Determining the effect of inducible protein phosphorylation on the DNA-binding activity of transcription factors

203,302-309

ANALYTICALBlclCHEMlSTRY

(1992)

Determining the Effect of Inducible Phosphorylation on the DNA-Binding Activity of Transcription Factors Athanasios

G. Papavassiliou,

Kerstin

Bohmann,

European Molecular Biology Laboratory, Differentiation Meyerhofstrasse 1, D-6900 Heidelberg, Germany

Protein

and Dirk

Bohmann’

Programme, Postfach 10.2209,

Received January IO, 1992

Inducible phosphorylation or dephosphorylation of transcription factors is an important mechanism of signal-dependent gene regulation in eukaryotic cells. This paper describes a combination of techniques that can he used to study the effect of this covalent protein modification on the DNA-binding activity of transcription factors. The protein of interest is genetically tagged with olinohistidine to allow ranid ourification on nickel-chelate columns after being-trz&ently overexpressed in eukaryotic tissue culture cells. Phosphorylation-dependent DNA-binding activity is determined in a blotting assay including an in situ dephosphorylation step. Studies on the protooncogene-encoded transcription factor c-Jun employing this assay revealed a TPA-inducible protein dephosphorylation event that strongly increases the DNA-binding potential of the protein. Our data confirm the results published recently by others and provide a rapid, efficient, sensitive, and well-controlled experimental system to analyze the phosphorylation-regulated modulations in the DNA-binding activity of transcription factors. a lssz *oademieprpsa,TIC.

While the importance of phosphorylation/dephosphorylation switches in regulating the transcriptional potential of a growing number of proteins on the level of DNA binding is becoming more and more apparent (11, the functional analysis of transcription factor phosphorylation is hampered by several technical difficulties. Phosphate groups on proteins are often extremely sensitive to phosphatase attack, especially in the course of the “conventional” purification of DNA-binding ’ To whom correspondence should be addressed. Fax: (0049) 6221 387 306. 302

transcription factors by either immunoprecipitation or DNA-affinity chromatography. A further impediment arises from the low abundance of many of these factors. To circumvent these technical problems, we devised s straightforward, rapid expression, isolation, and analysis method. Here we present studies on the post-translational modulation of AP-1 activity on the level of cJun phosphorylation using this new assay system. AP-1’ (activator protein-l) is the collective name for a group of sequence-specific DNA-binding transcription factors which recognize a common promoter element. The AP-1 family comprises both Fos- and Junrelated proteins whose functional form is a dimer that is composed of either two Jun monomers or one Jun and one Fos-related monomer (Z-6). AP-l-binding sites are frequently found in promoters or enhancers of genes that are inducible by s wide range of extracellular signals, most notably by phorbol esters (e.g., phorbol 1% myristate 13.acetate) (7,8), which act as stimulators of protein kinase C (a key mediator of intracellular signaling) and as tumor-promoting agents (TPA). Several lines of evidence indicate that the transcriptional activation in response to TPA is indeed exerted by AP-1 and that AP-l-binding sites function as TPA-responsive elements (TREs). The TRE-mediated inducibility of AP-1 target genes is, in part, independent of de noun protein synthesis (9,10), implying that preexisting AP-1 factor(s) can be activated by post-translational mechanisms in response to intracellular signals. Conceivably, phosphorylation events sre involved in the regulation of aAbbreviations used: AP-1, activator protein-l; TPA, tumor-promoting agents: TRE, TPA-responsive element; TK-, thymidine kinase negative; DTT, dithiothreitol; PMSF, pbenylmethyl sulfonyl fluoride; PAP, potato acid phosphatsse; SDS, sodium dodecyl sulfate: BSA, bovine ~emm albumin; NTA, nitrilotriacetic acid; PAGE, polyaerylamide gel electrophoresis.

