THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 14, Issue of May 15, pp. 10440-10447,1933 Printed in U. S. A.

Purification and Characterization of a Template-associated Protein Kinase That Phosphorylates RNA Polymerase 11*

(Received for publication, October 5, 1992, and in revised form, January 9, 1993)

Arik DvirS, Lisa Y. Stein, Briana L. Calore, and William S . Dynans From the Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309

We have recently shown that a template-associated protein kinase, which phosphorylates the carboxyl- terminal domain (CTD) of RNA polymerase 11, is a two-component system. We describe here the purifi- cation of these two components to apparent homoge- neity from human (HeLa) cell nuclear extract. Kinase component A has a 340-kDa native molecular mass, consists of a single large polypeptide, and contains the kinase active site. Kinase component B, which is iden- tical to the Ku autoantigen, has a 180-kDa native molecular mass, and consists of apparently equimolar 67- and 83-kDa polypeptides. Component B stimulates the activity of component A, and under some condi- tions, confers DNA dependence on the reaction. The purified kinase converts the CTD to the multiply phos- phorylated CTDo form. Conversion occurs proces- sively, and this processivity is an inherent property of component A. The in vitro phosphorylated CTDo form contains approximately equimolar phosphoserine and phosphothreonine, but no detectable phosphotyrosine.

The largest subunit of RNA polymerase I1 contains tandem heptapeptide repeats. These repeats, which have the consen- sus sequence SPTSPSY, form a discrete carboxyl-terminal domain (1,2). The repeat sequence is present in RNA polym- erase I1 in almost all eukaryotic organisms that have been examined (reviewed in Refs. 3 and 4). The CTD’ is essential for viability (5-a), and is involved in the response of RNA polymerase I1 to transcriptional activator proteins (9-11). In the mouse and the human CTD, there are 52 repeats, of which 21 are identical to the consensus, and the remainder have substitutions at one or more positions (2, 12).

The CTD exists in two forms that differ in their degree of phosphorylation. The CTD, form contains little or no phos- phate, whereas the CTDo form is phosphorylated a t multiple serine and threonine residues (13, 14). This phosphorylation causes a conformational change (15, 16) and a characteristic shift in SDS-PAGE mobility (13). RNA polymerase I1 con-

* This work was supported in part by National Institute of General Medical Science Grant GM 35866. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Fellow of the Israel Cancer Research Foundation. 5 To whom correspondence should be addressed Dept. of Chem-

istry and Biochemistry, Campus Box 215, University of Colorado, Boulder, CO 80309. Tel.: 303-492-1550; Fax: 303-492-3586.

The abbreviations used are: CTD, carboxyl-terminal domain; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; AdUAS, GAL4 upstream activating sequence-adenovirus major late promoter; HSP, heat shock protein; TF, transcription factor.

taining the CTD. preferentially binds in the preinitiation transcription complex (17,18). This binding may be mediated in part by a direct interaction between the CTD. and the general transcription factor (TF) IID (19). The CTD. is phosphorylated in the transcription complex by a template- associated protein kinase, converting it to the CTDo (20, 21). This conversion occurs at about the same time as the initia- tion of RNA synthesis, suggesting that it facilitates entry into the elongation phase of the reaction.

A number of protein kinases have been described that phosphorylate the CTD in vitro (22-30). We have previously identified a CTD kinase that is present in association with transcription complexes isolated using an immobilized tem- plate system (21). The kinase phosphorylates the endogenous RNA polymerase I1 that is present in the complexes, in a reaction that is promoter-dependent and closely associated with initiation.

The kinase can be eluted from the immobilized template and shown to phosphorylate a recombinant protein containing the murine CTD fused to the DNA binding domain of the yeast protein, GAL4 (31). The reaction is DNA-dependent, and the kinase shows a strong preference for substrate con- taining the GAL4 DNA binding domain, relative to a control substrate lacking the DNA binding domain.

We have recently reported that the kinase is a two-com- ponent system (32). One component is a 350-kDa polypeptide previously identified in preparations of the DNA-dependent protein kinase (33,34). The other component is identical with the human Ku autoantigen (32, 35). The Ku autoantigen binds DNA directly, and recruits the 350-kDa component to the DNA. Both components of the kinase are necessary for phosphorylation of purified RNA polymerase 11, in a reaction that is also highly dependent on transcription factors TFIIB, TFIID, and TFIIF (32).

In this paper, we describe the purification to apparent homogeneity of both kinase components. We show that the 350-kDa component indeed contains the active site. The Ku component stimulates the activity of the catalytic component, and under certain conditions, is responsible for the DNA dependence of the phosphorylation reaction. In addition, we report that the nucleotide substrate requirements and phos- phoaminoacid products of the purified kinase allow US to distinguish this enzyme from other recently described CTD kinases.

