From the Service de Biochimie et Génétique Moléculaire, CEA/Saclay, Gif sur Yvette 91191, France
Received for publication, November 8, 2000
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ABSTRACT |
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RNA polymerase II CTD kinases are key elements in
the control of mRNA synthesis. They constitute a family of
cyclin-dependent kinases activated by C-type cyclins.
Unlike most cyclin-dependent kinase complexes, which are
composed of a catalytic and a regulatory subunit, the yeast CTD kinase
I complex contains three specific subunits: a kinase subunit (Ctk1), a
cyclin subunit (Ctk2), and a third subunit (Ctk3) of unknown function
that does not exhibit any similarity to known proteins. Like the Ctk2
cyclin that is regulated at the level of protein turnover, Ctk3 is an
unstable protein processed through a ubiquitin-proteasome pathway.
Interestingly, Ctk2 and Ctk3 physical interaction is required to
protect both subunits from degradation, pointing to a new mechanism for
cyclin turnover regulation. We also show that Ctk2 and Ctk3 can each interact independently with the kinase. However, despite the formation of CDK/cyclin complexes in vitro, the Ctk2 cyclin is unable
to activate its CDK: both Ctk2 and Ctk3 are required for Ctk1 CTD kinase activation. The different specific features governing CTDK-I regulation probably reflect requirement for the transcriptional response to multiple growth conditions.
The C-terminal domain
(CTD)1 of the large subunit
of RNA polymerase II contains a repeated heptapeptide that is highly
phosphorylated in a portion of the molecules in the cell. This domain
plays an essential role in the control of mRNA synthesis, as well
as RNA processing, and its phosphorylation is a key feature of its
function (1). The formation of initiation complexes on promoter DNA involves RNA polymerase II molecules with unphosphorylated CTD, whereas
the CTD becomes phosphorylated during or after the transition to
elongation. Three CTD kinases are involved in the regulation of RNA
polymerase II transcription in the yeast Saccharomyces cerevisiae, all being cyclin-dependent kinases (CDKs).
The first yeast CTD kinase, Kin28 (2), is associated with cyclin Ccl1
(3) and is required for transcription of most genes (4). This complex,
like its human counterpart the Cdk7 kinase complex (5), is a component
of the general transcription factor TFIIH (6, 7). Kin28 is an essential
kinase that has been shown to phosphorylate the CTD only after the RNA
polymerase II is associated with promoter DNA, thus promoting the
elongation mode (8). Kin28 is also required for proper capping enzyme targeting in vivo (9). The second yeast CDK implicated in
CTD phosphorylation, the nonessential Srb10/Ume5/Ssn3 kinase and its cyclin partner Srb11/Ume3/Ssn8, is a component of the holoenzyme (10). The Srb10/11 complex is structurally related to mammalian Cdk8
kinase and its associated cyclin C (11). Several studies established
that the Srb10 kinase is implicated in the transcriptional repression
of specific sets of genes (4, 12-15). Hengartner et al. (8)
showed that Srb10 is uniquely capable of phosphorylating the CTD prior
to formation of the initiation complex on promoter DNA, with consequent
inhibition of transcription. Srb10 has also been implicated in Gal4
phosphorylation, thus modulating its activity (16).
The CTDK-I complex has not been found in the holoenzyme. It was
isolated by its ability to phosphorylate CTD-containing fusion proteins
(17). It is composed of three nonessential subunits: the
CTK1 and CTK2 genes encode a kinase and a cyclin
respectively, whereas the third subunit, Ctk3, does not exhibit any
similarity to known proteins (18, 19). In vitro studies
carried out in HeLa nuclear extracts have shown that CTDK-I can
modulate the elongation efficiency of RNA polymerase II (20).
Patturajan et al. (21) reported that deletion of the
CTK1 gene results in an increase in phosphorylation of
serine in position 5 of the CTD repeat, indicating that CTDK-I
negatively regulates CTD Ser5 phosphorylation.
CTK1 deletion also eliminates the transient increase in CTD
serine 2 phosphorylation observed during the diauxic shift, this latter
defect being correlated with a defect in transcriptional induction
during the diauxic shift, suggesting a role for CTDK-I in response to
nutrient depletion. In humans, the closest kinase to Ctk1 is Cdk9 (for
review, see Ref. 22), a CTD kinase that was first described as a
positive transcription elongation factor (23), and that interacts
specifically with the human immunodeficiency virus-1 transactivator
protein Tat, which acts to enhance the processivity of RNA polymerase
II (24, 25).
How are these CTD kinases regulated? CDKs were first described as
central regulators of the major transitions of the eukaryotic cell
division cycle. Their activity is determined by cyclin binding, by both
positive and negative regulatory phosphorylation and by CDK inhibitors
(CKIs) (26, 27). An important aspect of CDK regulation is the rapid
destruction of their cyclin partners (28). Several studies both in
yeast and in mammals have demonstrated that cell cycle cyclins are
selectively degraded by ubiquitin-dependent proteasome
pathways (29-32). Although exceptions have been described (33, 34),
proteasome substrates are usually marked for degradation by conjugation
with several molecules of ubiquitin, a highly conserved 76-amino
acid-long polypeptide (33, 35). The 26 S proteasome is a nuclear and
cytosolic multicatalytic proteinase that breaks down targeted
substrates to short peptides and recycles the ubiquitin molecules (36).
CDKs involved in transcriptional control are activated by C-type
cyclins that constitute a divergent group of cyclins (37, 38). C-type
cyclins exhibit sequence conservation for the cyclin box,
i.e. the kinase binding domain (39, 40), but their
expression does not seem to fluctuate during the cell cycle (41, 42),
and little is known about their regulation. Cdk8·cyclin C
phosphorylates cyclin H, resulting in down-regulation of TFIIH
activities (43). In yeast, it has been shown that the C-type cyclin
Srb11/Ume3 is regulated at the level of its turnover. Srb11/Ume3 is
destroyed by multiple and independent pathways when cultures are
subjected to diverse stresses (42, 44). We previously showed that the
Ctk2 cyclin subunit is phosphorylated and rapidly degraded, in a
growth-related fashion, by a ubiquitin-proteasome pathway (45).
Interestingly, unlike what has been described for the G1
cyclins, neither Ctk2 phosphorylation nor its destruction require its activated kinase.
