From the
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.
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
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)
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),
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)
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
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.
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.
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.
These results are in agreement with the reported
consensus sequence of ERK1, ERK2, and p44
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).
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.
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
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.
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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
kinases
(6) but also in vivo by a minor
form
(7) . Interestingly, MAP kinases are activated after
heat-shock
(8) .
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.
in
rat
(21) , and TFIIH in human cells
(24) .
. 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) .
. 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.
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.
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.
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) .
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.
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).
,
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.
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).
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.
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.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.