PHOSPHORYLATION-SENSITIVE

AP-1 and in particular c-dun activity. Indeed, Boyle et al. (11) present good evidence for a mechanism that modulates c-Jun activity by an inducible change of its phosphorylation state. After TPA treatment of HeLa TK- cells, a cluster of two serine and one threonine (S239, S243, and T249) residues located N-terminally adjacent to the basic DNA-binding domain becomes hypophosphorylated and the DNA-binding activity of the protein increases simultaneously. Using our assay system we were able to reproduce in a simpler manner this signal-dependent enhancement of c&n DNA-binding potential via dephosphorylation and demonstrate the validity of this analytical method in studying the correlation of the phosphorylation state of transcription factors with their DNA-binding properties. MATERIALS

Plasmid

AND

pHJmet5 was the vector used to express (from a promoter recognized by T’7 RNA polymerase) the complete human c-dun protein in Escherichio coli strain BL21 starting at methionine number 5, which is the probable initiation codon utilized in uivo (Fig. 1, ecJun1. A detailed description of this construct can he found elsewhere(6). The vector used for the expression of cmvJun, pKH6, was derived from pCMV-Jun (12). The first nine codons of the Jun open-reading frame in pCMVJun were replaced with an oligonucleotide encoding the N-terminal peptide MDPHHHHHHDPP (Fig. 1, cmvdun) Mutagenesis

of c-Jun

The A243 mutant cmvJun used in the experiment presented in Fig. 4 was created by substituting the codon for Ser-243 with a codon for Ala employing an oligonucleotide-directed in vitro mutagenesis system (Amersham RPN 1523). Ser-243 was mutated to Ala using the 25.bp oligonucleotide

5’-ACA CCG CCC CTG &

CCC ATC GAC A-3’.

Recombinant mutant clones were sequenced using the dideoxynucleotide chain termination method to confirm the presence of the S243 to A243 mutation. Cell Culture

from

the

plates,

303

BLOTTING

and

fresh

medium containing 50 -IV’ - ethanesulfonic acid (Hepes).KOH (pH 7.2) was added. Cells were transfected by the calcium phosphate coprecipitation method as described (13). The amount of C&l-purified wild-type or mutant pKH6 DNA transfected was 3 gg, adjusted to 20 Fg with pUC18 as carrier DNA. Approximately 48 h post-transfection, the cells were washed three times with ice-cold phosphate-buffered saline and scraped into lysis buffer (see below). For TPA treatment of the cultures, TPA [Sigma P-8139; stored at -20°C as a 100 pglml stock solution in dimethyl sulfoxide (Merck) prior to dilution] was added to the medium 48 h after transfection to a final concentration of 100 rig/ml, and cells were incubated for an additional 2 h prior to harvesting. mM N - 2 - hydroxyethylpiperazine

METHODS

Constructions

Site-Directed

“SOUTH-WESTERN”

and Transient

Tramfections

The HeLa thymidine kinase negative (TK-) cell line was propagated at 37°C in a humidified 10% CO, atmosphere in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum For transfections, 8 X 10’ cells were seeded on regular tissue culture plates (100 mm diameter) 24 h prior to transfection. Three hours prior to transfection the medium was removed

Purification

of Recombinant

c-Jun Proteins

(a) ecJun: the purification scheme of bacterially expressed c-dun has been described elsewhere(6). (b) Wild-type and A243 mutant cmvJun: mock-treated or TPA-treated transiently transfected HeLa TK- cells were lysed by the addition of 1 ml per lo-cm plate of 6 M guanidinium-HCl, 0.1 M Na,HPO,/NaH,PO, (pH 8.0). This lysate was centrifuged at 12,000g for 20 min at room temperature, and the supernatant sonicated with a Branson microtipped sonic&or at setting 1.5 for 3 X 10 s to break down chromosomal DNA and to reduce viscosity. The lysate of five plates was applied to a 0.2.ml nickel-chelate column (Ni’+-NTA-agarose, Qiagen); the column was washed three times with 10 ml of 6 M guanidinium-HCl, 0.1 M Na,HPO,/NaH,PO, (pH 5.91, and bound protein was eluted with 0.6 ml (2 X 0.3 ml) of 6 M guanidinium-HCI, 0.1 M Na,HP0,/NaH2P0, (pH 4.0). The &ate was then concentrated to 0.4 ml by centrifug&ion through a Centricon 10 microconcentrator unit (Amicon) and subsequently dialyzed at 4°C against 2000 vol of a buffer containing 10 rn~ Hepes * KOH (pH 7.81, 20% glycerol, 0.1% lauryldimethylamine oxide (Calbiochem), 20 mM KCl, 2 rn~ MgCl,, 0.2 rn~ EDTA, 1 rn~ DTT, 2 rn~ phenylmethylsulfonyl fluoride (PMSF, Sigma P-76261, 5 pglml aprotinin (Sigma A-6279),2 pg/ml pepstatin (Sigma P-42651, and 2 aglml leupeptin (Sigma L-2884); ecJun was also brought into this buffer. The yield of both wild-type and A243 mutant cmvJun per plate of HeLa TK- cells was in the order of 1 pg per dish.