CDK complexes are minimally composed of a catalytic and a regulatory
subunit. The vertebrate TFIIH kinase contains an additional component,
Mat1, that exhibits a zinc-binding motif typical of C3HC4 RING finger
domains likely to mediate protein·protein interactions (46). Mat1
functions as an assembly factor, promoting a stable interaction between
the Cdk7 kinase and the cyclin H that does not require Cdk7-activating
phosphorylation (47-49). Also, association with Mat1 and core TFIIH
modulates the activity of Cdk7 at the level of substrate specificity
(50). Although the yeast TFIIH complex contains Tfb3 that is 32.5%
identical to Mat1 (51, 52), a Kin28·Ccl1·Tfb3 complex has never
been identified (53). However, Tfb3 has been implicated in
transcription (51), and conditional alleles of TFB3 are
severely defective in Kin28T132A kinase activity,
suggesting that it might be the Mat1 ortholog (54).
The function of the third component of the CTDK-I complex is unknown.
The fact that deletion of each individual CTK gene generates cold-sensitive cells that display similar growth defects (19), suggests
that Ctk3 is essential for the function of the complex. We studied Ctk3
expression in vivo, and we show that it is regulated at the
protein level. Ctk3 contains a PEST motif involved in its degradation
by the ubiquitin-proteasome pathway. Furthermore, binding of Ctk3 to
the cyclin subunit is required for stabilization of both subunits. Ctk3
can directly interact with the kinase and is required, in addition to
the cyclin, for its activation. It thus appears that, in contrast to
other CDKs, the Ctk1 kinase is in fact regulated by a heterodimer
composed of two unstable subunits.
Yeast Strains and Growth Conditions--
The yeast strains used
in this study were WCG4a (MATa ura3 leu2-3,
112 his3-11, 15) and its isogenic pre1-1
pre2-2 derivative WCG4-11/22a constructed by Richter-Ruoff
et al. (55). The Plasmids--
For Ctk2 overexpression, the pFL44-CTK2 plasmid
was used (URA3 2 µm) (45). All the constructs were
made via several PCR cloning steps. Briefly, 500-bp PCR fragments
containing either the CTK1 or the CTK3 promoter
were cloned into pFL36 (LEU2 CEN) (57). The different coding
sequences were cloned downstream of sequences encoding two HA
influenza virus hemagglutinin tags or a single c-Myc epitope
tag prior to cloning downstream of their cognate promoter, yielding to
the final constructs CTK1-HA, CTK1-Myc, CTK3-HA, and CTK3-Myc. The
CTK2-HA construct (URA3 CEN) is described in Hautbergue and
Goguel (45). The Myc-tagged CTK2 coding sequence was
inserted into pFL38-PrCTK2 (45), yielding the CTK2-Myc
construct. The CTK3 Northern Blots Analyses--
Total RNA was extracted from 15 ml
of cells by hot acid-phenol treatment as described (59). 30 µg were
separated on a 1% agarose gel containing 6% formaldehyde, blotted to
a nylon membrane (Hybond N+, Amersham Pharmacia Biotech), and rRNA was
quantified by methylene blue staining. Hybridization to a PCR fragment
containing the CTK3 coding sequence labeled with
[ Yeast Cell Extracts and Western Immunoblotting--
Proteins
were extracted in trichloroacetic acid by mechanical lysis with glass
beads and analyzed by Western immunoblotting as previously described
(45).
Detection of Ctk3 Ubiquitin Conjugates--
Buffers described
previously by Willems et al. (63) were supplemented with
15% (v/v) glycerol. Yeast pellets from 1.5 liters of cultures, with or
without induction of the CUP1 promoter, were resuspended in
G buffer and subjected to cellular lysis in an Eaton press. After
ultracentrifugation at 100,000 × g for 1 h, total
ubiquitinated species from 10 mg of yeast proteins were loaded onto
50-µl nickel columns (Amersham Pharmacia Biotech) packed into
micro-tips with G buffer. After elution, ubiquitinated species were
analyzed by Western immunoblotting.
Bacterial Extracts--
Escherichia coli BL21(DE3)
cells (Novagen) were cotransformed with the pLysS vector (Novagen) and
the plasmid coding for the protein of interest. Typically, cells were
grown to an A600 of 0.4 in 100 ml of LB medium.
Expression of the T7 polymerase was then induced for 3 h with 250 µM isopropyl-1-thio- Coimmunoprecipitation Experiments--
Immunoprecipitations from
yeast extracts were performed as described previously (45). Briefly,
proteins extracted with glass beads from 10 ml of cells were subjected
to immunoprecipitation with polyclonal antibody HA.11 (1/125, Babco)
and protein G-immobilized on Sepharose beads (Amersham Pharmacia
Biotech). Immunoprecipitations from bacterial or wheat germ extracts
were performed in the BB100 buffer described above. Bacterial extract
containing Myc-Ctk3 and HA-Ctk2 synthesized with
[35S]methionine in coupled transcription/translation
wheat germ extract (Promega) were mixed overnight at 10 °C.
Immunoprecipitations were performed with HA-12CA5 antibody coupled to
20 µl of magnetic beads according to the manufacturer (Dynal). After
a 2-h incubation on a wheel, proteins were eluted with Laemmli buffer
and separated by SDS-PAGE prior to Western immunoblotting.
Pulse-chase Experiment--
50 ml of yeast cells transformed
with the CTK3-HA construct were grown in SD medium to an
A600 of 0.4. Cell labeling was achieved for 10 min at 30 °C with 1 mCi of Pro-Mix
[35S]methionine/cysteine (Amersham Pharmacia Biotech).
After different times of chase by addition of 10× chase medium (10 mM methionine, 2 mM cysteine, 4% yeast
extract, 25% glucose), 10-ml aliquots were added to 60 µl of STOP
solution (0.5 M sodium azide, 0.5 M sodium
fluoride) and centrifuged. Pellets were washed with water prior
to freezing in liquid nitrogen. Protein extracts and
immunoprecipitations were made following the in vivo
immunoprecipitation procedure described above. Totality of eluted
proteins were separated by SDS-PAGE before analysis by autoradiography.
Reconstitution and Immunopurification of Ctk
Complexes--
Equal amounts of different bacterial extracts each
containing a tagged protein were mixed and incubated for an hour at
15 °C. 15 µg of purified monoclonal HA-12CA5 antibodies were
coupled overnight at 10 °C to 30 µl of protein G-Sepharose beads
in phosphate-buffered saline buffer containing 0.1% to 0.5% bovine
serum albumin and 10% (v/v) glycerol. Typically, 30 µl of HA-coupled
beads were packed into micro-tips. After equilibration with BB100
buffer, the different mixes of proteins were loaded three times
consecutively on these mini-columns with one wash in between each.
