©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Different Carboxyl-terminal Domain Kinase Activities Are Induced by Heat-shock and Arsenite
CHARACTERIZATION OF THEIR SUBSTRATE SPECIFICITY, SEPARATION BY MONO Q CHROMATOGRAPHY, AND COMPARISON WITH THE MITOGEN-ACTIVATED PROTEIN KINASES (*)

Sylviane Trigon (§) , Michel Morange

From the (1) Unité de Génétique moléculaire, Ecole Normale Superieure, 46 rue d'Ulm, 75230 Paris cedex 05, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In response to heat-shock and chemical treatments, cells undergo profound biochemical changes such as modifications in protein phosphorylation in order to resist the new, unfavorable growth conditions. We have previously shown that in HeLa cells a protein kinase (HS-CTD kinase) activity is induced rapidly after a heat or sodium arsenite shock. This kinase activity is able to phosphorylate a synthetic peptide composed of four repeats of the motif Ser-Pro-Thr-Ser-Pro-Ser-Tyr, a motif highly repeated in the carboxyl-terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II. In this paper, we designed a new experimental procedure to characterize the substrate specificity of this kinase activity. We show that HS-CTD kinase activity phosphorylates a consensus sequence (-P-X-S/T-P-) which is similar to the sequence phosphorylated by extracellular regulated protein kinases (also called mitogen-activated protein kinases). However, there is a slight but reproducible difference between these kinases in their use of serine or threonine as the phosphate acceptor. Mono Q chromatography allows the separation of five stress-induced CTD kinase activities, two of which coelute with active mitogen-activated protein kinase forms revealed by Western blotting with anti ERK1-ERK2 antibodies. The other three CTD kinase activities induced after a stress are distinct from ERK1 and ERK2 and have different enzymatic properties. The molecular nature of these HS-CTD kinases and the physiological significance of their activation during stress remain to be determined.


INTRODUCTION

All cells respond to transient, unfavorable growth conditions such as small increases in temperature or the presence of toxic agents (sodium arsenite, ethanol, amino acid analogs) by a decrease in transcriptional activity and protein synthesis. In spite of this general decrease, the rate of expression of a restricted set of genes called heat-shock genes is significantly increased, and the corresponding proteins are preferentially translated. Rapid intracellular modifications are required to switch on the heat-shock response. These modifications, occurring in the interval between the stress and heat-shock protein synthesis, include exchanges of molecules between different cellular compartments, changes in chromatin structure (1), and modifications of proteins by phosphorylation, glycosylation, acetylation, etc. Changes in phosphorylation have been described for several proteins including translation initiation factors eiF2 and eIF4F (2, 3) and HSP 27 (4) . It has recently been shown that a protein kinase called MAPKAP kinase 2 is able to phosphorylate hsp27 in vitro(5) . This MAPKAP kinase 2 can be activated in vitro by the major MAP() kinases (6) but also in vivo by a minor form (7) . Interestingly, MAP kinases are activated after heat-shock (8) .

In a previous paper we described the enhancement of a protein kinase activity after heat or sodium arsenite shock in HeLa cells (9) . This activity, called HS-CTD kinase, is induced even in the presence of protein synthesis inhibitors. It is able to phosphorylate serines in a synthetic peptide (Ser-Pro-Thr-Ser-Pro-Ser-Tyr) corresponding to a 4-fold repeat of a highly repeated motif in the carboxyl-terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II.

RNA polymerase II CTD is required for cell viability and seems more important for transcription initiation than for elongation and termination (10, 11, 12, 13, 14) . Phosphorylation of RNA polymerase II occurs within the CTD which contains multiple phosphorylation sites. The highly phosphorylated form of RNA polymerase is called IIo and the nonphosphorylated form IIa. In vitro experiments indicate that RNA polymerase IIa form is recruited to the promoter during preinitiation complex assembly (13) , whereas RNA polymerase IIo seems to be essential for the elongation phase. RNA polymerase can be phosphorylated on serine and threonine residues, but also on tyrosines (15).

Protein kinases able to phosphorylate the CTD in vitro have been partially purified or purified to homogeneity from various organisms. Two kinases have been purified from yeast (16, 17) , one from Aspergillus nidulans(18) , two from mouse (both containing a cdc2 subunit) (19, 20) , one from rat (21) , and at least four from man (22-25). Some of these protein kinases correspond to the same essential transcription initiation factor, called factor b in yeast (17), in rat (21) , and TFIIH in human cells (24) .