MATERIALS AND METHODS

Growth of Cells and Preparation of Nuclear Extract-HeLa cells were grown to a 0.5-0.7 X IO6 cells/ml in Joklik minimal essential medium supplemented with 5% calf serum and 1% fetal calf serum. Cells were harvested by centrifugation, washed in phosphate-buffered saline containing 1 g/liter MgC12, resuspended in 4 packed cell vol- umes of 10 mM KCl, 10 mM Tris-HC1, pH 7.9, 1 mM DTT, 1 g / m l

10440

Template-associated RNA Polymerase 11 CTD Kinase 10441

pepstatin A, leupeptin, and soybean trypsin inhibitor, and 10 gg/ml phenylmethylsulfonyl fluoride, incubated for 10 rnin on ice, and lysed by Dounce homogenization. The resulting cell nuclei were collected by centrifugation, washed in the same buffer, and resuspended in 4 packed cell volumes of 50 mM Tris-C1, pH 7.9, 0.42 M KCl, 5 mM MgC12, 0.1 mM EDTA, 20% glycerol, 10% sucrose, 2 mM DTT, and protease inhibitors as above. The nuclei were stirred for 30 min on ice and centrifuged for 30 min at 26,500 X g. The supernatant was collected and proteins were precipitated by addition of 0.33 g/ml (NH4),S04 and centrifugation for 10 min at 20,500 X g. The pellet was resuspended in 50 mM Tris-HC1, pH 7.9, 0.1 M KC1, 12.5 mM MgCl,, 1 mM EDTA, 20% glycerol, 1 mM DTT, and protease inhibi- tors as above (1.5 rnl of buffer/liter of original cell culture). The sample was dialyzed overnight against the same buffer and centri- fuged for 10 min at 9,000 X g, and the supernatant was aliquoted and frozen at -70 "C until use.

CTD Kinase Assay-CTD kinase assays were performed using a recombinant protein substrate, consisting of glutathione S-transfer- ase, amino acids 1-147 of yeast GAL4, and the murine CTD. This protein is referred to in the text as GAL4-CTD (formerly GC147) (31). GAL4-CTD was expressed in Escherichia coli and purified as previously described (31). Phosphorylation reactions contained, in a final volume of 50 pl: 25 mM Tris-C1, pH 7546.25 mM MgC12, 0.5 mM EDTA, 2.5% (v/v) glycerol, 0.5 mM DTT, 12.5 p M [Y-~'P]ATP (Du Pont-New England Nuclear, specific activity 30-40 Ci/mmol), 20 ng/ ml EcoRI-BglI AdUAS DNA fragment (31), 2 pg/ml GAL4-CTD, and 1-4 pl of CTD kinase fractions. It was necessary to control for KC1 present in the column fractions that were assayed. The KC1 optimum for the GAL4-CTD phosphorylation reaction is approximately 10-25 mM, and concentrations above 50 mM resulted in significant inhibi- tion. Reactions were assembled on ice, initiated by addition of ATP, incubated at 30 "C for 60 min, and stopped by the addition of SDS- PAGE sample buffer. Radiolabeled products were analyzed by 6% SDS-PAGE, using 10 cm X 10-cm X 0.75-mm gels in an Idea Scientific apparatus, and visualized by autoradiography or Molecular Dynamics PhosphorImager analysis.

Purification of CTD Kinase Components A and B-Nuclear extract (50 ml) was cleared by centrifugation for 30 min at 20,000 X g and passage through a 0.22-pm filter. The filtrate was loaded on a phos- phocellulose column (Whatman P11, 2.5 X 4 cm) equilibrated with column buffer (50 mM Tris-C1, pH 7.9, 1 mM EDTA, 5% glycerol, 0.02% Tween 20, 1 mM DTT, 1 pg/ml pepstatin A, leupeptin, and soybean trypsin inhibitor, and 10 gg/ml phenylmethylsulfonyl fluo- ride) containing 0.1 M KCl. The column was eluted with a KC1 step gradient (0.1 M increments) in the same buffer. Fractions eluting between 0.2 and 0.3 M KC1 were pooled, diluted to 0.1 M KCl, and applied to a DEAE-Sephacel column (Pharmacia LKB Biotechnology Inc., 2.5 X 4 cm) equilibrated in column buffer containing 0.1 M KCI. The column was eluted with a step gradient as above. Fractions eluting between 0.1 and 0.2 M KC1 were pooled, diluted to 0.1 M KCl, and applied to a 1.5 X 6-cm heparin-agarose column (36). Fractions eluting between 0.2 and 0.3 M KC1 were pooled, adjusted to 0.1 M KC1 and 0.75 M (NH4)2S04, and a portion (typically half, to avoid overloading) was applied to a phenyl-Superose column (Pharmacia, 0.5 X 5.0 cm) equilibrated in the same buffer. The flow-through was collected as kinase component B. The column was eluted with a decreasing step gradient of (NH4)$04 (0.25 M increments). Proteins eluting between 0.5 and 0.25 M (NH4)2S04 were pooled as kinase component A. The two components were loaded separately on a Superdex 200 column (Pharmacia, 1.6 X 60 cm) equilibrated with column buffer containing 0.2 M KCl. The column was eluted with the same buffer, and active fractions were pooled, diluted to 0.1 M KC], and applied to a Mono S column (Pharmacia, 0.5 X 5.0 cm) equili- brated with column buffer containing 0.1 M KCl. The column was eluted with a 30 ml of 0.1-0.5 M linear gradient of KCI. Active

at -70 "C. fractions were adjusted to 20% (v/v) glycerol, aliquoted, and stored

Phosphoamino Acid Analysis-GAL4-CTD was phosphorylated using the standard reaction conditions except that [y-32P]ATP was present at 3.2 p M (60 Ci/mmol). The reaction products were resolved by SDS-PAGE as above, electroblotted to an Immobilon poly(viny1idene fluoride) membrane (Millipore), and visualized by autoradiography. CTDO- and CTD,-containing segments were excised separately, placed in 200 pl of 5.7 N HCl, and incubated at 110 "C for 2 h. The sample was dried in uacuo, resuspended in 10 pl of H20, and analyzed by two-dimensional thin-layer chromatography on cellulose plates (37, 38). Radiolabeled phosphoaminoacids were visualized by

autoradiography or Molecular Dynamics PhosphorImager analysis.