Native elutions of immunopurified complexes were performed in batch
with 50 µl of BB100 buffer supplemented with 0.1 µg/µl HA peptide
for 20 min at 37 °C. Extracts were either analyzed by SDS-PAGE or
Western immunoblotting after addition of Laemmli buffer.
Gel Filtration Analysis--
Bacterial extracts containing
HA-Ctk2 and Myc-Ctk3, respectively, were mixed and immunopurified on an
HA mini-column as described above. 5 µl of bacterial extracts
containing either HA-Ctk2 or Myc-Ctk3, or 50 µl of Ctk2·Ctk3
immunopurified complex, were applied on Superdex 75 PC 3.2/30 columns
(SMART system, Amersham Pharmacia Biotech). The flow rate during the
chromatography was 40 µl/min of BB100 buffer, and at 0.95 ml of
elution, 30-µl fractions were collected and analyzed by Western Immunoblotting.
Purification of Recombinant Ctk Complexes--
Purification of
high amounts of Ctk complexes was performed by ion exchange and
affinity chromatographies on a fast protein liquid chromatography
system (Amersham Pharmacia Biotech). Bacterial extract from 500 ml of
cells expressing (His)6-HA-Ctk1 was loaded onto a 2-ml
Bio-Rex 70 column (Bio-Rad) with PC In Vitro CTD Kinase Assays--
Extracts from High5 cells
(Invitrogen) infected with baculoviruses expressing the GST-CTD fusion
protein (a gift from J. Acker) were treated with the PP2A phosphatase
prior to inhibition with okadaic acid and loaded onto a
Hi-TRAP/glutathione column (Amersham Pharmacia Biotech). GST-CTD
elution was achieved by competition with 20 mM reduced
glutathione in BB100 buffer. Activities from 40 µl of each purified
complex (fraction 7, Fig. 7C), or Ctk1 alone,
were assayed by incubation of 2 µg of GST-CTD with 3 µCi of
[ Ctk3 Is an Unstable Protein--
The subunits of the yeast CTDK-I
complex are encoded by three genes: CTK1, encoding a
cyclin-dependent kinase subunit; CTK2, encoding
a C-type cyclin subunit; and CTK3, encoding a 296-amino acid
subunit of unknown function. We have previously shown that Ctk2 is
processed through a ubiquitin-proteasome pathway (45). To analyze Ctk3
expression in vivo, its coding sequence was cloned on a
single-copy plasmid under the control of its own promoter. The protein
was fused at its N-terminal to either a c-Myc or two-HA epitope tags.
The resulting constructs, CTK3-Myc and CTK3-HA, allowed expression of
functional proteins, because each could restore
A number of proteins with a fast turnover contain PEST sequences (60).
PEST regions are enriched in proline, glutamate, serine, threonine, and
aspartate, these regions being uninterrupted by positively charged
residues and flanked by lysine, arginine, or histidine residues (61).
Several studies have shown that these peptide motifs are involved in
protein turnover. Examination of Ctk3 sequence with the PESTfind
algorithm revealed a strong potential PEST sequence (score of +14.41)
composed of a stretch of 18 amino acids located at the N terminus of
the protein (Fig. 1D). We made a 13-amino acid deletion
inside the potential PEST motif of the Myc-Ctk3 protein. The resulting
protein, Ctk3 Ctk3 Is Degraded by a Ubiquitin-dependent Proteasome
Pathway--
In eukaryotic cells, PEST sequences have been implicated
in ubiquitination processes required for vacuolar degradation (62), as
well as degradation by the proteasome (36). Biochemical and genetic
evidence indicates that the ubiquitin-proteasome pathway is involved in
the degradation of abnormal proteins and regulatory proteins with
naturally short half-lives, such as cell cycle cyclins (35), and the
C-type cyclin Ctk2 (45). To establish whether Ctk3 is a substrate for
the proteasome degradation pathway, we examined its stability in the
conditional pre1 pre2 mutant strain, which is
defective in two of the proteasome catalytic subunits at 37 °C (55).
The CTK3-Myc construct was introduced into pre1 pre2 and
wild type isogenic cells. Cell cultures were shifted to the
nonpermissive temperature to block proteasome function and analyzed for
Ctk3 levels after cycloheximide addition. In wild type cells, Ctk3
turnover was slightly different from that previously observed (Fig.
2A; half-life, 15 min),
indicating that, like Ctk2 turnover (45), it is dependent on genetic
background and/or temperature. In pre1 pre2 cells, Ctk3
levels remained almost unchanged for 6 h after cycloheximide
addition (Fig. 2A). In conclusion, Ctk3 is stabilized in a
mutant strain that exhibits defects in the activity of the proteasome,
showing that its degradation is mediated by the 26 S proteasome
pathway.
Although the 26 S proteasome degrades an unknown number of proteins
that are recognized without undergoing ubiquitination, the ubiquitin
system constitutes the major targeting process leading to selective
degradation (33). Despite the fact that Ctk3 was readily observed by
immunodetection, we did not observe any ubiquitinated forms. Indeed,
due to deubiquitination and degradation by the proteasome, steady-state
levels of ubiquitin species are often very low. In an attempt to detect
these forms, wild type cells were cotransformed with the CTK3-HA
construct and a multicopy plasmid expressing a
(His)6-Myc-tagged variant of ubiquitin from the
CUP1 inducible promoter (32). After elution of total
ubiquitin species retained on a nickel column under denaturing
conditions (to avoid the recovering of proteins interacting with Ctk3),
proteins were analyzed by Western immunoblotting. Hybridization with
Myc antibodies allowed detection of equal amounts of ubiquitin species when expression from the CUP1 promoter was induced (Fig.
2B, lower panel). Ctk3 species were further
analyzed by hybridization with HA antibodies (Fig. 2B,
upper panel). We could observe high-molecular-weight bands
corresponding to Ctk3 ubiquitin conjugates. Taken together, our results
demonstrate that Ctk3 is a substrate for a ubiquitin-proteasome pathway.
Ctk3 Turnover Is Influenced by the Ctk2 Cyclin--
The turnover
of cyclins involved in cell cycle progression has been described as
being under the control of their associated CDKs (63, 64). Because Ctk3
is an unstable protein that is part of a CDK complex, we wondered
whether the Ctk1 kinase was implicated in the control of its turnover.
The CTK3-Myc construct was introduced into
We next studied the stability of the Myc-tagged Ctk3 protein in
Because Ctk2 levels seemed to influence Ctk3 turnover, we next examined
the effect of Ctk2 overexpression on Ctk3 stability. When the
CTK2 gene was carried by a multicopy plasmid, Ctk3 was significantly stabilized (Fig. 3E; half-life, 120 min,
compare with Fig. 1A). In conclusion, the data show that
Ctk2 levels strongly influence Ctk3 turnover.