Another protein kinase called T669 kinase, responsible for the phosphorylation of threonine 669 in the epidermal growth factor receptor and induced by interleukin-1 (26) , is also able to phosphorylate the peptide Arg-Arg-Arg-(Tyr-Ser-Pro-Thr-Ser-Pro-Ser). This kinase is a member of the extracellular signal-regulated kinase family (or mitogen-activated protein kinases). p44, a MAP kinase purified from maturing sea star oocytes, also phosphorylates in vitro a peptide derived from the CTD sequence (27) .

We previously demonstrated that HS-CTD kinase activity has no affinity for the yeast suc 1 gene product p13 or for anti-cdc2 antibody, which indicates that HS-CTD kinase(s) probably does not contain a cdc2 subunit. In this paper, we further characterize this activity by determining the consensus sequence for phosphorylation. Since this sequence appears similar to the consensus sequence described for MAP kinases (27, 28, 29) , we compared HS-CTD kinase activity to the main members of the MAP kinase family: ERK1, ERK2, and p44. HS-CTD kinase activity appears to result from at least five different protein kinase activities with different substrate specificities, which can be separated by Mono Q chromatography. Only two of these kinase activities correspond to the MAP kinases ERK1 and ERK2.


MATERIALS AND METHODS

Culture of HeLa Cells and Stress Conditions

Cells were cultured at 37 °C in a 10% CO water-saturated atmosphere in 175-cm flasks using Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal calf serum, 2 mM glutamine, 50 µg/ml streptomycin, and 50 units/ml penicillin.

Serum induction was carried out 24 h after replacing the normal medium with serum-free medium, by addition of a 20% serum-containing medium for 15 min.

Heat-shocks were performed by immersing closed flasks in a water bath at 45 °C for 15 min. Chemical shocks were performed by treating the cells with 800 µM sodium arsenite for 1 h at 37 °C in the presence of 10 mM MOPS.

Preparation of Cell Extracts

Crude Extracts and Extracts for Mono Q Chromatography

Stressed cells and control cells were immediately washed in buffer A (50 mM glycerophosphate, 10 mM MgCl, 1 mM EGTA, 10% glycerol, pH 7.3), and then lysed in Buffer A containing 1% (v/v) Nonidet P-40, 1 µg/ml pepstatin, 1 µg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 50 mM NaF, and 0.01% (v/v) -mercaptoethanol. Lysates were centrifuged for 10 min at 10,000 g and supernatants (about 1.5 mg protein/ml) were stored at -80 °C.

Extracts for DEAE Chromatography

Arsenite-treated and control Hela cells were lysed in Buffer B (15 mM glycerophosphate, 10 mM MgCl, 1 mM EGTA, 10% glycerol, 1 µg/ml pepstatin, 1 µg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, and 0.01% (v/v) -mercaptoethanol, pH 7.4) containing 1% (v/v) Nonidet P-40 and centrifuged as described above.

Separation from Endogenous Substrates by DEAE Chromatography

After thawing, lysates from HeLa cells were centrifuged at 100,000 g for 15 min at 4 °C to remove cellular debris.

Supernatants (50 mg of proteins) were loaded onto a 10-ml DEAE-trisacryl M (IBF) column previously equilibrated in Buffer B containing 1 µg/ml pepstatin, 1 µg/ml aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride. After washing with equilibration buffer, proteins were eluted by a gradient of 15-200 mM glycerophosphate in buffer B. The flow rate was 0.25 ml/min during loading, washing, and elution. Fractions of 1.0 ml were collected. Hepta-4 kinase activity was eluted as a broad peak between 30 and 100 mM glycerophosphate. Fractions containing this activity were pooled and frozen at -80 °C. This pool contained 14% of total proteins and glycerophosphate concentration was 65 mM.

Kinase Assays

Reaction mixtures (24 µl) contained (final concentrations): 60 mM glycerophosphate, 10 mM EGTA, 10 mM MgCl, 7.5% glycerol, 1 mM sodium orthovanadate, 50 mM NaF, [-P]ATP (0.2 mM, 1 µCi), 0.1% -mercaptoethanol, myelin basic protein from bovine brain (Sigma) (0.3 mg/ml), peptides hepta-4 (SPTSPSY) (0.4 mg/ml), hepta-2 (SPTSPSY) or modified hepta-2 peptides (0.4 mg/ml), hepta-1 (SPTSPSY) or modified hepta-1 peptides (1.6 mg/ml). Four µl of partially purified HS-CTD kinase, human ERK1 (agarose-conjugated ERK1, UBI), murine recombinant ERK2 (0.4 unit/ml, kindly provided by Dr. P. Cohen, University of Dundee, United Kingdom), or sea star p44 (UBI) were added, and in vitro reactions were performed in initial rate conditions at 30 °C for 2 h for DEAE-enriched HS-CTD kinase, 1.5 h for ERK1 and p44 and 30 min for ERK2 and Mono Q fractions. Peptides were synthesized by Dr. O. Siffert from the Organic Chemistry Laboratory at Institut Pasteur. Reactions were stopped by adding 1 volume of Laemmli sample buffer containing 5% -mercaptoethanol (30).