RESULTS

Purification of a Template-associated CTD Kinase-In an earlier study (31), we characterized preparations of CTD kinase that were obtained from transcription complexes iso- lated on a small scale. To obtain sufficient amounts of kinase for more detailed studies, it was necessary to develop a puri- fication procedure based on conventional column chromatog- raphy. Pilot experiments were performed to determine the elution patterns of kinase activity from a variety of columns. Kinase activity was assayed using the GAL4-CTD fusion protein, as described under "Materials and Methods." Using this assay, only one major DNA-dependent CTD kinase ac- tivity was detected in HeLa cell nuclear extracts.

An activity profile from Superdex 200 gel filtration chro- matography is shown in Fig. 1. There appears to be a single peak of CTD kinase activity, centered at fraction 31. T h e upper radiolabeled band corresponds to the mobility-shifted CTDo form. Previous studies suggest that a minimum of approximately eight phosphate groups is required to give a full mobility shift on SDS-PAGE (16). T h e lower radiolabeled band corresponds to the CTD, form, which contains one or a few phosphate groups and comigrates with the input sub- strate. The peak of kinase activity eluted at approximately 300 kDa (panel A ) , consistent with our previous results (31).

Further analysis showed that this apparent peak of activity actually represented the overlap of two separate, partially resolved kinase components. These components were identi- fied by complementation assays (panels B and c). Fraction 33 had little activity on its own, but was able to complement an activity peaking in fraction 29, which we shall refer to as kinase component A. Similarly, fraction 29 had little activity on its own, but was able to complement an activity peaking in fraction 33, which we shall refer to as kinase component B. These data demonstrate that kinase activity can be readily separated into two essential components under relatively mild fractionation conditions.

Based on the results of these and other experiments, we devised the kinase purification scheme presented in Fig. 2. Kinase components A and B cofractionated in the initial three chromatographic steps. The two components were then resolved by phenyl-Superose hydrophobic interaction chro- matography. Kinase component B flowed through the phenyl- Superose column, whereas kinase component A was retained and eluted at lower (NH4)2SOd concentration. Each compo- nent was further purified by Superdex 200 gel filtration and Mono S cation exchange chromatography.

Fig. 3 shows an analysis of fractions obtained during the final two chromatographic stages of kinase component A purification. Superdex 200 column fractions contained a sin- gle, large, predominant polypeptide, as determined by SDS- PAGE with silver staining (Fig. 3A). This polypeptide pre- cisely coeluted with component A enzymatic activity (Fig. 3B). Activity eluted at an apparent native molecular mass of 340 kDa, relative to marker proteins. The same large polypep- tide was visible in fractions from Mono S chromatography (Fig. 3C) and coeluted with component A activity (Fig. 3 0 ) . This polypeptide appears to be identical to the approximately 350-kDa polypeptide previously identified as a component of DNA-dependent protein kinase (33,34).

Fractions obtained during the final two chromatographic stages of kinase component B purification are analyzed in Fig. 4. The Superdex 200 fractions contained only two poly- peptides visible on SDS-PAGE with silver staining (Fig. 4A). These had apparent molecular masses of 67 and 83 kDa, and

10442 Template-associated RNA Polymerase 11 CTD Kinase

A. 23 24 25 26 27 28 29 30 31 32 -- 33 34 35 37 39

B. A - 23 26 27 28 29 30 31 c. A(29) + + + + + + + B(33) + + + + + + + + 0 - 31 32 33 34 35 36

KINASE COMPONENT A KINASE COMPONENT B FIG. 1. Separation of the two components of the template-associated CTD kinase by Superdex 200 gel chromatography.

CTD kinase was purified from 10 ml of nuclear extract by successive DEAE-Sephacel and heparin-agarose chromatography and concentrated with an Amicon ultrafiltration cell using a 30,000-dalton cutoff membrane. The retentate was applied to a Pharmacia Superdex 200 gel filtration column equilibrated with column buffer containing 0.2 M KCI. The column was eluted with the same buffer and 2-ml fractions were collected. A, CTD kinase activity (2 pl/assay) determined under standard assay conditions with GAL4-CTD substrate (see “Materials and Methods”). B, reassay of the same fractions with 2 pl of kinase component l3 (fraction 33) added to all reactions. C, reassay of the same fractions with 2 p1 of kinase component A (fraction 29) added to all reactions.

NUCLEAREXTRACT

PHOSPHO CELLULOSE F.T.

F.T.