Ctk3 Influences the Ctk2 Cyclin Turnover--
A striking feature
of the CTDK-I complex is that, unlike that of cell cycle cyclins, the
Ctk2 cyclin turnover is not under the control of the kinase that it
activates (45). Ctk3 turnover does not require the Ctk1 kinase but is
dependent on Ctk2 expression. These observations prompted us to study
whether Ctk3 was involved in the control of the Ctk2 cyclin expression.
The CTK2-HA construct that allows expression of a functional
tagged-cyclin from a single-copy vector (45) was introduced into
In conclusion, Ctk2 and Ctk3 mutually control their respective
turnover, a process that is independent of their associated kinase.
Ctk2·Ctk3 Heterodimerization--
To study potential physical
interaction between Ctk2 and Ctk3, we first performed
coimmunoprecipitation experiments from yeast extracts.
To test a direct interaction between these two proteins, constructs
were made to express tagged versions of the Ctk2 and Ctk3 proteins from
the T7 promoter. HA-tagged Ctk2 was synthesized in wheat germ extracts,
whereas Myc-tagged Ctk3 was bacterially expressed. Extracts were
incubated together prior to subjecting the HA-tagged proteins to
immunoprecipitation. We observed that Ctk2 immunoprecipitation resulted
in coimmunoprecipitation of the Ctk3 protein (Fig. 5B),
demonstrating a direct interaction between these two subunits.
To further examine Ctk2·Ctk3 complexes, gel filtration experiments
were performed. Bacterial extracts containing either the HA-Ctk2 or the
Myc-Ctk3 protein were subjected to gel filtration chromatography on a
Superdex 75 column. HA-Ctk2 and Myc-Ctk3 were eluted in fractions whose
apparent molecular mass corresponded to 44 and 33 kDa,
respectively, consistent with their theoretical size (41 and 37 kDa,
respectively) (Fig. 5C, upper and middle panels). In addition, Ctk3 aggregates were found in the void
volume. When both extracts were preincubated and HA-Ctk2 was further
immunopurified prior to loading on the gel filtration column, elution
resulted in the presence of both factors in the same fractions
corresponding to an apparent molecular mass of 78 kDa (Fig.
5C, lower panel). These data demonstrate that,
in vitro, Ctk2 and Ctk3 interact to form heterodimers.
Ctk2·Ctk3 Interaction Is Required for Their Mutual
Stabilization--
Taken together, our data suggest that Ctk2 and Ctk3
turnover are controlled by their heterodimerization. To investigate
further the role of Ctk2·Ctk3 interaction, we constructed several
deletion mutants of the Ctk3
protein.2 We concentrated our
study on a C-terminal 50 amino acid truncation. The resulting protein,
Ctk3 Activation of the Ctk1 Kinase Requires Both Ctk2 and Ctk3
Subunits--
In higher eukaryotes, the third component of the TFIIH
kinase complex has been described as an assembly factor that promotes in vitro the association of the CDK with its cyclin. To
analyze whether Ctk3 is required for Ctk1·Ctk2 complex formation,
constructs were made to express tagged versions of the Ctk1 protein
from the T7 promoter. Different combinations of proteins expressed in
E. coli were mixed to allow protein·protein interaction to occur, prior to immunopurification on HA columns. We observed that the
Ctk1 kinase copurified with the Ctk2 cyclin, regardless of Ctk3
presence (Fig. 7A, compare lane 3 to lane
2). The addition of the third subunit is not required for this
interaction, suggesting that Ctk3 is not an assembly factor in
vitro. Interestingly, we also observed that Ctk1 copurified with
Ctk3 (Fig. 7A, lane 4), showing a direct
interaction between the kinase and the third subunit.
Our results show that the CTDK-I complex contains two unstable subunits
that can both interact independently with the kinase. We thus wondered
whether the cyclin alone, or the third subunit alone, was able to
activate Ctk1. This question was difficult to address in
vivo because when Ctk2 (or Ctk3) is not expressed, as a
consequence, Ctk3 (or Ctk2) is very quickly degraded. To analyze the
CTD kinase activity of different complexes, we reconstituted and
purified Ctk complexes by ion exchange and affinity chromatography (Fig. 7B). The coelution of the HA-tagged subunits was
evidenced by Western immunoblotting (Fig. 7C). The Ctk
proteins contain identical epitope tags, thereby showing identical
reactivity to anti-HA antibodies. They coelute in nearly stoichiometric
amounts within the different complexes. CTD kinase activity was assayed using as substrate a purified GST/human-CTD fusion protein expressed in
insect cells. No contaminant CTD kinase activity was detected with
GST-CTD alone (Fig. 7D, lane 1) or by addition of
protein fractions eluted after purification from an E. coli/empty vector extract (Fig. 7D, lane 2).
When GST-CTD was incubated with the kinase subunit alone, no labeling
was detected (Fig. 7D, lane 4). On the other
hand, CTD kinase activity was recovered when the three subunits were
present (Fig. 7D, lane 3). Interestingly, neither
Ctk2 nor Ctk3 alone could activate Ctk1 (Fig. 7D,
lanes 5 and 6). In conclusion, Ctk2 and Ctk3 can
each interact independently with the kinase, however, both proteins are
required for its activation.
In principle, CDK activation is a process achieved by cyclin
binding and CDK activating phosphorylation. We show in this report that
activation of Ctk1, a CDK involved in the control of RNA polymerase II
transcription, requires its association with two unstable subunits: a
C-type cyclin and a subunit displaying no homology to known proteins.
Interestingly, the turnover of both subunits is controlled by their
direct interaction, pointing to a divergent mechanism for CDK regulation.
Activation of the Ctk1 CDK Requires Its Association with Two
Distinct Subunits--
CDKs are flexible proteins that are regulated
in several different ways. In addition to cyclin binding, their
complete activation is generally achieved by phosphorylation of the CDK
by a CDK-activating kinase. We do not know whether the Ctk1 CDK is
phosphorylated in vivo. However, Ctk1 expressed in E. coli can support CTD phosphorylation, suggesting that, like the
two other yeast CTD kinases (54, 65), this CDK can be active without
itself becoming phosphorylated.