Gel Electrophoresis Conditions and Quantification

Hepta-4 Peptide and Myelin Basic Protein Analysis

Reaction samples were analyzed by 15% SDS-polyacrylamide gel electrophoresis performed according to Laemmli (30) .

Hepta-2 Peptide Analysis ((SPTSPSYSPTSPSY) and Modified Peptides)

Reaction samples were analyzed by 22% SDS-polyacrylamide gel electrophoresis performed according to Laemmli (30).

Hepta-1 Peptide Analysis ((SPTSPSY) and modified peptides)

Reaction samples were subjected to electrophoresis on 10% denaturing phosphate buffer gels at pH 6.0 in conditions previously described by Weber and Osborn (31) and modified by us (32) .

Gels were fixed in ethanol/acetic acid/trichloroacetic acid/water (3:1:1:5, v/v), dried, and submitted to autoradiography with intensifying screens at -70 °C. Signals were quantified with PhosphorImager (Molecular Dynamics), and measurements were made with ImageQuant software version 3.3.

Western Blot Analysis

Fractions were heated for 5 min at 95 °C in Laemmli sample buffer and electrophoresed on 10% polyacrylamide gels. Proteins were electrotransferred on nitrocellulose (0.45 Micron, Hybond-ECL, Amersham Corp.). Western blots were performed as described in the ECL kit. Proteins were probed with antibody ERK1 (956/837) (Santa Cruz Biotechnology) diluted 100-fold. This antibody is a rabbit polyclonal antibody raised against a peptide which corresponds to amino acids 352-367 within the carboxyl-terminal part of the rat ERK1 protein. This antibody is able to react with the ERK1 and also ERK2 proteins of different species.

Chromatography on Mono Q

Crude cell extracts were prepared in buffer A as described above. They were centrifuged at 100,000 g for 15 min at 4 °C to clarify lysates. Supernatants (8 mg of proteins in 6 ml) were applied to an HR 5/5 Mono Q column (Pharmacia) previously equilibrated in Buffer A, essentially as described by Northwood et al.(33) . After washing with 10 ml of buffer A, proteins were eluted with a 30-ml linear salt gradient from 0 to 300 mM NaCl in Buffer A. The proteins remaining on the column were eluted by a 10-ml gradient from 300 mM to 1 M NaCl in Buffer A. The flow rate was 0.5 ml/min during loading and 1.0 ml/min during washing and elution. Fractions of 1.0 ml were collected.


RESULTS

HS-CTD Kinase Activity Present in HeLa Cells and MAP Kinases Phosphorylate One Unique and Identical Serine in the Heptapeptide Motif

HS-CTD kinase activity phosphorylates serines in a peptide composed of four repeats of the CTD consensus sequence (Ser-Pro-Thr-Ser-Pro-Ser-Tyr), but it does not phosphorylate either threonines or tyrosines in this peptide (9) . A set of different peptides was synthesized to determine which serines were phosphorylated by HS-CTD kinase activity. These peptides contain one (hepta-1) or two (hepta-2) CTD motifs in which each serine was successively substituted by alanine to remove the phosphate acceptor groups. Hepta-1 peptides are smaller than the hepta-4 peptide and cannot be analyzed with the same electrophoretic conditions. Thus, a new electrophoresis protocol was designed by replacing the Tris-glycine buffer by a phosphate buffer. When crude extracts were used, phosphorylation of an endogenous protein interfered with observation of the phosphorylated hepta-1 peptides. To eliminate this contaminant, HeLa cell crude extracts were loaded on a DEAE column, which binds HS-CTD kinase activity while the unwanted protein remains in the flow through (see ``Materials and Methods''). By using the set of hepta-1 peptides, we showed that replacement of serine 1 or 6 by an alanine did not disturb peptide phosphorylation whereas substitution of serine 4 led to a complete loss of phosphorylation. We concluded that only serine 4 of the CTD motif was phosphorylated by HS-CTD kinase activity (Fig. 1A). When the same peptides were used with ERK1, ERK2, and p44 the results were identical (Fig. 1, B-D). These experiments strongly suggest that ERK1, ERK2, and p44 do indeed preferentially phosphorylate serine 4 in the CTD motif, as does the HS-CTD kinase activity.