F.T. 7 1 HEPARIN-AGAROSE

PHENYL-SUPEROSE

F.T. 0.5 0.25 0.0

SUPERDEX 200 SUPERDEX 200 vo vt vt vo

t t MONO S MONO S

0.1 1 .o 1 .o 0.1

t t COMPONENT B COMPONENT A

FIG. 2. Purification scheme. 50 ml of nuclear extract prepared as described under “Material and Methods,” containing about 500 mg of protein, were used for a typical kinase preparation. Kinase com- ponents A and B co-chromatographed through the first three steps and separated in the fourth. Each component was further purified by two additional fractionation steps. The yield of the final products was between 0.5 and 1.0 pg of protein for each component. F.T., salt concentrations in elution buffers are indicated; Vo, void volume; Vt, total volume.

were apparently present in equimolar amounts. They precisely coeluted with activity (Fig. 4B). Kinase component B activity eluted at an apparent native molecular mass of 180 kDa, relative to the markers. The simplest interpretation of these data is that kinase component B is a 1:l heterodimer. The same 67- and 83-kDa polypeptides were visible in fractions from Mono S chromatography (Fig. 4C) and again coeluted with activity (Fig. 4 0 ) . We have reportedelsewhere that these polypeptides are identical to the human Ku autoantigen (32).

Significantly, the polypeptide composition of components A and B was nearly unchanged in the final three chromato- graphic steps (Figs. 3 and 4, and data not shown), suggesting that the major polypeptides present in these fractions are, in fact, associated with kinase activity.

Dependence on DNA-Fig. 5 shows the effect of DNA concentration on GAL4-CTD phosphorylation. Consistent with the results of earlier studies (31), GAL4-CTD phos- phorylation is increased in the presence of DNA. There is a marked optimum a t approximately 1 ng per reaction. At DNA concentrations above the optimum, the kinase and its sub- strate may partition onto different DNA fragments or onto different regions of the same DNA fragment, thus becoming less available to one another for reaction. This is supported by our finding that the DNA optimum shifts toward higher values when higher concentrations of kinase and substrate are used in the reaction (data not shown). Inhibition of phosphorylation a t high DNA concentration has recently been described for human p53 and Spl (35, 39).

In order to better understand the basis of the DNA depend- ence of the reaction, we performed kinase assays with com- ponent A, in the presence and absence of kinase component B, DNA, and protein substrate. The results of these experi- ments are shown in Fig. 6. Isolated component A was capable of autophosphorylation (panel A), indicating that it, and not component B, contains the kinase active site. Interestingly,

Template-associated RNA Polymerase 11 CTD Kinase 10443

A.

205 -

116- 97 -

SUPERDEX 200 FRACTION # 25 26 27 28 29 30 31 C. MONO S FRACTION #

15 16 17 18 19 20 21 22

205 .

116- 97 -

67 - 67 -

45 - 45 -

B.

2

I-

-I > P 0 X n v) 0 X n

0 a

0 I- o

30 -

25 -

Chymotrypsinogen

D.

2 P t -I > a 0 X n v)

X 0 n 0 I- o

2o 18 I ”*

X D

2 0 2 5 30 3 5 4 0 4 5 50 5 5 6 0 0 5 1 0 1 5 20 2 5 3 0 35

FRACTION t FRACTJOM I

FIG. 3. Purification of kinase component A. A, the 0.25 M (NH&SO4 activity peak from phenyl-Superose chromatography (Fig. 2) was applied to a Superdex 200 gel filtration column equilibrated with column buffer containing 0.2 M KC1 (see “Materials and Methods”). The column was eluted with the same buffer and 2-ml fractions were collected. Proteins were analyzed by SDS-PAGE with silver staining. B, the same fractions were assayed for CTD kinase activity in a standard reaction supplemented with 0.4 ng of purified component R. Radiolabeled CTDo product was quantitated by PhosphorImager analysis. Arrows indicate the elution positions of protein molecular mass standards that were run separately on the same column. C, fractions 27-30 from the Superdex 200 column were pooled, diluted to 0.1 M KC1, and applied to a Mono S cation exchange column. The Mono S column was developed with a 0.1-0.5 M linear gradient of KC1 (see “Materials and Methods”). Fractions of 1.5 ml were collected and analyzed by SDS-PAGE with silver staining. D, the same fractions were assayed for CTD kinase activity as above (closed circles). KC1 concentration was determined by measurement of conductivity (open circles).

the autophosphorylation reaction with isolated component A Somewhat different results were obtained with the GAL4- was not DNA-dependent. When purified component B was CTD substrate (panel C). When reactions were run in the present in the reaction, there was a small increase in the presence of component A only, GAL4-CTD phosphorylation phosphorylation of component A, which occurred only in the presence of DNA. Both polypeptides of component B were phosphorylated in a reaction that was strongly dependent on DNA.

We next tested the phosphorylation of HSP 90, which has been widely used as a substrate for DNA-PK (33, 34, 40). When reactions were run in the presence of kinase component A only, HSP 90 phosphorylation was relatively weak and DNA-independent (panel B ) . In separate experiments, no effect of DNA was seen a t concentrations as high as 250 times the standard amount (data not shown). By contrast, when component B was present in the reaction, HSP 90 phos- phorylation was enhanced and became DNA-dependent. Thus, it appears that the DNA dependence was conferred by the regulatory component B.

was DNA-dependent. When component B was present in the reaction, GAL4-CTD phosphorylation was enhanced, but con- tinued to show about the same degree of dependence on DNA. Thus, it appears that here, the DNA dependence is inherent in the component A-substrate system, and not specifically conferred by component B. It is possible that GAL4-CTD phosphorylation represents a special case, and this will be taken up further under “Discussion.”