So far, of the three yeast CDKs implicated in RNA polymerase II
transcription control, only CTDK-I has been described as a heterotrimeric complex. We show that Ctk3, the third component of the
CTDK-I complex, is not required for the association of the
unphosphorylated Ctk1 kinase with its cyclin subunit in
vitro. These results suggest that, unlike Mat1, Ctk3 is not an
assembly factor. Also, we observed a direct interaction between Ctk3
and the CDK. In terms of protein interaction, Ctk3 thus behaves like CKIs that have been shown to bind both to cyclin and CDK subunits (for
review see Ref. 66). However, Ctk3 is a specific factor that does not
share any structural similarity with CKI proteins. The Ctk3 sequence
does not appear to contain a cyclin box, and as yet we do not know the
Ctk1·Ctk3 binding domains.
Based on sequence similarity and functional properties, Cdk9 has been
proposed as a potential human ortholog of Ctk1 (24). The transactivator
Tat recruits the Cdk9 CTD kinase by binding to the RNA TAR element (for
review see Ref. 22) and modifies the substrate specificity of the Cdk9
complex (67). It has been suggested that genes regulated by CTDK-I may
share common cis-acting RNA elements that, similar to the
TAR element, might attract the CTDK-I complex (21). It is interesting
to note that Ctk3 shares with the Tat protein the ability to bind to
both the CDK and the cyclin subunit, raising the possibility that Ctk3
and Tat might be functionally related.
Strikingly, despite the formation of Ctk1·Ctk2 complexes in
vitro, the Ctk2 cyclin is unable to activate its CDK. Indeed, both
Ctk2 and Ctk3 subunits are required for Ctk1 CTD kinase activation. Because Ctk2 and Ctk3 expression are mutually dependent on each other,
it is difficult to study in vivo, when the CDK might be phosphorylated, whether a single subunit could activate Ctk1. When Ctk2
was overexpressed from the strong PGK1 promoter, we observed
a high steady state of the cyclin in Ctk3 Is a Substrate for a Ubiquitin-Proteasome Pathway--
Our
study of Ctk3 expression revealed that, in exponentially growing cells,
it is a relatively unstable protein (half-life, 30 min). Ctk3 turnover
is markedly affected in a mutant strain deficient for two of the
proteasome catalytic subunits (pre1 pre2) (55), indicating
that its destruction is mediated by the proteasome pathway. We
identified a nonessential PEST-region located at the N-terminal of the
protein involved in Ctk3 turnover. PEST regions are usually composed of
phosphorylation sites involved in protein targeting to the
ubiquitination machinery. Several studies indicated that
phosphorylation of G1 cyclin PEST regions by their
associated CDKs was the signal that triggered their degradation (63,
68, 69). As yet, we do not know the phosphorylation state of Ctk3 nor
its potential role in the regulation of its turnover. However, it is
clear that the Ctk3 PEST motif is not a target for the CDK of the
complex, because Ctk3 turnover does not require CTDK-I complex assembly
nor its activity. This feature is different from what has been
described for G1 cyclins, consistent with the fact that the
CTDK-I kinase is subject to a distinct type of regulation.
Ctk3 joins the growing list of naturally short-lived proteins targeted
to the proteasome. Interestingly, of the three subunits composing the
CTDK-I complex, the two activating subunits Ctk2 and Ctk3 are regulated
at the level of protein turnover, whereas the catalytic subunit is a
stable protein.
Ctk2·Ctk3 Heterodimerization Controls Their Respective
Turnover--
The results presented here demonstrate that the Ctk2
cyclin plays an essential role in the control of Ctk3 turnover. In
cells lacking Ctk2, Ctk3 turnover is dramatically increased, whereas in
contrast, Ctk2 overexpression generates a substantial decrease in Ctk3
turnover. Strikingly, Ctk3 steady-state levels also influence Ctk2
turnover. In other words, changing the relative levels of Ctk2 (and
Ctk3) leads to important changes in the degradation kinetics of Ctk3
(and Ctk2). Furthermore, Ctk2 and Ctk3 coimmunoprecipitate in the
absence of the kinase subunit, and in vitro, they form heterodimers. In cells expressing a Ctk3 mutant protein lacking the
cyclin binding domain, both Ctk3 and Ctk2 are very unstable. The fact
that both proteins are quickly degraded when they cannot bind to each
other strongly suggests that their heterodimerization protects both
subunits from degradation by the proteasome.
Ubiquitination of the p27 CKI requires trimeric complex formation with
the cyclin and CDK subunits (70). It has also been reported that
polyubiquitination of cyclin B is likely to target the complex to the
proteasome where Cdc2 is released from cyclin B (71). Because
phosphorylated G1 cyclin bound to Cdc28 has been
observed,3 it is probable
that yeast G1 cyclin is recognized by the ubiquitination machinery while complexed with the CDK. Thus, high affinity association of a CKI or a cell cycle cyclin with its CDK would necessitate regulated proteolytic degradation to break down the complex. In contrast, our data indicate that neither Ctk2 nor Ctk3 destruction occurs within the CTDK-I complex.
Protein interactions have often been described as a means to regulate
transcription factor turnover, but this is the first description of the
regulation of cyclin turnover by heterodimerization with a specific
factor. Interestingly, Johnson et al. (72) also reported
mutual stabilization of yeast proteins: the MAT
transcription factors. Coexpression of a1 and
What are the regulatory signals that could induce Ctk2·Ctk3
dissociation? Interaction with another factor (such as a component of
the ubiquitination machinery for example) could bring conformational changes leading to Ctk2·Ctk3 dissociation. Alternatively, Ctk2 and/or
Ctk3 protein modification could trigger this dissociation. Several
lines of evidence suggest that Ctk2 phosphorylation is a signal for its
rapid destruction (45). In vivo, Ctk3 immunoprecipitation from wild type as well as The CTDK-I Complex: A New Mechanism for CDK Regulation?--
Most
CDKs interact sequentially with multiple cyclin subunits (27), cyclin
binding thus regulating kinase temporal activity, as well as substrate
specificity. In contrast, only one cyclin has been characterized per
yeast CTD kinase. Interestingly, Ctk1 activation supposes a balanced
concentration of two specific regulatory subunits. Control of protein
degradation by regulating protein·protein interaction could establish
modulations in CTDK-I activity rather than the classic on/off switch
observed for other CDKs. In addition, Ctk2 and Ctk3 might respond to
distinct signals thus allowing a larger spectrum of signals to be
integrated. The different specific features governing CTDK-I regulation
probably reflect requirement for the transcriptional response involved
in cell adaptation to multiple growth conditions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ctk strains
(MAT
ade2-1 can1-100 his3-11, 15 leu2-3, 112 trp1-1 ura 3-1,
ctk::TRP1) are described in work by Hautbergue and
Goguel (45). Yeast cultures were grown at 30 °C to an
A600 of 0.8, unless otherwise stated, in SD
medium containing 0.67% yeast nitrogen base without amino acids
(Difco) supplemented with appropriate nutrients for auxotrophy and 2%
glucose as a carbon source. For induction of the CUP1
promoter, CuSO4 was added at 0.1 mM overnight.