Preparation of DNA Probes for “Southand Dot-Blotting/Binding Assays

Western”

Blots

The AP-l-binding site probe was generated starting with the following two complementary, synthetic oligonucleotides:

304

PAPAVASSILIOU,

BOHMANN,

5’.GATCGTGACTCAGCGCG-3’ 3’.CACTGAGTCGCGCCTAG-5’. Five micrograms of each oligonucleotide was phosphorylated with 200 &i of [y-32P]ATP (sp act 5000 Gil mmol; 10 &i/*1, Amersham) and 20 units of T4 polynucleotide kinase (New England Biolabs) in 50.~1 reactions under standard conditions (14). The two oligonucleotides were then mixed (still in the polynucleotide kinase reaction buffer) and annealed by incubating for successive 20-min intervals at 68, 37, 24, and 4°C. The reaction was subsequently supplemented with 10 units of T4 DNA ligase (New England Biolabs) and left overnight at 15°C. Concatenated DNA, with an average length of -190 bp, was purified by phenol/chloroform/ isoamyl alcohol (25/24/l, v/v) and chloroform/isoamyl alcohol (24/l, v/v) extractions and concentrated by two rounds of ethanol precipitation in the presence of 2 M ammonium acetate. After two sequential washes of the DNA pellet with 70 and 90% ethanol, respectively, the probe was dissolved in 10 rn~ Tris. HCl (pH 7.2) to a final concentration of -0.5 wg/pl. The obtained specific activity for a typical labeling reaction was in the order of -10’ cpmlrg of probe.

Acid Phosphatase Blotting

Treatment

and South-Western

Potato acid pbosphatase (PAP; Grade I, BoebringerMannheim) was centrifuged out of (NH,),SO, suspension (10,OOOg for 1 min), and a total of 60 units was dissolved in 0.6 ml of buffer E [40 rn~ piperazine-l\i,iV’bis(2-ethanesulfonic acid). KOH (pH 6.0)] and dialyzed against two changes of 500 vol of buffer E. Enzyme (0.2 units) was employed in a total reaction volume of 25 pl containing -0.1 pg of protein and buffer E, and incubations were performed at room temperature for 1 h in the presence or absence of a mixture of competitive and noncompetitive phosphatase/kinase inhibitors [ 100 rn~ NaF (Merck), 15 rn~ Na?MoO, (Sigma S-6646). 20 rn~ (Na,)p-nitrophenyl phosphate (Sigma 104-O), and 10 rn~ (Na,)B-glycerophosphate (Sigma G-625111. After the incubation, these inhibitors (in a total volume of 5 ~1) were added to reactions lacking them, and the mixtures quenched in 0.33 vol of 4~ gel sample buffer to yield 2% sodium dodecyl sulfate (SDS), 10% glycerol, 5% /3-mercaptoethanol, 5 rn~ EDTA, and 62.5 rn~ Tris . HCl (pH 6.8), vortexed vigorously, and loaded on a SDS-lo% polyacrylamide gel without prior boiling. Following electrophoresis, proteins were electroblotted onto nitrocellulose membrane (BA85, Schleicher and Schuell) in a buffer containing 20 rn~ Na,HPO,/ NaH,PO, (pH 7.0) at 50 V for 1 h. After transfer, the nitrocellulose sheet was placed in a hybridization bag

