf 24K ot Raf C'

200 -

:

!!! ii i!!!~¸iiii?,!~!iiiiii~i~iiiiiii!!!ill 5!~ii i i!!!~!!!i~i i i i 'i!!~!!i~i i !i i i ~i i i !!!!'~i i !i ~

97- iiiiiiiiii!ii!iiiiii!iii!ii!! 5 iiiiiiiii!i!ili!iiiiii!!!!i!iiii!!iiii: : 68 -

iiiii!iiiii~iiiiiiiii!i!!iiii!i!!iiiiiiiiiiii iiiiiii! -~,.-Raf-1 ~,,~i:!!ii!!iiii.iii!!i!ii:iiiiiii~ii~iiii!i!!iiiiiiiii!!ii!~iii'ill

MKK 45-

Frci. 2. Comparison of anti-Raf-1 antibodies for use in immunoprecipitation and in vitro kinase assays. Untreated ( ) or PDGF-treated (+) NIH 3T3 cells were lysed and Raf-1 proteins were immunoprecipitated using l /xg of either of two commercially available antiRaf-1 antibodies. In vitro kinase assays were performed on the Raf-I immunoprecipitates and the assays were terminated by the addition of gel loading buffer. The samples were resolved on a 7.5% SDS-polyacrylamide gel and the phosphoproteins were visualized by autoradiography. The two antibodies used for this comparison were the anti-Raf-1 mouse monoclonal antibody generated using a 24-kDa protein fragment corresponding to amino acid residues 162-378 of human Raf-I as an immunogen (o~Raf 24K; Transduction Laboratories) and the affinity-purified anti-Raf-1 peptide antibody generated against the last 12 Cterminal amino acid residues of Raf-I (c~ Raf C': Santa Cruz Biotechnology, Inc.). Molecular size markers (in kilodaltons) shown on the left.

308

CELLEXPRESSIONAND ANALYSISin Vitro

[31]

a 1.5-ml microfuge tube, 1 ml of RIPA lysis buffer, 25 /zl of protein ASepharose beads (50:50 slurry in RIPA lysis buffer), and 1-5/zg of antiRaf-1 antibody are mixed and incubated on a rocking platform for 1 hr at 25 ° or overnight at 4 °. When using the anti-Raf-1 monoclonal antibody, it is necessary either to add 1-5 tzg of affinity-purified rabbit anti-mouse IgG or to use protein G-agarose (Santa Cruz Biotechnology, Inc.) instead of protein A-Sepharose. Following the binding reaction, the anti-Raf-l-coated beads are gently pelleted and washed twice with RIPA lysis buffer. Cell lysates prepared as described above are then added to the washed beads and the samples are incubated on a rocking platform at 4 ° for 2-4 hr. The immunoprecipitated complexes are collected by centrifugation in a microfuge at 2000 rpm for 1 min at 4 °. The supernatant is removed and discarded (being careful not to disturb the pelleted beads), and the pelleted beads are washed three times with I ml of cold NP-40 lysis buffer containing 20 mM (pH 8.0), 137 mM NaC1, 10% (v/v) glycerol, 1% (v/v) NP-40, 2 mM EDTA, 1 mM PMSF, 1 mM aprotinin, 20/~M leupeptin, and 5 mM sodium vanadate, repeating the centrifugation step as described above. I n Vitro Kinase Assay

Raf-I proteins are specifically immunoprecipitated and washed as described above. Following the final wash, the immunoprecipitated Raf-1 complexes are resuspended and incubated for 20 min at 25 ° in 40/xl of kinase buffer containing 30 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, pH 7.4), 7 mM MnC12 (made fresh), 5 mM MgC12, 1 mM dithiothreitol (DTT), 15 ~M ATP, 20 ~Ci of [y-32p]ATP (3000 Ci/millimol; Amersham); [y--~2P]ATP should be less than 1 week old and carefully freeze-thawed), and 0.1 ~g of purified 5'-p-fluorosulfonylbenzoyladenosine (FSBA)-treated MKK. The M K K (kindly provided by P. Dent and T. Sturgill, University of Virginia, Charlottesville, VA) used in our assay is purified from Sf9 cells infected with a recombinant baculovirus encoding MKK and is treated with FSBA to inactivate the autokinase activity of MKK. Is To terminate the kinase assays, 15 /xl of 4× gel sample buffer containing 33% iv/v) glycerol, 0.3 M DTT, and 6.7% (w/v) SDS is added directly to the samples. The samples are heated for 5 min at 100 ° and then analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. C o m m e n t s on M e a s u r e m e n t of Raf-1 Activity 1. The conditions for cell lysis are critical for the proper determination of Raf-1 activity. The use of RIPA lysis buffer that contains SDS, NP-40, Is p. Dent, Y. H. Chow,J. Wu, D. K. Morrison, R. Jove, and T. W. Sturgill, Biochem. J. 303, 105 (1994).