When indicated, cycloheximide (Sigma) was added to 100 µg/ml. Yeast
transformations were performed by the LiCl method (56).
PEST construct is identical to the wild type CTK3
construct except that 13 amino acids (see Fig. 1) are replaced by a
leucine. CTK3
C50 contains a STOP codon in position 247 of the Ctk3
protein. The +C50 construct encodes a 100-amino acid peptide composed
of 25 amino acids encoding two HA-tags/Ctk3 24 N-terminal amino
acids/one leucine/Ctk3 50 C-terminal amino acids. All constructs
containing PCR fragments were sequenced (the Ctk3
PEST and Myc-Ctk1
proteins each contain a single mutation, His58
Leu and
Pro35
Leu, respectively). For expression of the
Ctk proteins from the T7 promoter, the different epitope-tagged coding
sequences were cloned either into the pRSET5d vector (58) or the pET14b (Novagen) to express the (His)6-HA tagged Ctk1 protein. The
plasmid pMT1089 (TRP1 2 µm) encodes a
(His)6-Myc-tagged ubiquitin under the control of the
copper-inducible CUP1 promoter (32).
-32P]dATP by random priming (Appligene) was performed
under standard methods.
-D-galactopyranoside. Cell pellets were resuspended in 2 ml of BB100 (25 mM
HEPES, pH 7.5, 100 mM potassium acetate, 10 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 15% (v/v) glycerol) containing 1 mM
phenylmethylsulfonyl fluoride and Complete protease inhibitor mixture
(Roche Molecular Biochemicals). Cell disruption was completed by
sonication. After ultracentrifugation at 100,000 × g,
levels of tagged proteins from the extracts were estimated by Western
blot analyses.
(100) buffer (25 mM
HEPES, pH 7.5, 10 mM MgCl2, 15% glycerol;
supplemented with 100 mM potassium acetate). After 5 column-volumes of washing, an excess of recombinant HA-Ctk2, HA-Ctk3,
or HA-Ctk2+HA-Ctk3 extracts, were loaded on the column. After washing
with 10 column-volumes of PC
(100) buffer, proteins eluted with a
linear gradient (20 column-volumes) from 0.1 to 2 M
potassium acetate. Fractions containing Ctk proteins were determined by
Western immunoblotting with HA antibodies, pooled, and loaded onto 1-ml
Hi-TRAP/Ni2+ columns (Amersham Pharmacia Biotech) with
PC
(650) buffer. After 5 column-volumes of washing with PC
(650)
buffer, a linear gradient (5 column-volumes) from 650 to 100 mM potassium acetate was performed, and washing was
achieved with 5 column-volumes of PC
(100) buffer. Proteins were
eluted by step with BB100 buffer without dithiothreitol, supplemented
with 0.3 M imidazole.
-32P]ATP in BB100 buffer. Enzymatic reactions took
place for 1 h at 25 °C, before concentration on Strataclean
beads (Stratagene) and further elution with Laemmli buffer. The
totality of the eluted proteins was separated by SDS-PAGE before
analysis by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ctk3 cell growth at the nonpermissive
temperature (data not shown). Western blot analyses of
ctk3 cell extracts expressing the Myc-tagged
Ctk3 protein revealed a specific band of the expected size (Fig.
1A, lane 0'). To
analyze Ctk3 stability, cycloheximide was added to cultures grown to an
A600 of 0.8 at 30 °C to block cytoplasmic
protein synthesis, and Ctk3 level was monitored by immunodetection at
various times after cycloheximide addition (Fig. 1A). The
half-life of Ctk3 was found to be relatively short (half-life, 30 min).
Identical results were observed with the CTK3-HA construct (Fig.
1B). Because cycloheximide treatment might cause a stress
affecting Ctk3 stability, we also performed a pulse-chase experiment.
Cells were labeled with a [35S]methionine/cysteine mix
for 10 min prior to HA-Ctk3 immunoprecipitation after different times
of chase. We observed a decrease in Ctk3 levels similar to that
observed after cycloheximide addition (Fig. 1C). We conclude
that, like the Ctk2 cyclin subunit, Ctk3 is an unstable protein in
exponentially growing cells.
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Fig. 1.
Ctk3 is an unstable protein. Cells were
grown in SD medium to an A600 of 0.8 at
30 °C. A and B, aliquots of
ctk3 cells transformed with either the
CTK3-Myc (A) or the CTK3-HA (B) construct, were
taken at different times (minutes) after the addition of cycloheximide
(100 µg/ml). Cellular extracts were analyzed by immunoblotting using
anti-Myc (A) and anti-HA (B) antibodies.
C, pulse-chase experiment. After cell labeling with a
[35S]methionine/cysteine mix for 10 min, aliquots were
taken at different times as indicated during the chase. Ctk3 was
immunoprecipitated with HA polyclonal antibodies, and proteins were
separated on SDS-PAGE before autoradiography. D, a 13-amino
acid deletion (boxed) was made inside a potential PEST
region located at the N-terminal of Ctk3 as indicated. E,
ctk3 cells were transformed with the
CTK3
PEST-Myc construct. After the addition of cycloheximide,
extracts were analyzed by immunoblotting.
PEST, restored
ctk3 cell
growth similar to wild type, indicating that this region is not
required for Ctk3 function (data not shown). Ctk3
PEST stability was
determined after cycloheximide addition. We observed a substantial
stabilization of the protein, because its levels were only slightly
decreased 6 h after cycloheximide addition (Fig. 1E).
Consistently, we also observed a higher level of Ctk3
PEST steady
state as compared with wild type Ctk3 (data not shown). These results
show that Ctk3 contains a functional PEST motif that is involved in its degradation.
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Fig. 2.
Ctk3 is degraded by a
ubiquitin-dependent proteasome pathway. A,
WCG4a (wild type, WT) and isogenic WCG4-11/22a (pre1
pre2) cells transformed with the CTK3-Myc construct were grown in
SD medium to an A600 of 0.8. Cultures were then
shifted to the nonpermissive temperature (37 °C) for 40 min prior to
the addition of cycloheximide, and aliquots were taken at different
times (minutes) of incubation at 37 °C. Resulting cellular extracts
were analyzed by Western immunoblotting. B, W303-1B (wild
type) cells were cotransformed with the pMT1089 plasmid
(2µ/(His)6-Myc-Ub) and
either a control plasmid (0) or the CTK3-HA construct
(Ctk3). Cells were grown in SD medium to an
A600 of 0.8 without ( ) or with (+)
CuSO4 to induce expression of the tagged ubiquitin.