AND

BOHMANN

containing 0.1 ml/cm* membrane of buffer A [lo rn~ Tris.HCl (pH 7.21, 10% glycerol, 2.5% Nonidet P-40 (BDH), 5% bovine serum albumin (BSA, Sigma A-2153), 10 pglml sonicated (to a mean length of 200 bp) herring testes DNA (Sigma D-6898), 0.02% NaN,, 100 rn~ NaF, 15 rn~ Na,MoO,, 20 rn~ (Na,)p-nitrophenyl phosphate, and 10 rn~ (Na,)P-glycerophosphate], and proteins were allowed to renature by incubating for 6-12 h at 4°C with gentle agitation on a tumbling platform. With regard to the composition of buffer A we have found that the widely used blocking agent nonfat dry milk from various distributors is highly enriched in phosphomonoesterases and kinases, whereas the aforementioned grade of BSA is either free or contains amounts of these enzyme activities that are completely inactivated by the employed concentrations of inhibitors. The filter was removed from the bag, briefly rinsed in buffer B [IO rn~ Tris. HCl (pH 7.2), 0.125% BSA, 10 pglml sonicated herring testes DNA, 0.02% NaN,, 40 rn~ NaF, 7.5 rn~ Na,MoO,, 10 rn~ (Na,)p-nitrophenyl phosphate, and 5 mM (Na,)B-glycerophosphate], placed in a new hybridization bag containing 0.1 ml/cm’ membrane of buffer B and a total of 0.5 wg (-5 x 10’ cpm) “‘P-labeled (AP-I), DNA, and incubated for 5 h at 4°C as before. The membrane was then washed four times (15 min per wash) in 30 ml of buffer B (lacking BSA) at 4”C, briefly air dried, and exposed to x-ray film for 30 min (step a). The filter was subsequently washed at 37°C with three 20.ml changes (30 min each) of buffer C [lo rn~ Tris. HCl (pH 7.2), 0.6 M N&l, 5 rn~ MgCl,, and 2.5 rn~ CaC12] containing freshly added DNase I (5 fig/ml; DPFF, Worthington). rinsed in four 30.ml changes (15 min each) of buffer D [lo rn~ Tris’HCl (pH 7.2), 1.5 M N&l, 20 rn~ (Na,) EDTA, and 10 /&I ATP] at 37”C, briefly air dried, and exposed to x-ray film for 3 h to confirm complete removal of the probe (step b). Following autoradiography, the membrane was equilibrated in two 30.ml changes (30 min each) of buffer E at room temperature, placed in a hybridization bagcontaining0.1 ml/cm’membrane ofbufferE supplemented with 8 units/ml PAP, and gently rocked for 6 h at room temperature. The filter was then briefly rinsed in 10 rn~ Tris. HCl (pH 7.2), washed by three 30-ml changes (20 min each) of buffer A at 4”C, placed in a hybridization hag containing 0.1 ml/cm’ membrane of buffer A, and gently incubated for 6 h at 4°C. Reprobing with the same amount of DNA (0.5 pg), washing, and exposing to x-ray film for the same period of time (30 min, step c) were performed exactly as described above (step a). Finally, the filter was immunostained with a polyclonal anti-Jun serum raised in rabbits against ecJun (step d), as previously described (15). In addition to the components described above, all buffers contained the following concentrations of protease inhibitors: 2 rn~ PMSF, 5 pglml aprotinin, 2 fig/ml pep&tin, and 2 pg/ml leupeptin. All protease and phosphatase/kinase

PHOSPHORYLATION-SENSITIVE inhibitors were added to the corresponding nrior to use. Dot-Blat

Filter-Binding

buffers

just

“SOUTH-WESTERN”