[31]

ACTIVATIONOF R a M BY Ras IN INTACTCELLS

309

and sodium deoxycholate is required for maximal solubilization of the Raf-1 protein and to prevent the coimmunoprecipitation of associating kinases. Contaminating kinase activities in the anti-RaM immunoprecipirates have been observed when less stringent cell lysis conditions are used (lysis buffers containing only 1% NP-40 or Triton X-100 as detergents). The presence of these contaminating kinase activities can be misleading and can obscure the determination of authentic Raf-1 activity. 2. The choice of an exogenous substrate for determining RaM activity is another important consideration. In signaling pathways in vivo, activated Raf-1 phosphorylates and activates M A P K kinase (MKK). 1'~ Raf-1 has been demonstrated to associate directly with MEK in the yeast two-hybrid system 2° and to copurify with M K K when coexpressed in inse,ct cells. 2~ Because MKK is a target of Raf-1 in vivo, MKK is the substrate of choice for the assessment of Raf-1 kinase activity in vitro. Furthermore, MKK is the only known well-characterized physiologically relevant substrate of RAF-1. However, because M K K is a kinase capable of autophosphorylation, kinase-inactive MKK (generated by mutation or by treating with FSBA) must be used for these assays. Interestingly, in our studies we have consistently found that activation of R a M as measured by MKK phosphorylation correlates with activation as measured by Raf-1 autophosphorylation or by phosphorylation of a synthetic peptide containing the Raf-1 autophosphorylation site at Thr-268.16 However, while Raf-1 autophosphorylation is an accurate assay for assessing Raf-1 activity, the levels of MKK phosphorylation are usually much greater than those of autophosphorylation. Finally, other substrates that have been previously used to measure Raf-1 activity were often assayed using less stringent cell lysis conditions, and most likely represent phosphorylation by contaminating kinase activities. 3. A third variable to consider is the stability of the activated R a M enzyme. In our studies, we have found that once Raf-1 becomes activated, the enzymatic activity is quite stable. Activated Raf-1 can be freeze-thawed, incubated for several days at 4 °, and heated to 37 ° for 30 min without significant loss of activity. Furthermore, the enzyme is stable in lysis buffers containing SDS and can function in kinase assay buffers containing 1% NP40 or Triton ×100. 4. Finally, when assaying for activators or novel substrates of Raf-1 by using the baculovirus system, it is always advisable to include kinase-inactive 1~)C. M. Crews and R. L. Erickson, Cell (Cambridge, Mass.) 74, 215 (1993). 2o L. Van Aelst, M. Barr, S. Marcus. A. Polverino, and M. Wigler, Proc. Natl. Acad. Sci. U.S.A. 90, 6213 (1993). 21 W. D. Huang, C. M. Crews, A. Alessandinni, and R. L. Erickson, Proc. Natl. Acad. Sci. U.S.A. 90, 10947 (1993).

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[32]

Raf-1 as a control. This control helps to ensure that activity measurements are of Raf-1 and not of contaminating kinases. The kinase-inactive Raf-1 that we use encodes a lysine-to-methionine mutation at amino acid 375 in the ATP-binding site.

Acknowledgment This work was supported by the National Cancer Institute (DHHS) under Contract No. N01-CO-74101 with A B U

[321 R a s - R a f

By

Complexes: Analyses of Complexes F o r m e d in V i v o

ROBERT FINNEY a n d DESIREE HERRERA

Introduction A key pathway for transduction of proliferative, developmental, and oncogenic stimuli from receptors at the cell surface to transcription factors located in the nucleus involves activation of Ras and Raf-1 (Fig. 1). The receptors at the cell surface are typically protein tyrosine kinases themselves [e.g., the epidermal growth factor receptor (EGF receptor) or the T cell receptor (TCR)] or they are associated with the nonreceptor class of protein tyrosine kinases (e.g., proteins encoded by the src gene family). In the signal transduction pathway, activation of Ras and Raf-1 results in the sequential activation of serine/threonine protein kinases that include MEK (or mitogen-activated protein kinase kinase, MKK) and MAPK (mitogenactivated protein kinase, also known as ERK), and ultimately in activation of nuclear transcription factors that include Fos, Jun, and MycJ 5 The pathway has been found to function in many cell types and in a diverse number of organisms including Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, and mammals/~ ~ i W. J. Fantl, D. E. Johnson, and L. T. Williams, Annu. Rev. Biochem. 62, 453 (1993). 2 G. L. Johnson and R. R. Vaillancourt, Curr. Opin. Cell Biol. 6, 230 (1994). 3 L. A. Feig, Science 260, 767 (1993). 4j. R. Woodgett. Curr. Biol. 2, 357 (1992). J. Blenis, Proc. Natl. Acad. Sci. U.S.A. 90, 5889 (1993). B. F. Dickson, F. Sprenger, D. Morrison, and E. Hafcn, Nature (London) 360, 600 (1992). 7 X. Lu, T. B. Chou, N. G. Williams, T. Roberts, and N. Pcrrimon, Genes Dev. 7, 621 (1993). M. Han, A. Golden, Y. Ham and P. W. Sternberg, Nature (London) 349, 426 (1993).

METHODSIN ENZYMOLOGY,VOL.255

Copyright t5 1995by AcademicPress, Inc. All rightsof reproductionin any formreserved.