Proteins were retained on a nickel column under denaturing conditions.
Eluted proteins were analyzed by Western immunoblotting with HA
(upper panel) and Myc (lower panel)
antibodies.
ctk1 cells, and Ctk3 levels were followed after cycloheximide addition. In the absence of the kinase, Ctk3 stability was similar to that observed in wild type cells (Fig. 3A; half-life, 30 min),
indicating that Ctk3 turnover is a process that requires neither CTDK-I
complex assembly nor its activity.
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Fig. 3.
Ctk3 turnover is affected by Ctk2
expression. A, B, and E, cells
were grown in SD medium to an A600 of 0.8, and
aliquots were taken at different times (minutes) of incubation at
30 °C after the addition of cycloheximide. Resulting cellular
extracts were analyzed by Western immunoblotting with either Myc or HA
antibodies. The CTK3-Myc construct was introduced into
ctk1 (A) and
ctk2 cells (B). C,
ctk1,
ctk2, and
ctk3 cells, respectively, were transformed
with CTK3-HA. The asterisk indicates a nonspecific band that
shows lanes loading. D, Northern blot analysis. Total RNA
from W303-1B (wild type, WT) and isogenic
ctk2 cells were separated on an agarose gel
and hybridized with a CTK3 probe (upper panel).
rRNA staining shows that equal amounts of RNA were loaded (lower
panel). E,
ctk3 cells carrying
the CTK3-Myc construct and a 2µ-CTK2 plasmid.
ctk2 cells. Strikingly, the Ctk3 steady-state
level was very low, because the protein could be detected only when the
blots were overexposed (Fig. 3B). We further compared the
steady-state level of the HA-tagged Ctk3 in these different strains in
the same experiment (Fig. 3C). Indeed, Ctk3 levels were
dramatically reduced in
ctk2 cells as compared
with
ctk1 cells. Unexpectedly, Northern blot
analyses revealed that
ctk2 cells contained
substantial amounts of CTK3 mRNA (Fig. 3D).
Furthermore, the levels of CTK3 mRNA were similar in
ctk2 and
ctk1 cells
(data not shown). Taken together, these results suggest that Ctk3 is
synthesized and very quickly degraded in the absence of the cyclin.
ctk1,
ctk2, and
ctk3 cells. As previously described (45), Ctk2
steady-state level was only slightly lower in
ctk1 as compared with pseudo wild type cells (
ctk2 cells, Fig.
4A). In contrast, Ctk2 could
only be observed in
ctk3 cells when the blots
were overexposed (Fig. 4A), suggesting that Ctk2 turnover
was very fast in these cells. Also, in exponential cells, Ctk3
overexpression resulted in a significant stabilization of the cyclin
(data not shown). We conclude that Ctk3 expression influences the Ctk2
cyclin degradation. To test whether this effect was specific, we
analyzed the expression of the catalytic subunit of the CTDK-I complex,
namely, the Ctk1 kinase. Unlike the two other subunits, Ctk1 appeared
to be a stable protein in exponentially growing cells (half-life > 6 h, data not shown). Importantly, its steady-state level was
not significantly affected in either
ctk2 or
ctk3 cells as compared with pseudo wild type
cells (
ctk1, Fig. 4B). These
results indicate that Ctk3 plays a specific role in the control of Ctk2
destruction.
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Fig. 4.
Ctk3 expression influences the Ctk2 cyclin
turnover. Cells were grown in SD medium to an
A600 of 0.8, and resulting cellular extracts
were analyzed by Western immunoblotting. ctk1,
ctk2, and
ctk3 cells,
respectively, were transformed with the CTK2-HA (A) or the
CTK1-HA plasmid (B). Asterisks indicate a
nonspecific band that shows lanes loading.
ctk1 cells were cotransformed either with the
CTK2-Myc and CTK3-HA constructs or with the CTK2-HA and CTK3-Myc
constructs. HA-tagged proteins were immunoprecipitated. As revealed by
hybridization with Myc antibodies, Ctk2 was coimmunoprecipitated with
Ctk3 (Fig. 5A, left
panel); and reciprocally, Ctk3 was coimmunoprecipitated with Ctk2
(Fig. 5A, right panel). These results demonstrate
that, even in the absence of the kinase subunit, Ctk2 and Ctk3 are part of the same complex in vivo.
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Fig. 5.
Ctk2·Ctk3 heterodimerization.
A, ctk1 cells cotransformed with
the CTK2-Myc (left panel) or CTK3-Myc (right
panel) construct, and either a control plasmid (
) or the CTK3-HA
(+, left panel) or CTK2-HA (+, right panel)
plasmid, were grown in SD medium to an A600 of
0.8. After immunoprecipitation with polyclonal HA antibodies, proteins
were analyzed by Western blotting with Myc (upper panel) and
HA (lower panel) antibodies successively. B,
wheat germ extract expressing HA-Ctk2 labeled with
[35S]methionine and bacterial extract containing Myc-Ctk3
were incubated together prior to immunoprecipitation with monoclonal HA
antibodies. Proteins were analyzed by Western blotting with Myc
antibodies (upper panel) or by autoradiography (lower
panel). Asterisks indicate bands corresponding to
immunoglobulins. C, gel filtration analysis. Bacterial
extracts containing HA-Ctk2 (upper panel) or Myc-Ctk3
(middle panel) were applied to a gel filtration column as
described under "Experimental Procedures." On the other hand,
HA-Ctk2- and Myc-Ctk3-containing extracts were incubated together prior
to immunopurification of HA proteins. The resulting eluate was applied
to a gel filtration column (lower panel). A sample of every
other fraction was analyzed by Western blotting with HA (upper
panel), Myc (middle panel), or a mixture of HA and Myc
antibodies (lower panel).
C50, was unable to restore
ctk3 cell growth at the nonpermissive temperature (data not shown), indicating that this region is essential for Ctk3 function. The
CTK3
C50-HA and CTK3-HA genes
carried on single-copy plasmids were separately introduced into wild
type and
ctk2 cells, and the resulting
extracts were analyzed. In wild type cells, Ctk3
C50 steady-state
levels were extremely low as compared with wild type Ctk3, whereas, in
ctk2 cells, both Ctk3 and Ctk3
C50 amounts
were extremely low (Fig. 6A).