Figure 1: Determination of the serine phosphorylated by HS-CTD kinase activity, ERK1, ERK2, and p44 within the CTD motif by using hepta-1 peptides. A set of four peptides containing 7 amino acids was chemically synthesized. Their sequence is derived from the CTD motif by successively replacing each serine by an alanine: SPTSPSY (H-1), APTSPSY (A1), SPTAPSY (A4), and SPTSPAY (A6). Kinase assays were performed by adding DEAE-enriched HS-CTD kinase activity, ERK1, ERK2, or p44 to these synthetic peptides (1.6 mg/ml) as described under ``Materials and Methods.'' Phosphorylated peptides were separated by electrophoresis on denaturing phosphate-buffered polyacrylamide gels. Gels were dried and autoradiographed.



To exclude the possibility that the lack of phosphorylation of serines 1 and 6 could be due to their external position in hepta-1 peptides, similar alanine replacement studies were performed with hepta-2 peptides (Fig. 2). Phosphorylated hepta-2 peptides separated by electrophoresis on a 22% polyacrylamide gel gave rise to two separate bands, supposedly corresponding to the phosphorylation of serine 4 in the right or left part of the peptide (positions 4 and 11 in hepta-2; since the extent of peptide phosphorylation was low, the probability of a doubly phosphorylated peptide is close to zero). To verify this, different analogs of the hepta-2 peptide were synthesized and assayed as substrates of HS-CTD kinase activity. In these peptides either one or two of the serines present in a central position of the hepta-1 motif were replaced by alanines. Replacement of serines 4 and 11 led to the disappearance of the two phosphorylated bands, whereas replacement of serine 4 or serine 11 led to the selective disappearance of the lower or upper phosphorylated bands. Replacement of serine 8 had no effect on the number of radioactive bands, showing that this position is not phosphorylated by HS-CTD kinase.


Figure 2: Determination of the serine phosphorylated by HS-CTD kinase activity, ERK1, ERK2, and p44 within the CTD motif by using hepta-2 peptides. Extracts from control (C) or sodium arsenite-treated (As) cells were assayed for phosphorylation of hepta-2 or hepta-2 modified peptides (0.4 mg/ml). Serines 4 (A4), 8 (A8), and 11 (A11) were successively substituted by alanine residues. In peptide A4 A11, both serines 4 and 11 were substituted. Phosphorylated peptides were subjected to electrophoresis on 22% denaturing polyacrylamide gels.



These experiments show that only 1 of the serines found in a serine-proline sequence present in the CTD motif is a potential site of phosphorylation by HS-CTD kinase activity.

HS-CTD Kinase Present in HeLa Cells and MAP Kinases Share a Very Similar Consensus Sequence

Another set of peptides was used to determine the consensus sequence phosphorylated by HS-CTD kinase. Their sequence differs from the motif SPTSPSY by one amino acid. All of the amino acids surrounding the phosphorylated serine were replaced or moved. Fig. 3A shows the phosphorylation of these different peptides by HS-CTD kinase from HeLa cells, analyzed as described previously. When proline 5 was replaced by alanine (A5 peptide), HS-CTD kinase activity was no longer able to phosphorylate the peptide, demonstrating that proline is indispensable. This classifies HS-CTD kinase as a member of the serine-proline kinase family which absolutely requires the presence of a proline on the COOH-terminal side of the phosphorylated residue. By moving proline 2 to position 1 or 3 (P3 or P1 peptide), phosphorylation was completely lost. Moreover, the peptide Ser-Thr-Ser-Pro-Ser-Tyr was not phosphorylated by HS-CTD kinase (data not shown). This indicates that proline 2 is also absolutely required. Phosphorylation of peptide A7, in which tyrosine is substituted by an alanine, was weak compared with hepta-1 phosphorylation, showing that this tyrosine must be present for optimal phosphorylation of the CTD motif. Position 3 was substituted with three amino acids: leucine, glutamic acid, and arginine. Phosphorylation by HS-CTD kinase was different with each peptide. The highest phosphorylation was obtained with leucine, then arginine, hepta-1 (threonine), and glutamic acid, which is a poor substrate compared with the others (Fig. 4).


Figure 3: Nature of the site phosphorylated by HS-CTD kinase (A) and ERK2 (B). A third set of synthetic peptides was synthesized. Amino acids surrounding the phosphorylated residue were substituted or displaced in the hepta-1 motif to determine their importance in the recognition of the peptide. The different peptides were assayed with partially purified HS-CTD kinase (A) or ERK2 (B). Phosphorylated peptides were analyzed as in Fig. 1.