Nucleotide Substrate Requirements-Previously, we have shown that the template-associated kinase phosphorylates the GAL4-CTD substrate processively. Radiolabeled CTD, prod- uct appears at the earliest times in the reaction, followed after a brief lag by the appearance of fully mobility-shifted CTDo. Intermediately shifted forms do not accumulate (31).

Here we report the effect of ATP concentration on the

10444 Template-associated RNA Polymerase 11 CTD Kinase

A. SUPERDEX 200 FRACTION #

29 30 31 32 33 34 35 36 37 38 205 -

B.

116- 97 -

67 -

45 -

1; 10 1

C. MONO S FRACTION # 15 16 17 18 19 20 21 22 23

205 -

116 - 97 - +

+ 67 -

Catalase

Thyroglobulin

.1 A l b i i n .1

Ovalbumin

Chymotrypsinogen

20 25 3 0 3 5 4 0 4 5 5 0 5 5 6 0

FRACTION It

45 -

l o 0.9

0 + 0

8 -

6 -

4 -

2 -

- 0.8

- 0.7 - 0.6

- 0.5

- 0.4

- 0.3 - 0.2 - 0.1

0 I . . . . , . . . . , . . . . , . . . . , . . . . ! o . o 1 0 15 20 2 5 30 3 5

FRACTION I

a 2 a

FIG. 4. Purification of kinase component B. A, the flow-through from phenyl-Superose chromatography (Fig. 2) was concentrated with an Amicon ultrafiltration cell using a 30,000-dalton cutoff membrane. The retentate was applied to Superdex 200 gel filtration column equilibrated with column buffer containing 0.2 M KC1 (see “Materials and Methods”). The column was eluted with the same buffer and 2-ml fractions were collected. Proteins were analyzed by SDS-PAGE with silver staining. B, the same fractions were assayed for CTD kinase activity in standard reaction supplemented with 0.2 ng of purified component A. Radiolabeled CTDo product was quantitated by Phosphor- Imager analysis. Arrows indicate the elution positions of protein molecular mass standards that were run separately on the same column. C, fractions 31-35 from the Superdex 200 column were pooled, diluted to 0.1 M KC1, and applied to a Mono S cation exchange column. The Mono S column was developed with a 0.1-0.5 M linear gradient of KC1 (see “Materials and Methods”). Fractions of 1.5 ml were collected and analyzed by SDS-PAGE with silver staining. D, the same fractions were assayed for CTD kinase activity as above (closed circles). KC1 concentration was determined by measurement of conductivity (open circles).

phosphorylation reaction. At ATP concentrations below 1 PM, most of the radiolabeled product migrated at the CTD, posi- tion (Fig. 7). By contrast, a t high concentrations of ATP, most of the radiolabeled product migrated at the CTDo posi- tion. Qualitatively similar results were observed in 5-, 15-, 30-, and 60-min reactions (data not shown).

The data indicate that the concentration of ATP affects the ability of the enzyme to convert the CTD, to the CTDo. One possibility is that ATP stabilizes the interaction between the enzyme and the partially phosphorylated substrate, thereby increasing the processivity of the reaction.

Previous studies have shown that the endogenous CTD kinase present in isolated preinitiation transcription com- plexes has a strong preference for ATP over GTP (21). To determine whether the purified kinase shared this same char- acteristic, GAL4-CTD phosphorylation was tested in the pres- ence of increasing amounts of nonradioactive GTP. Very little competition was observed with more than a 100-fold excess

of GTP, in contrast to the strong competition observed with ATP (Fig. 8). This nucleotide specificity is different from the results recently reported for a different CTD kinase, the rat 6 factor (29), where labeling is almost totally competed by nonradioactive GTP under similar conditions. The human TFIIH-associated CTD kinase has also been reported to use GTP as a substrate (28).

Phosphoaminoacid Products-RNA polymerase I1 is phos- phorylated in vivo at serine and threonine residues, but not at tyrosine residues (13, 14, 41). To determine which amino acids are phosphorylated in vitro by the kinase that we have purified, the products of the GAL4-CTD phosphorylation reaction were analyzed. Radiolabeled CTD. and CTDo were resolved by SDS-PAGE, electroblotted to an Immobilon mem- brane, and hydrolyzed separately in the presence of HCl. The hydrolysate was analyzed by two-dimensional thin-layer elec- trophoresis (37, 38). The results are shown in Fig. 9. The GAL4-CTDo contained approximately equal amounts of phos-

Template-associated RNA Polymerase 11 CTD Kinase 10445

0 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2

DNA (n9) FIG. 5. DNA dependence of GALI-CTD phosphorylation.

Kinase assays were performed as described under “Materials and Methods” using 0.2 ng of component A and 0.4 ng of component B. The amount of DNA in each reaction was varied as indicated. The relative amount of label incorporated into the CTD, form was quan- titated.

A. AUTOPHOSPHORYLATION

COMPONENTB: - - + + DNA: + - + - .-

- c 4 - A

205 -

116 - 97 - 67 -

45 -

2.00

1 .oo

0

B.

phoserine and phosphothreonine. No detectable phosphoty- rosine was present. This differs markedly from the rat 6 factor, which produces exclusively phosphoserine (29).