Importantly, unlike wild type Ctk3, Ctk3
C50 levels were identical in
both strains, indicating that the mutant protein was very rapidly
degraded, regardless of the presence or the absence of the cyclin
encoding gene. We next analyzed Ctk2 expression by introducing the
CTK2-Myc construct in
ctk3 cells expressing Ctk3 and
ctk3 cells expressing Ctk3
C50,
respectively. In the presence of wild type Ctk3, we could easily detect
Ctk2 species (Fig. 6B, Ctk3), whereas, in marked
contrast, Ctk2 was difficult to detect in the sole presence of the
Ctk3
C50 mutant protein, even when blots were overexposed (Fig.
6B,
50). Altogether, these results
show that both Ctk2 and Ctk3
C50 are very unstable proteins. A
possible explanation to account for this observation was the absence of
interaction between Ctk2 and Ctk3
C50. To test Ctk2·Ctk3
C50 interaction directly, the tagged proteins were expressed in E. coli and incubated overnight prior to loading on an HA column and
subsequent HA peptide elution. Remarkably, although the Ctk3
C50 protein was immunopurified as efficiently as the wild type Ctk3 (Fig.
6C, compare lane 2 to lane 3,
lower panel), Ctk2 did not copurify with the mutant protein
(Fig. 7C, lane 3). In
contrast, Ctk1 was efficiently copurified with Ctk3
C50 (Fig.
6D, lane 3), indicating that the mutant protein
is still correctly folded. These results show that the Ctk3 50 C-terminal amino acids are specifically required for Ctk2 binding. In
the absence of this interaction, both Ctk2 and Ctk3 become very
unstable.
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Fig. 6.
Ctk2·Ctk3 interaction is required in
vivo for their respective stabilization. Cells were
grown in SD medium to an A600 of 0.8, and
resulting extracts were analyzed by Western blotting with HA
(A) and Myc (B) antibodies. A, wild
type (WT) and ctk2 cells were
transformed with either the CTK3-HA (Ctk3) or the
CTK3
C50-HA (
50) construct. B,
ctk3 cells were cotransformed with the
CTK2-Myc construct and either the CTK3-HA (Ctk3) or the
CTK3
C50-HA (
50) construct. C and
D, extracts made from E. coli cells expressing
either Myc-Ctk2 (C) or Myc-Ctk1 (D) were further
incubated with control extracts (lane 1), or extracts
containing either HA-Ctk3 (lane 2) or HA-Ctk3
C50
(lane 3), prior to loading on HA columns. Proteins were
eluted with HA peptide and analyzed by Western blotting with Myc
(upper panel) and HA (lower panel)
antibodies.
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Fig. 7.
Ctk1 activation requires both Ctk2 and Ctk3
subunits. A, protein complex formation. Different
combinations of E. coli extracts expressing each a single
tagged protein were incubated at 15 °C prior to immunopurification
on HA columns. Proteins were eluted with HA peptide and analyzed by
Western blotting with a mixture of HA and Myc antibodies. B,
fractionation scheme used to purify recombinant Ctk complexes by ion
exchange and affinity chromatography (see "Experimental
Procedures"). C, protein fractions eluted from
Hi-TRAP/Ni2+ columns were analyzed by Western blotting with
HA antibodies. Ctk1+2+3, Ctk1+2, and Ctk1+3 coelutions are shown after
purification. D, CTD kinase assays. Activities of the
different complexes were assayed by incubation with GST-CTD and
[ -32P]ATP: Ctk1+2+3 (lane 3), Ctk1 alone
(lane 4), Ctk1+2 (lane 5), and Ctk1+3 (lane
6). Negative controls were provided by incubation of GST-CTD alone
with [
-32P]ATP (lane 1), and addition of
proteins eluted from the column after isolation from an E. coli/empty vector extract (lane 2). The
arrow points to labeled GST-CTD detected by
autoradiography.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ctk3
cells (data not shown). However,
ctk3 cell
growth was not rescued at the nonpermissive temperature. This
observation supports the in vitro data and suggests that the
cyclin cannot by itself activate its CDK. It thus appears that, within
the CTDK-I complex, the cyclin function requires two distinct factors,
pointing to a divergent mechanism for activation of a CDK implicated in
RNA polymerase II CTD phosphorylation.
2 which
occurs in a/
diploids results in a dramatic stabilization
of both proteins. The authors further demonstrated that a1
and
2 turnover is controlled by their direct physical interaction
that masks proteolytic signals. Ctk2 and Ctk3 are both constitutively
expressed in the cell, implying that the degradation of these two
unstable regulatory factors requires either prevention of their
interaction or promotion of the dissociation of Ctk2·Ctk3 complexes.
ctk1 cells allowed
recovery of both phosphorylated and unphosphorylated species of the
cyclin, indicating that Ctk2 phosphorylation is not a signal for
Ctk2·Ctk3 dissociation. Because Ctk2·Ctk3 binding prevents their
respective degradation, it is tempting to speculate that, similar to
the case of a1 and
2, target sites for the ubiquitination
machinery are not exposed when the two proteins are associated, and
that only their dissociation would expose, at least transiently, these
degradation sites. It will be important to determine whether
degradation determinants in Ctk2 and Ctk3 overlap (or are closely
juxtaposed to) the interaction surfaces of these two molecules.
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ACKNOWLEDGEMENTS |
---|
We are grateful to C. Jacq for his support while this work was initiated, and to A. Sentenac for welcoming us in his laboratory. Special thanks are due to M. Riva and C. Carles for stimulating discussions and friendly support. We also thank G. Peyroche for numerous fruitful discussions. A. Sentenac, C. Mann, and E. Ceccarelli are thanked for critical reading of the manuscript. We thank J. Acker for helpful discussions and for the generous gift of the GST-CTD construct, and E. Favry for friendly technical advice.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a French Ministère de la Recherche et des
Technologies fellowship and by the Association pour la Recherche contre le Cancer.
§ To whom correspondence should be addressed: Tel.: 33-1-6908-9921; Fax: 33-1-6908-4712; E-mail: goguel@jonas.saclay.cea.fr.
Published, JBC Papers in Press, December 15, 2000, DOI 10.1074/jbc.M010162200
2 V. Goguel, unpublished data.
3 E. Ceccarelli and C. Mann, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CTD, C-terminal domain; CDK, cyclin-dependent kinase; CKI, cyclin-dependent kinase inhibitor; HA, hemagglutinin; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; PCR, polymerase chain reaction; bp, base pair(s).
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