Figure 4: Influence of different substitutions at position 3 of the CTD motif on the rate of substrate phosphorylation by HS-CTD kinase activity, ERK1, ERK2, and p44. Threonine at position 3 was replaced by leucine, glutamic acid, or arginine to determine the influence of this residue which precedes the phosphorylated amino acid. Phosphorylation assays were performed as described under ``Materials and Methods'' with DEAE-enriched HS-CTD kinase activity, ERK1, ERK2, or p44. The amount of phosphorylated (SPLSPSY) peptide was arbitrarily set to 100 in order to compare the relative activity of the different kinases on the four peptides. The activity was quantified with a PhosphorImager (see ``Materials and Methods'').



Identical experiments were performed with ERK1, ERK2, and p44. Fig. 3B illustrates the phosphorylation of the same peptides by ERK2. The results obtained with MAP kinases appear very similar to those obtained with HS-CTD kinase activity. As shown with peptide A5, removal of the COOH-terminal proline is critical for phosphorylation: MAP kinases are SP kinases. The results with the P1 and P3 peptides show that a proline on the NH-terminal side is also very important. Substitution of tyrosine 7 by an alanine decreased the level of phosphorylation by these kinases. Substitution at position 3 led to different levels of phosphorylated peptides according to the type of amino acid used to replace threonine. The highest levels of phosphorylation were obtained with leucine whereas the peptide containing glutamic acid at position 3 was a poor substrate. Identical data were obtained with the two other MAP kinases (data not shown).

These results are in agreement with the reported consensus sequence of ERK1, ERK2, and p44, described as -P-X-(S/T)-P- (28, 29). In some cases, when whole proteins were used as substrates, the proline at position -2 was not absolutely necessary, in which case the minimal consensus sequence was then -(S/T)-P- (34, 35) . In order to obtain optimal phosphorylation, X must be basic or neutral for ERK2 and ERK1, whereas p44 has been shown to tolerate an acidic residue at this position (27) . The effect resulting from the substitution of this amino acid would enable distinction between different members of the MAP kinase family. However, this was not the case in our hands. When these three MAP kinases were compared with HS-CTD kinase using four peptides containing different amino acids at this position (threonine (hepta-1 without any substitution), leucine, glutamic acid or arginine (Fig. 4)), the three MAP kinases exhibited the same relative activity as HS-CTD kinase.

Threonine and Serine Are Phosphorylated Differentially by HS-CTD Kinase Activity Extracted from HeLa Cells and MAP Kinases

Fig. 3A shows that HS-CTD kinase activity was able to phosphorylate threonine 4 of peptide SPTTPSY with the same efficiency as serine 4 of the hepta-1 peptide. The three MAP kinases are also serine-threonine kinases, but threonine is a poorer phosphate acceptor than serine (29) , as shown in Fig. 3B. Phosphorylation reactions were performed with different concentrations of peptide SPTSPSY or SPTTPSY (Fig. 5). These experiments showed that a serine-containing peptide is a better substrate than a threonine-containing peptide for ERK1 and ERK2 whereas HS-CTD kinase activity seems to phosphorylate threonine as efficiently as serine within the CTD motif. The high concentration of peptide necessary to saturate the enzymes makes it difficult to determine whether the difference between these protein kinases results from their different affinity for these amino acids or from their differential catalytic efficiency.


Figure 5: Serine 4 and threonine 4 of hepta-1 peptides are differentially phosphorylated by HS-CTD kinase and MAP kinases. Two hepta-1 peptides, with a serine () or a threonine () at position 4, were compared as substrates for HS-CTD kinase activity from HeLa cell crude extracts, ERK1 and recombinant ERK2. Different concentrations of peptides were used (0.4-4 mg/ml, final concentrations). Protein kinase activities are reported in arbitrary units (au).



Separation of HS-CTD Kinase Activities by Chromatography on Mono Q: Comparison with the CTD Kinase Activities Induced by Serum Addition

Mono Q chromatography is currently used to purify MAP kinases. We chromatographed HeLa cell extracts stimulated by heat shock or sodium arsenite on Mono Q. Each fraction was assayed with hepta-4 peptide, and we obtained five separate peaks of hepta-4 kinase activity induced after a stress treatment, one which eluted in the flow through (corresponding to 40% of total proteins) and the four others at 100 mM NaCl (peak I, 2% of total proteins), 175 mM (peak II, 1%), 200 mM (peak III, 1%), and 550 mM (peak IV, 5%) (Fig. 6). The activity found in peak I was low but always present. Extracts from serum-stimulated cells were also analyzed on Mono Q chromatography. All fractions were assayed with hepta-4 peptide, and only three separate hepta-4 kinase activities were observed, one which eluted at 175 mM, another at 200 mM NaCl, and the third at 550 mM NaCl, corresponding to peak II, peak III and peak IV of HS-CTD kinases (Fig. 7).