BLOTTING

305

echm

Analyses

Dot-blotting was carried out using an aspirator pump on a minifiltration apparatus (SRC 96 Minifold I, Schleicher and Schuell) according to the instructions of the manufacturers through prewet (in water) BA85 0.45~urn nitrocellulose membrane (Schleicher and Schuell) backed with two sheets of prewet blotter paper (Whatman 3MM). Treatment with PAP in the presence or absence of phosphataselkinase inhibitors was performed where indicated as described above. The final volume of the reaction mixtures applied to each well was 100 pl (30 pl of the actual reaction containing -0.03 jrg of protein, plus 70 ~1 of buffer E supplemented with the mixture of phosphatase/kinase inhibitors). The nitrocellulose membrane was then processed exactly as detailed under the South-Western blotting procedure (steps a to d). RESULTS To characterize the functional consequences of alterations in the phosphorylation state of transcription factors that occur in eukaryotic cells, for example after receipt of an extracellular stimulus, it is necessary to obtain a sufficiently concentrated preparation of the factor that is (a) free of contaminating phosphatase or kinase activities, (b) devoid of related transcription factors which might obscure functional assays, and (c) derived from a cell type which is competent for the signaling pathway of interest. As a source for such material we employed transiently transfected mammalian tissue culture cells. A cytomegalovirus enhancerlpromoterdriven expression vector was constructed which, after transfection into HeLa TK- cells, directs the synthesis of a recombinant c-dun protein (in the following called cmvJun) that bears, in addition to the wild-type amino acid sequence, a run of six histidine residues attached to the N-terminus (Fig. 1, cmvJun). This His, tag mediates a tight interaction of the fusion protein with a Nie+NTA-agarose resin and allows its efficient and rapid purification in a single step that does not rely on the native structure of the protein (16,17). In this way cmvJun can be purified from transfected cells after lysis under strongly chaotropic conditions (6 M guanidinium-HCl), which precludes any disturbances in the phosphorylation pattern of c-dun by denaturing all cellular kinases and phosphatases. cmvJun obtained from control and TPA-treated cells as well as a bacterially expressed c-Jun preparation (Fig. 1, ecdun), which served as unphosphorylated control, were treated with PAP (a nonspecific and potent protein dephosphorylating enzyme) in the presence (Fig. Za, lanes 1,3, and 5) or absence (Fig. Za, lanes 2,4,

/ ... METTFYGDALNAS.... cmvJun CMV enhancer Pmmot- c M Hs c+ +++ LLLLL SV40~01~A .--. /: / ‘-.. ..: --; ~DP~~HHHHHD~~NAS.... FIG. 1. Vectors used for the expression ofecJun and wild-type or A243 mutant cmvJun. The vector employed for the expressian of recombinant e-dun in two different systems are schematically depicted. Methionine (M), bistidine (H,), and leucine (LLLLL) codons indicated above the c-dun open-reading frame mark the position of the initiation cadon. the histidine, tag, and the leueine-zipper motif, respectively. + + + demarcates the basic DNA-binding domain of cJun. The N-terminal amino acids of the expressed proteins me shown in single-letter code with c-Jun-derived sequences printed in bold. and6) of a specific mixture ofphosphatase/kinase inhibitors (see Materials and Methods). The mock treatment in the presence of phosphatase inhibitors should ensure that any biochemical differences detected between the treated and the mock-treated material can be attributed specifically to dephosphorylation and not to other components that potentially contaminate commercial PAP preparations. After terminating the incubations by the addition of phosphatase inhibitors to reactions lacking them, the samples were run on an SDS-polyacrylamide gel and electroblotted onto nitrocellulose membrane. The DNA-binding activity of the different immobilized c-Jun proteins was then monitored by probing the blot with a “P-labeled multimerized oligonucleotide containing high-affinity AP-l-binding sites (Fig. 2a). The sequence specificity of the assay was demonstrated by competition with excess unlabeled multimerized probe; furthermore, a double-point mutation within the AP-l-binding site of the probe almost completely abrogates the obtained signal (data not shown). While the DNA-binding activity of ecJun was high regardless of whether it had been PAP-treated or not (Fig. 2a, lanes 5 and 6), the binding activity of cmvJun isolated from the control cells that were not exposed to TPA was barely detectable in the mock-treated sample, but was dramatically enhanced (more than 20-fold) after dephosphorylation (Fig. 2a, compare lane 1 to lane 2). In contrast, no PAP treatment was necessary to obtain full DNA-binding competence for cmvJun purified from the TPA-induced cells. This result indicates that c-dun exists in a predominantly inactive state maintained by the presence of inhibitory phosphate groups,

306

PAPAVASSILIOU. cmvJun

a.