DISCUSSION

We have described the purification to homogeneity of the major DNA-dependent CTD kinase in HeLa cell extracts. The enzyme consists of two components, A and B. Component A contains the active site. It phosphorylates both itself and exogenous substrates to low levels. Component A is appar- ently composed of a monomer of a single large polypeptide. A monoclonal antibody against a similar sized polypeptide has previously been reported to inhibit the activity of the enzyme DNA-dependent protein kinase (33). Component B, which regulates the activity of component A, is apparently composed of a heterodimer of 67- and 83-kDa polypeptides. We have reported elsewhere that component B is identical to Ku pro- tein, a human autoantigen that binds tightly to DNA (32).

Previously, we showed that kinase component B binds to DNA directly and that kinase component A binds to DNA only in the presence of component B. Here, we have examined more closely the way in which kinase component B affects the reaction. It appears that component B has two distinct effects. In the absence of DNA, component B increased the phosphorylation of two exogenous substrates, HSP 90 and GAL4-CTD. Thus, component B appears to function as a direct allosteric activator of component A.

Component B also regulates the phosphorylation reaction

C. HSP 90 PHOSPHORYLATION CTD PHOSPHORYLATION

” + + + - + -

4- HSP 90

4 0

4 - 0

2.0c

1 .oc

0

- - + + + - + - r

I I

i I

FIG. 6. DNA dependence of phosphorylation in the presence and absence of component B. A , autophosphorylation. Reactions were performed with 0.2 ng of component A in the presence or absence or 0.4 ng of component B and DNA. Exogenous substrate was not present, and [y3*P]ATP concentration was adjusted to 1.25 PM (300 Ci/mmol). Products were analyzed by SDS-PAGE and autoradiography. Arrows denote radiolabeled kinase components A and B. Bottom portion of figure shows PhosphorImager quantitation. Solid bars, component A; hatched bars, 67-kDa subunit of component B. B, phosphorylation of HSP 90 substrate. Reactions were performed with 0.2 ng of component A, 0.4 ng of component B, and 0.25 pg of HSP 90 under standard conditions as described under “Materials and Methods.” Products were analyzed by SDS-PAGE and autoradiography. Bottom portion of figure shows PhosphorImager quantitation. C , phosphorylation of GAL4- CTD substrate. Reactions were performed as in panel B except with GAL4-CTD. Products were analyzed by SDS-PAGE and autoradiography. Arrows denote CTD, and CTD. forms. Bottom portion of figure shows PhosphorImager quantitation of label incorporated into CTDo form.

10446 Template-associated RNA Polymerase 11 CTD Kinase

ATP(pM): 0.06 0.13 0.26 0.52 1.7 2.7 4.8 1 5 23

CTD, .--)

CTDA-

-.” i

1

ATP, pM FIG. 7. Effect of ATP concentration on GAL4-CTD phos-

phorylation. Reactions were performed with 0.2 ng of component A, 0.4 ng of component B, and GAL4-CTD substrate under standard conditions as described under “Materials and Methods,” except that reactions were performed for 30 min and the concentration of non- radioactive ATP was varied as indicated. Products were analyzed by SDS-PAGE and autoradiography. Bottom portion of figure shows PhosphorImager quantitation, normalized to correct for changes in specific activity of ATP. Open circles, relative incorporation of phos- phate into CTDo form; closed circles, relative incorporation of phos- phate into CTD, form.

by altering the DNA dependence. Phosphorylation of HSP 90 substrate, and to some extent phosphorylation of component A itself, became DNA-dependent only when component B was present. We envision two possible mechanisms by which component B might cause the phosphorylation of a non-DNA- binding substrate such as HSP 90 to become DNA-dependent. A conformational change may occur in component B when it binds DNA, increasing its ability to function as an allosteric activator of component A. In this model, there need not be any direct contact between component A and DNA. Alterna- tively, component B might recruit DNA to the complex, where it can interact with a separate allosteric site in component A. In this case, component A would contact the DNA directly.

The results obtained with the GAL4-CTD substrate provide some support for the second model. Phosphorylation of GAL4-CTD was DNA-dependent in the presence of compo- nent A alone. This may be because the GAL4-CTD substrate is bound to a fragment of DNA, which is brought into close proximity to component A whenever the substrate binds the active site. This would facilitate the binding of DNA to an allosteric site, obviating the need for the separate DNA- tethering activity of component B. However, we cannot ex- clude other interpretations, for example, that the DNA de- pendence observed with GAL4-CTD substrate reflects a spe-

200

z

3 150 0

& P 9 a 0 100

a n 0 50 t-

0

1 -

I -

I -

l l -

i 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

ADDED GTP OR ATP (mM) FIG. 8. Competition with nonradioactive ATP and GTP.

Reactions were performed with 0.2 ng of component A, 0.4 ng of component B, and GAL4-CTD substrate under standard conditions as described under “Materials and Methods” except that the concen- tration of nonradioactive ATP (closed circles) and G T P (open circles) was varied as indicated. Products were analyzed by SDS-PAGE and the relative amount of radiolabeling was determined by Phosphor- Imager analysis.