Figure 6: Separation of several HS-CTD kinase activities on Mono Q. Control cell extracts (), arsenite-treated (), or heat-shocked () cell extracts containing an equal amount of proteins (8 mg) were chromatographed on Mono Q as described under ``Materials and Methods.'' After a 10-ml wash, the columns were eluted with a 30 ml 0 to 300 mM NaCl gradient, followed by a second gradient from 300 mM to 1 M NaCl (- - - - -) and assayed for CTD kinase activity with the hepta-4 peptide (0.4 mg/ml). Phosphorylated peptides were analyzed by SDS 15% polyacrylamide gel electrophoresis and quantified with PhosphorImager (au, arbitrary units). The experiments with arsenite and heat- shock were repeated three times, and the curve shown is an average of these experiments. Five induced CTD kinase activities were resolved, one in the flow-through (FT), the second eluted at 100 mM NaCl (peak I), two others at 175 mM (peak II), and 200 mM (peak III) and the fifth at 550 mM (peak IV).




Figure 7: Separation of CTD kinase activities induced by serum on Mono Q. Control () or serum-treated () cell extracts containing an equal amount of proteins (8 mg) were chromatographed on Mono Q as described under ``Materials and Methods.'' Chromatography and analysis conditions were the same as described for stressed cells in Fig. 6. The curve shown is an average of three experiments. Two major induced CTD kinase activities were resolved, one which eluted at 175 mM NaCl and the other at 200 mM NaCl. A minor induced activity was also eluted at 550 mM NaCl.



Characterization of the MAP Kinases by Western Blotting

Immunoblotting with antibodies raised against both ERK1 and ERK2 revealed two species in control cell extracts. Their elution was centered around fraction 36 for the higher mobility form (p42 ERK2) and fraction 37 for the other one (p44 ERK1) (Fig. 8Aa). In serum-treated cell extracts four species were observed: the two forms present in control cell extracts, corresponding to the inactive MAP kinases, and two additional forms. One of them had intermediate mobility and was centered around fraction 37 whereas the other migrated like p44 ERK1 and was centered around fraction 41 (Fig. 8Ab). These two additional species were eluted exactly at the same position as the two major hepta-4 activities induced by serum (Fig. 7). Gomez and Cohen (36) demonstrated previously that the inactive forms of MAP kinases were eluted before the active forms on Mono Q. MAP kinases are activated by a double phosphorylation, and this could explain the difference in elution and/or migration between the active forms and the inactive forms. Immunoblotting of Mono Q fractions from stressed cell extracts showed the same four species of MAP kinases with identical electrophoretic migration and similar conditions of salt elution (Fig. 8Ac). The presence of modified forms of ERK2 (p42) and ERK1 (p44) corresponds precisely to the hepta-4 kinase activity of peaks II and III (Fig. 6). Immunoreactivity was not detected in flow-through or peaks I and IV (Fig. 8B), confirming that these fractions do not contain MAP kinases. Activated forms of MAP kinases were less abundant than inactive forms: the induction of MAP kinase activity by stress in the conditions we used was weaker than their induction by serum.


Figure 8: Characterization of the MAP kinases by Immunoblotting of Mono Q fractions with anti-ERK1-ERK2 antibody. A, Mono Q fractions 32-44 were analyzed by SDS gel electrophoresis, transferred to nitrocellulose, and immunoblotted with an anti-ERK1-ERK2 antibody (Santa Cruz Biotechnology). a, control cell extracts; b, serum-treated cell extracts; c, sodium arsenite-treated cell extracts. B, the fractions corresponding to the different peaks of hepta-4 kinase activity induced by arsenite were collected, electrophoresed, and analyzed by immunoblotting with the anti-ERK1-ERK2 antibody.



Mono Q chromatography allows separation of five hepta-4 kinase activities induced after stress treatments, only two of which are due to MAP kinases ERK1 and ERK2.

Substrate Specificity of the Five Hepta-4 Kinase Activities Separated by Mono Q

Fig. 5 illustrates that the specificity of the CTD kinase activity contained in DEAE-enriched or crude extracts differs from that of ERK2. As far as the nature of the phosphorylated residue is concerned, ERK1 and ERK2 phosphorylate serine better than threonine at position 4 whereas HS-CTD kinase activity phosphorylates both equally well. Interestingly, when these same peptides were assayed with the different activities isolated on the Mono Q column, two opposite specificities were observed (Fig. 9). The activities contained in the flow-through and in peak I phosphorylated threonine better than serine. Peak II and peak III activities, which have been shown to be due to MAP kinases, actually phosphorylated serine better than threonine, as described previously in Fig. 5 for recombinant ERK2 and ERK1. Peak IV activity had an intermediate behavior. Therefore the specificity of HS-CTD kinase observed in crude extracts or DEAE-enriched fractions resulted from the addition of opposite specificities of at least five different protein kinases.