BOHMANN.

eCJUn

Probe with 32P-(AP.l),

b.

Strip

AND BOHMANN

tional round of probe stripping revealed that the same amount of protein was present in all lanes and that the PAP treatment in solution had not caused any detectable degradation of the protein (Fig. 2d). Because all signal of the South-Western blots originated from probe binding to the cmvJun band, it was possible to simplify the procedure and to replace the SDS gel electrophoresis and electroblotting steps by applying the cmvJun samples directly onto the nitrocellulose filter using a dot-blot apparatus (Fig. 3). If the PAP treatment in solution may be omitted, the procedure can be even further abbreviated by loading the sample(s) (still in the guanidinium-containing &ate) directly onto the nitrocellulose filter. The results described above are in agreement with cmvJun

c.

in situ PAP treatment Reprobe

d.

Immunostain with anti-lun

m- -++ mm + _ +

mv

Probe with -w-L-L

-

1234

5 6

FIG. 2. The DNA-binding activity of c-dun IS dependent on dephospborylation that foollowsTPA induction. cmvJun protein (-0.1 eg) purified from transiently transfected HeLa TK- cells, which had been exposed (lanes 3 and 4) or not (lanes 1 and 21 to TPA for 2 h prior to harvesting, or ecJun protein C-0.1 pg) isolated from expressing E. colt cells (lanes 5 and 6) were treated with potato acid phosphatase (PAP) in the presence (lanes 1, 3, and 5) or absence (lanes 2.1. and 6) of a specific combination of phosphatase/kinase inhibitors. The reaction mixtures were run on an SDS~polyacrylam~de gel and proteins electroblotted onto a nitrocellulose membrane. The membrane was (a) probed with39labeled multimerized AP-l-binding oligonucleotides, (b) stripped of the probe, Cc) treated with PAP and reprobed. and (d) immunostained with anti-dun serum.

until cell stimulation (in our experiment mimicked by TPA) occurs. This apparently causes a removal of these phosphates, thus eliciting the DNA-binding potential of c-Jun with the concomitant activation of AP-1 target genes. To confirm this conclusion, the blots were stripped of the probe (Fig. 2b), subjected to PAP treatment in situ, and reprobed. This procedure rescued the DNA-binding activity of cmvJun (Fig. 2 steps a and c, compare lane 1 to lane 2). The restoration of DNAbinding activity of the filter-bound protein is PAP dependent, as mock treatment of the filters and reprobing reproduces the pattern seen in Fig. 2a (data not shown). Immunostaining with anti-dun serum without an addi-

aeo

3*P-(AP.l),

Strip

in situ PAP treatment Reprobe

t

*r

Immunostain with anti-Iun

.

.

*

I

.

1234 FIG. 3. A dot-blot permutation of the assay shown in Fig. 2. cmdun protein (-0.03 ~8) pun&d from transiently transfected HeLa TIC cells which had been exposed (dots 3 and 4) or not (dots 1 and 2) to TPA for 2 h prior to harvesting were treated with potato acid phosphatase (PAP) in the presence (dots 1 and 3) or absence (dots 2 and 4) of a mixture of phosphataselkinase inhibitors. Samples were subsequently dot-blotted onto a piece of nitrocellulose membrane and the filter was processed exactly aa described in the Legendto Fig. 2.