- - pH 1.9 pH 1.9

FIG. 9. Phosphoaminoacid analysis of GAL4-CTDo. GAL4- CTD was phosphorylated as described under “Materials and Meth- ods” and analyzed for phosphoaminoacid content by two-dimension thin-layer electrophoresis as described under “Materials and Meth- ods.” Left panel, products visualized by autoradiography; right panel, positions of phosphoaminoacid markers stained with ninhydrin ( p - ser, p-thr, p-tyr) and expected position of incompletely hydrolyzed peptides ( p e p ) (37).

cia1 interaction between the CTD and DNA (42). The purified kinase phosphorylates the GAL4-CTD sub-

strate in a processive manner. The bulk of the radiolabel in our assays migrates at the CTD. and CTDo positions, with relatively little label in intermediate forms. This processivity is inherent in component A, since it occurs even when com- ponent B is absent from the reaction (Fig. 6). The same degree of processivity was seen when up to 20 Fg/ml GAL4-CTD was present in the reaction, 10 times the normal amount (data not shown). This suggests that the processivity is unlikely to be attributable to the presence of limiting amount of sub- strate, but rather to an interaction between the CTD and component A that is stable on the time scale of the reaction. The stability of this interaction is probably dependent on the presence of enzyme-bound ATP, as the ability to convert the

Template-associated RNA Polymerase I I CTD Kinase 10447

substrate to the fully mobility-shifted CTDo form was much reduced at low ATP concentrations.

The kinase that we have purified has many similarities to a DNA-dependent protein kinase described by others. TWO recent papers describe the partial purification DNA-depend- ent protein kinase (33, 34), and report the presence of a predominant 350-kDa polypeptide. Interestingly, one of these groups reported that Ku protein was present during the early stages of the DNA-dependent protein kinase preparations and that it was a substrate for the kinase. Although the role of Ku in regulating the phosphorylation of other substrates was not recognized, these authors noted that the 350-kDa component did not necessarily co-chromatograph with kinase activity (34). It is interesting that, in at least one instance, the peak of activity eluted midway between the 350-kDa polypeptide and two polypeptides resembling Ku antigen.

Recent reports describe CTD kinases that have been puri- fied in association with the general transcription factors b obtained from yeast (27), 6 obtained from rat liver (29), and TFIIH obtained from cultured human cells (28). These factors resemble each other and factor BTFZ from HeLa cells (43), and all may be homologues (44). They are composed of mul- tiple polypeptides and have biochemical functions in addition to kinase activity (27, 28, 45).

The kinase we have purified differs in both polypeptide composition and enzymatic properties from the factors de- scribed above. It appears that none of these factor prepara- tions contain major polypeptides that are as large as our kinase component A. Moreover, although some of the factor preparations contain polypeptides similar in size to those in our kinase component B, in two cases these polypeptides have been cloned and sequenced (43,44) and have no similarity to the Ku protein. In addition, there are differences in enzymatic properties between the other kinases and ours. The yeast factor b-associated kinase is not DNA-dependent. The mam- malian kinases are DNA-dependent for RNA polymerase I1 phosphorylation, but DNA-independent with CTD peptide (29) or GAL4-CTD substrates (28). The mammalian kinases also have a greater ability to use GTP as a substrate (28, 29). Finally, the ratio of phosphothreonine to phosphoserine in the CTDo product differs with the other kinases. They either do not produce phosphothreonine (29) or produce it as a minor product (28), whereas our kinase produces phospho- threonine and phosphoserine approximately equally.

The extent to which different CTD kinases have distinct roles in transcription remains to be determined. We have noted (32) that the unique properties of the Ku autoantigen, and especially its ability to slide along the DNA, may have a physiological significance in terms of delivering the kinase catalytic component to substrate bound at fixed positions on the template. It is known that P,r hydrolyzable ATP or dATP is required for RNA polymerase I1 to form the initial phos- phodiester bonds in RNA (46, 47). However, recent evidence suggests that the requirement for ATP in initiation is separate from the requirement for ATP for CTD phosphorylation (20). These findings suggest that CTD phosphorylation may be important at a postinitiation phase of the reactions, such as release of the CTD from contacts with TFIID or transcrip- tional activator proteins (19, 48). The availability of CTD kinases in pure form will facilitate a more detailed investiga- tion of their roles in the transcription reaction.

Acknowledgments-We thank M. Kissinger for production of HeLa cells, C. Anderson and S. Lees-Miller for HSP 90 protein, and N. Ahn for advice on phosphoaminoacid analysis. We also thank R. Kuchta and R.-B. Markowitz for helpful discussions and comments on the manuscript.

REFERENCES

1. Allison. L. A., Moyle, M., Shales, M., and Ingles, C. J. (1985) Cell 42,599- GI n

2.

3. 4. 5. 6.

7.

8.

9.

10.

11.

12.

14. 13

15, 16 17.

18,

19

20.

21.

22. 23. 24.

25.

26.

27.

28.

29.

30. 31.

32.

33.

34.

36. 35.

37.

38. 39.

40.

41. 42. 43.

44.

45.

46.

Corden, J. L., Cadena, D. L., Ahearn, J. M., Jr., and Dahmus, M. E. (1985)

Corden, J. L. (1990) Trends Biochern. Sci. 16,383-387 Chao, D. M., and Young, R. A. (1991) Gene Expression 1 , l - 4 Nonet, M., Sweetser, D., and Young, R. A. (1987) Cell 60,909-915 Allison, L. A., Wong, J. K. C., Fitzpatrick, V. D., Moyle, M., and Ingles, C.

Bartolomei, M. S., Halden, N. F., Cullen, C. R., and Corden, J. L. (1988)

Zehring, W. A,, Lee, J. M., Weeks, J. R., Jokerst, R. S., and Greenleaf, A.