Figure 9: The HS-CTD kinase activities present in the flow-through, peak I, and peak IV fractions have a substrate specificity different from ERK1 and ERK2. SPTSPSY () and SPTTPSY () hepta-1 peptides (1.6 mg/ml) were assayed with the different CTD kinase activities eluted from the Mono Q column. Phosphorylated peptides were separated by electrophoresis on denaturing phosphate buffer gels. The amount of phosphorylated (SPTSPSY) peptide was arbitrarily set to 1.




DISCUSSION

Characterization of the Residue Phosphorylated by HS-CTD Kinase Activity within the CTD Motif

We designed an experimental system to follow the phosphorylation reaction with only one repeat of the motif. With it, we demonstrated that HS-CTD protein kinase activity exclusively phosphorylates serine 4 within the CTD motif (S P T S P S Y). This result was confirmed by performing the same analysis with hepta-2 peptides. Protein kinases from the MAP kinase family (ERK1, ERK2, and p44 ) also exclusively phosphorylate serine 4 in the same motif. In fact, serine 4 is in an environment already shown to be favorable to phosphorylation by MAP kinases (see Ref. 37 for review).

We therefore decided to compare the properties of the HS-CTD kinase activity with the properties of the major MAP kinases ERK1 (p44) and ERK2 (p42).

Substrate Specificity of the HS-CTD Kinase Activity and Comparison with the Specificity of Different Members of the MAP Kinase Family

By successively replacing the amino acids surrounding the phosphorylated serine in the CTD motif, we deduced the following consensus sequence for phosphorylation by HS-CTD kinase: -P-X-(S/T)-P-. This sequence is identical to that established for ERK1 and ERK2 with peptides based on the primary sequence of the epidermal growth factor receptor (29) . No differences were found between the activity of ERK1, ERK2, and p44 on the different peptides. In the case of p44, this is rather surprising. This protein kinase has been purified from maturing sea star oocytes (38) . It is highly homologous to ERK1 and ERK2 and shares a similar consensus sequence phosphorylation site -P-X-(S/T)-P- (25) . However X must be neutral or basic for ERK1 and ERK2 whereas p44 has been suggested to tolerate every amino acid except a proline at this position. Our data show that a peptide containing a glutamic acid instead of a threonine at position 3 is a poor substrate for HS-CTD kinase, ERK1, ERK2, but also for p44. Previous results concerning p44 had been obtained with a peptide substrate (APRTPGGRR) derived from the myelin basic protein sequence, which is different from the one used here. The nature of the phosphorylated residue or of the amino acids surrounding this phosphorylated residue may interfere with substrate recognition and explain the difference between our results and those reported in the literature.

HS-CTD Kinase Activity Appears to be Different from the Major Members of the MAP Kinase Family

A closer examination of the results reveals a clear difference between HS-CTD kinase and MAP kinases: phosphorylation reactions with hepta-1 peptide or with a peptide in which serine 4 has been substituted by a threonine show that serine is a better phosphate acceptor than threonine for ERK1, ERK2, and p44 whereas DEAE-enriched HS-CTD kinase activity phosphorylates threonine as well as serine within the CTD motif. These results show that threonine and serine are recognized differentially by HS-CTD kinase and MAP kinases.

Separation of the Different HS-CTD Kinase Activities by Mono Q Chromatography

Five different induced CTD kinase activities were separated by Mono Q chromatography, one in the flow-through, one which eluted at 100 mM NaCl (peak I), two others at 175 mM and 200 mM (peak II and III), and one at 550 mM NaCl (peak IV). Peak I activity was very low but the induction was always present. In contrast, two hepta-4 activities were strongly induced by serum corresponding exactly to the positions of peak II and peak III. Another activity was induced to a minor extent and corresponded to peak IV. Immunoblotting with a broad specificity antibody targeted against ERK1 and ERK2 revealed the inactive forms of ERK1 and ERK2 and the activated forms which eluted slightly later on the Mono Q column in our experimental conditions. Peak II corresponds to the fractions containing the activated form of ERK2 (p42) and peak III to the activated form of ERK1 (p44). Regarding specificity, peak II and peak III activities reproduced recombinant ERK2 and ERK1 behavior since serine at position 4 of the CTD motif was more intensively phosphorylated than threonine at the same position. In contrast, the CTD kinase activity present in the flow-through and peak I phosphorylated the motif found in the carboxyl-terminal domain of RNA polymerase II with a higher efficiency when serine was replaced by threonine. Peak IV activity was indifferent to the nature of the phosphorylated residue. Interestingly, we previously observed that the HS-CTD kinase activity present in crude or DEAE-purified stressed cell extracts had equal activity on the two peptides. This confirms that HS-CTD kinase activity is a mixture of different protein kinases activated by stress treatment, two of which are MAP kinases while the others are different and have different properties. The properties of the crude HS-CTD kinase activity result from the combination of the different isolated activities.