PHOSPHORYLATION-SENSITIVE

those obtained by Boyle et al. (11) using a different experimental approach. Based on phosphoanalysis of cJun tryptic peptides generated from immunoprecipitated material and mobility-shift electrophoresis assays these authors reported an activation of c-dun DNAbinding potential after TPA-induced dephosphorylation of a cluster of two serine and one threonine residues located at positions 239, 243, and 249 of the amino acid sequence of the protein. If our observations rely on the dephosphorylation mechanism proposed by Boyle et al., one would expect that a c-dun point mutation that precludes phosphorylation in the TPA-responsive sites will render the DNA-binding activity of such a mutated protein refractory to stimulation by TPA in viva and by PAP in uitro. To test this prediction, the central serine residue (S243) of the TPA-responsive phosphoacceptor target sites of cmvJun was mutagenized to alanine (A243). This mutant protein was overexpressed in HeLa TK- cells and analyzed as shown before. Clearly, cmvJun A243 binds to DNA strongly without the requirement for dephosphorylation and therefore does not display phosphorylation-mediated changes in DNA-binding activity (Fig. 4). These data unambiguously demonstrate that the observed behavior of c-Jon in our system is dependent on an already identified regulatory phosphorylation site. The fact that the same conclusions were reached both by the present analysis and the “classical” studies presented in (11) confirms the fidelity of our assay and the correct interpretation of the results. DISCUSSION Reversible protein phosphorylation seems to play a major role in mediating intracellular signals that direct dynamic changes of gene activity. The experimental system we have developed and presented here should facilitate investigations on the influence of phosphate moieties on the activity of DNA-binding transcription factors. Employing this system, it should be possible to gain information not only about the mechanisms by which protein phosphorylation affects the function of transcription factors, but also about the upstream signal transducing events that regulate the activation or inactivation of these factors through alterations in their phosphorylation state. As an example, we show that the transcription factor and protooncogene product c-dun is converted from an inactive, phosphorylated form to a DNA-binding-competent underphosphorylated species after exposing cells to TPA. Hence, this signal-dependent switch in the phosphorylation status and activity of c-dun implicates protein kinase C as an intracellular intermediary. Moreover, a c-Jun point mutant in a critical phosphoacceptor amino acid (S243) abolishes the observed effect of TPA, proving that differential protein phosphorylation is involved in this regulation and

“SOUTH-WESTERN”

307

BLOTTING

clllVJUll

cmvJun w43 toAu3 +

Probe with ‘*P-CAP-l),

in sim PAP treatment Reprobe

0

with anti-Iun

1 2

*

3 4

FIG. 4. DNA-binding properties of wild-type and AZ43 mutant cJun from untreated and TPA-stimulated cells. Wild-type cmvJun (WT) or A243 mutant cmvJun (5243 to A2431 proteins were purified from parallel transient trsnsfections of HeLa TK-cells that had been exposed (dots 2 and 4) or not (dots 1 and 3) to TPA for 2 h prior to harvesting. Aliquots ofthe four protein preparations (-0.03 pg in 100 pl of buffer E supplemented with the mixture of phospbataselkinase inhibitors) were immobilized onto nitrocellulose filter by dot-blotting, and the membrane strips were processed exactly aa described in the leeend to Fie. 2. PAP treatment of the A243 mutant cmvJun sampies i the presence or absence of phosphataselkinase inhibitors prior to filter blotting generated an identical pattern (data not shown,.

neatly corroborating the results published by Boyle et al. (11). The assay system that we present here is a combination of several techniques: expression of proteins in eukaryotic cells by transient transfection with plasmid vectors, affinity purification by nickel-chelate chromatography, and South-Western blotting before and after in situ dephosphorylation of the immobilized proteins. Some of these methods are modifications of preexisting procedures, while others are newly developed. One important new reagent is the pKH vector which directs the expression of Hiss-tagged proteins from the extremely strong and universally active cytomegalovirus en-

308

PAPAVASSILIOU.

BOHMANN.