Allison, L. A,, and Ingles, C. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 8 6 ,

Scafe, C., Chao, D., Lopes, J., Hirsch, J. P., Henry, S., and Young, R. A.

Liao, S. M., Taylor, I. C., Kingston, R. E., and Young, R. A. (1991) Genes

Wintzerith, M., Acker, J., Vicaire, S. , Vigneron, M., and Kedinger, C. (1992)

Zhang, J., and Corden, J. L. (1991) J. Biol. Chem. 266,2290-2296 Cadena, D. L., and Dahmus, M. E. (1987) J. Biol. Chem. 262,12468-12474

Laybourn, P. J., and Dahmus, M. E. (1989) J. Biol. Chem. 264,6693-6698 Zhang, J., and Corden, J. L. (1991) J. Biochem. 266,2297-2302 Lu, H., Flores, O., Weinmann, R., and Reinberg, D. (1991) Proc. Natl. Acad.

Chesnut, J. D., Stephens, J. H., and Dahmus, M. E. (1992) J. Bid. Chem.

Y A Y

Proc. Natl. Acad. Sci. U. S. A. 8 2 , 7934-7938

J. (1988) Mol. Cell. Biol. 8 , 321-329

Mol. Cell. Biol. 8, 330-339

L. (1988) Proc. Natl. Acad. Sci. U. S. A. 86, 3698-3702

2794-2798

(1990) Nature 347,491-494

& Den 6,2431-2440

Nucleic Acids Res. 20,910

Sci. U. S. A. 88, 10004-10008

267. 10500-1050fi Usheva, A., Maldonado, E., Goldring, A., Lu, H., Houbavi, C., Reinberg,

Laybourn, P. J., and Dahmus, M. E. (1990) J. Biol. Chem. 2 6 5 , 13165-

Arias, J., Peterson, S . R., and Dynan, W. S. (1991) J. Biol. Chem. 2 6 6 ,

Cisek, L. J., and Corden, J. L. (1989) Nature 339,679-684 Guilfoyle, T. J. (1989) Plant Cell 1,827-836 Lee, J. M., and Greenleaf, A. L. (1989) Proc. Natl. Acad. Sci. U. S. A. 8 6 ,

Payne, J. M., Laybourn, P. J., and Dahmus, M. E. (1989) J. Biol. Chem.

Stevens, A., and Maupin, M. K. (1989) Biophys. Res. Commun. 169 , 508-

Feaver, W. J., Gileadi, O., Li, Y., and Kornberg, R. D. (1991) Cell 67,1223-

~ . ~ ~ .

D., and Alonl, Y. (1992) Cell 69,871-881

13173

8055-8061

3624-3628

2 6 4 , 19621-19629

515

1 3 m L;H, Zawel, L., Fisher, L., Egly, J.-M., and Reinberg, D. (1992) Nature

Serizawa, H., Conaway, R. C., and Conaway, J. W. (1992) Proc. Natl. Acad. 368,641-645

Sci. U. S. A. 89. 7476-7480 Stone, N.,~and Reinberg, D.-(1992) J. Biol. Chem. 2 6 7 , 6353-6360 Peterson, S. R., Dvir, A,, Anderson, C. W., and Dynan, W. S. (1992) Genes

Dvir, A., Peterson, S. R., Knuth, M. W., Lu, H., and Dynan, W. S. (1992)

Carter, T., Vancurova, I., Sun, I., Lou, W., and DeLeon, S. (1990) Mol. Cell.

Lees-Miller, S. P., Chen, Y.-R., and Anderson, C. W. (1990) Mol. Cell. Biol.

Davison, B. L., Leighton, T., and Rabinowitz, J. C. (1979) J. Bid. Chem. Gottlieb, T. M., and Jackson, S. P. (1993) Cell 7 2 , 131-142

Cooper, J. A., Sefton, B. M., and Hunter, T. (1983) Methods Enzymol. 9 9 ,

Kamps, M. P., and Sefton, B. M. (1989) Anal. Biochern. 176,22-27 Lees-Miller, S. P., Sakaguchi, K., Ullrich. S. J., Appella, E., and Anderson,

Lees-Miller, S. P., and Anderson, C. W. (1989) J. Biol. Chem. 2 6 4 , 17275-

Dahmus, M. E. (1981) J. Biol. Chem. 266,3332-3339 Suzuki, M. (1990) Nature 344,562-565 Fischer, L., Gerard, M., Chalut, C., Lutz, Y., Humbert, S., Kanno, M.,

Gileadi, 0.. Feaver, W. J., and Kornberg, R. D. (1992) Science 267,1389-

& Deu. 6,426-438

Proc. Natl. Acad. Sci. U. S . A. 89,11920-11924

Biol. 10,6460-6471

10,6472-6481

264,9220-9226

387-402

c. w. (1992) Mol. Cell. Biol. 1 2 , 5041-5049

17280

Cambon, P., and Egly, J.-M. (1992) Science 2 6 7 , 1392-1395

lR91 Conaway, R. C., and Conaway, J. W. (1989) Proc. Natl. Acad. Sci. U. S. A.

Bunick, D., Zandomeni, R., Ackerman, S., and Weinmann, R. (1982) Cell

""_ 86,7356-7360

29.877-886 47. Sawadogo, M., and Roeder, R. G . (1984) J. Biol. Chem. 269,5321-5326 48. Sigler, P. B. (1988) Nature 333,210-212