A significant proportion of the HS-CTD kinase activity is due to protein kinases distinct from the major members of the MAP kinase family, ERK1 and ERK2. Activation of MBP and MAP kinases by heat-shock has already been observed in Chinese hamster cells (CCL39) (4) , human cells (HeLa), mouse cells (3T3), and rat cells (H56) (8) . However, the increase in MBP kinase activity and the kinetics of activation of ERK1 and ERK2 seemed to be poorly correlated in these experiments: after 15 min of a 45 °C shock, MBP kinase activity had already reached its highest level whereas ERK1 was unchanged compared to control cells and ERK2 had only very partially shifted toward the active phosphorylated form. This discrepancy can now be explained since HS-CTD kinase (and MBP kinase) activity is only partially due to the MAP kinases. It would be interesting to see whether the induction of the different protein kinase activities follows the same kinetics. It should also be noted that the induction of MAP kinases by stress, although contributing to a significant fraction of the CTD kinase activity, remains limited (also see Ref. 39 for a similar conclusion). Immunoblotting experiments show that the activated forms of ERK1 and ERK2 are minor species in comparison with the inactivated forms. The level of activation of these MAP kinases by stress in the conditions we use is weaker than their level of activation by serum.

The Molecular Nature of the HS-CTD Kinase Activities

Three HS-CTD kinase activities distinct from ERK1 and ERK2 contribute to the activity present in crude extracts. These three activities displayed substrate specificities distinct from that of the main MAP kinases. Two questions remain open. What is the nature of these three CTD kinases? They can be distinguished from the major MAP kinases by enzymatic, but also immunological and chromatographic criteria. Are they due to the distantly related members of the MAP kinase family which seem to be involved in parallel pathways of intracellular signaling? Recently, new protein kinase families induced by cellular stresses have been described. Hog kinase is induced by heat and sodium arsenite and phosphorylates in vivo MAPKAP kinase 2 (7) . The SAPKs family contains at least four members (I, II, , ) (39) which are distant relatives of the MAP kinase group. JNK1 (c-Jun NH-terminal kinase 1), a protein kinase described as being stimulated by UV irradiation (40) , is a member of SAPKs family. Proline at position -2 is not required for phosphorylation by the SAPKs (34, 40, 41) . Consequently, HS-CTD kinase activities present in the flow-through, peak I and peak IV do not seem to be members of the SAPKs family, although the site specificity of the SAPKs has not been determined with the CTD sequence. We also showed previously that HS-CTD kinase is insensitive to protein synthesis inhibitors (9) , whereas SAPKs are activated by cycloheximide treatment.

These CTD kinases might also be related to the previously characterized CTD kinases. However, we previously demonstrated that the stress-induced CTD kinase activities do not contain a cdc2 subunit as do some other CTD kinases (9) . They are also distinct from the CTD kinase activity tightly associated with RNA polymerase II: the substrate specificity of the protein kinase activity present in the subunit is clearly different from the specificity of stress-induced protein kinases.() The nature of these three HS-CTD kinase activities therefore remains unknown at present.

The physiological meaning of the induction of these protein kinase activities also remains unclear. It has been shown that RNA polymerase II is over-phosphorylated during stress in some cells (42) . However, the fact that MAP kinases can be also considered as CTD kinases suggests that the new HS-CTD kinases characterized here might have very different physiological substrates.


FOOTNOTES

*
This work was supported by grants from the Université Paris VI. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33-1-44-32-39-45. Fax: 33-1-44-32-39-41.

The abbreviations used are: MAP, mitogen-activated protein; CTD, carboxyl-terminal domain; MOPS, 4-morpholinopropanesulfonic acid.

H. Serizawa and S. Trigon, unpublished results.


ACKNOWLEDGEMENTS

We are very indebted to Pr. P. Cohen for the gift of recombinant ERK2 protein and fruitful discussions. We thank R. Sousa-Yeh and P. Murphy for critical reading of the manuscript, M. Schapira for help with the fast protein liquid chromatography method, and S. Dimon and G. Keryer for help in immunoblotting experiments.


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