hancer/promoter. pKH vectors allow the rapidconstruction, expression, and purification of recombinant proteins in many different mammalian cell backgrounds, without special hiosafety precautions. In no in uitro or in viva experiment could we detect functional alterations of the expressed protein that might have been caused by the addition of the oligohistidine tag. The yield of protein, in the range of 1 ~g per lo-cm dish of HeLa cells, is sufficient for many biochemical analyses. If higher quantities of protein are required, liposome-mediated transfection protocols with DOTAB (BoehringerMannheim) can he employed (C. Chavrier, unpublished data). An improved series of Hiss-tag vectors for versatile cloning strategies has been constructed (M. Treier and D. Bohmann, manuscript in preparation). Nickelchelate chromatography of guanidinium lysates of the transfected cells makes purification of the pKH-derived protein feasible under strongly denaturing conditions, thus both protecting very short-lived or labile protein phosphorylations and preventing artifact& rephosphorylation during protein isolation. Assuming that only one protein subunit or a homodimer is required for specific DNA binding and/or regulation by phosphorylation, a successful South-Western blotting step relies on (i) the capability of the studied transcription factor to adopt its functional (at least within and around the DNA-binding domain) conformation or to form functional homodimeric complexes while attached to a nitrocellulose membrane and (ii) the preservation of the phosphorylation status of the protein during renaturation/filter blocking and probe binding. The first is an inherent problem of the South-Western technique itself, but omitting boiling of the sample(s) prior to SDS-PAGE, fast electroblotting in the suggested low-salt, methanol/SDS-lacking transfer buffer, and employing the specified renaturation mix seem to be quite favorable for this prerequisite. So far all proteins analyzed in this assay [c-Jun (this study), c-Ets-1 (unpublished results from our laboratory), HSV ICP4 (18), and TTF-2 (19)] have been successfully renatured as judged by their specific DNA-binding activity. The second requirement can be satisfied by supplementing the renaturation/blocking and DNA-binding buffers with the recommended concentrations of competitive and noncompetitive phosphatase inhibitors, two of which (NaF and Na,MoO,) can simultaneously inactivate any contaminating kinases. Another novel aspect of the described method is the in situ dephosphorylation of the transcription factor after SDS gel electrophoresis and electrotransfer to nitrocellulose. Using the nickel-chelate chromatography in conjunction with the South-Western blotting technique, it is possible to keep the phosphoprotein of interest in denaturing buffers until it has been immobilized on the filter and thus separated from contaminating phosphatases, kinases, and proteases by two powerful

AND BOHMANN

purification steps, namely affinity chromatography and SDS-polyacrylamide gel electrophoresis. After removal of denaturing agents, a controlled on-filter dephosphorylation with potato acid phosphatase can be performed. We note that activation of the DNA-binding potential of the filter-bound protein by dephosphorylation without a subsequent denaturationlrenaturation step (see Materials and Methods) renders preferential renaturation of the dephosphorylated form of the protein an unlikely explanation for the obtained effects. It is useful to quantitate the amounts of assayed protein after the various enzymatic treatments and analytical steps of the method. The nitrocellulose filter that is generated to be probed with radioactive DNA can be subsequently immunostained with antibodies against the factor under study. This option will ensure that the putative differences in DNA-binding activity are due to variations in the phosphorylation state of the protein and not to discrepancies in the amounts between the samples caused by uneven loading or protein degradation. Inclusion of the appropriate mixture of protease inhibitors with a broad spectrum of specificities (see Materials and Methods) in all buffers and PAP preparations after the purification procedure, almost completely eliminates the latter possibility. Our assay is generally applicable to all cloned DNAbinding transcription factors, if one wants to compare their activity in different cellular contexts both before and after dephosphorylation. It cannot, however, he used in its described form for factors that need two or more heterologous subunits for DNA binding. The presented combined analytical method may also be expanded to the study of the effects of phosphorylation on the activity of endogenous transcription factors ohtained from souxes such as immunoprecipitates, partially purified fractions, or even total cellular extracts [an example showing the sensitivity and successful application of our blotting/protein renaturation/DNAbinding protocol for crude nuclear extracts is given in (19)]. In these cases, however, care should be taken to avoid artifactual changes in the phosphorylation state of the protein after cell lysis. This may be achieved by maintaining high concentrations of the specific mixture of phosphatase/kinase inhibitors in all buffers during the initial steps of the assay. Finally, the method, especially in its dot-blot variation could be used for the identification and characterization of the kinases and phosphatases that modulate the DNA-binding activity of signal-dependent transcription factors, and which therefore are themselves candidate components of the cellular signal transduction machinery. ACKNOWLEDGMENTS We thank Catherme Chavner for technical assistance. We are grateful to Catherine Ovitt and Iain Mattaj for comments on the manuscript.

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