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Molecular and Cellular Biology, July 1999, p. 4774-4787, Vol. 19, No. 7
Department of Molecular Biophysics and
Biochemistry, Yale University School of Medicine, New Haven
Connecticut 06520-8024,1 and Department
of Biology, Massachusetts Institute of Technology, Cambridge,
Massachusetts 021392
Received 7 December 1998/Returned for modification 9 February
1999/Accepted 1 April 1999
Cyclin-dependent kinase (CDK)-activating kinases (CAKs) carry out
essential activating phosphorylations of CDKs such as Cdc2 and Cdk2.
The catalytic subunit of mammalian CAK, MO15/Cdk7, also functions as a
subunit of the general transcription factor TFIIH. However, these
functions are split in budding yeast, where Kin28p functions as the
kinase subunit of TFIIH and Cak1p functions as a CAK. We show that
Kin28p, which is itself a CDK, also contains a site of activating
phosphorylation on Thr-162. The kinase activity of a T162A mutant of
Kin28p is reduced by ~75 to 80% compared to that of wild-type
Kin28p. Moreover, cells containing kin28T162A
and a conditional allele of TFB3 (the ortholog of the
mammalian MAT1 protein, an assembly factor for MO15 and cyclin H) are
severely compromised and display a significant further reduction in
Kin28p activity. This finding provides in vivo support for the previous biochemical observation that MO15-cyclin H complexes can be activated either by activating phosphorylation of MO15 or by binding to MAT1.
Finally, we show that Kin28p is no longer phosphorylated on Thr-162
following inactivation of Cak1p in vivo, that Cak1p can phosphorylate
Kin28p on Thr-162 in vitro, and that this phosphorylation stimulates
the CTD kinase activity of Kin28p. Thus, Kin28p joins Cdc28p, the major
cell cycle Cdk in budding yeast, as a physiological Cak1p substrate.
These findings indicate that although MO15 and Cak1p constitute
different forms of CAK, both control the cell cycle and the
phosphorylation of the C-terminal domain of the large subunit of RNA
polymerase II by TFIIH.
The eukaryotic cell division cycle
is regulated by a series of kinase activities that increase and
diminish with periodicity. These kinases, which belong to the
cyclin-dependent kinase (CDK) family, are themselves positively
regulated by cyclin binding partners and activating phosphorylations
and are negatively regulated by inhibitory binding proteins and
inhibitory phosphorylations (for general reviews, see references
36, 46, and 59). Full kinase
activity of most CDKs requires activating phosphorylations of a
threonine located in a flexible region termed the T loop. Cdc2 and Cdk2
both require this activating phosphorylation for functional activity in
vivo and in vitro (14, 26, 27, 57, 60). Other CDKs,
including Cdk4 (33), Cdk6 (30), and Cdk7 (23, 35), are also activated by similar phosphorylations. Crystallographic studies of Cdk2 suggest that this phosphorylation may
help organize an acidic patch and thereby enhance protein substrate
binding at the Cdk2 active site (52; reviewed in
reference 47). In addition to stimulating substrate
binding, activating phosphorylations may also stabilize interactions
between certain CDKs and their cyclin partners (12, 14, 26,
43).
In mammals, activating phosphorylations of Cdc2 (11, 60),
Cdk2 (21, 50, 58), Cdk4 (44), and Cdk6
(30) are mediated by a CDK-activating kinase (CAK).
Purification of CAK activity from starfish and Xenopus
revealed that CAK contains the protein kinase MO15 (21, 50,
58), which is also called Cdk7 (23). The activity of
MO15 requires binding of its cyclin partner, cyclin H, to form a
dimeric complex (23, 41) and either an activating phosphorylation (on threonine-170 in human MO15) (23, 35, 43) or binding of the assembly factor MAT1 to form a trimeric complex (13, 22, 63). In addition to their roles as a CAK, MO15, cyclin H, and MAT1 are subunits of the RNA polymerase II (Pol II)
basal transcription factor TFIIH (1, 51, 54, 55). MO15
contained in TFIIH phosphorylates the heptapeptide repeat located
in the carboxy-terminal domain (CTD) of the large subunit of RNA Pol
II, stimulating transcriptional elongation (2, 40; reviewed in references 9, 42, and
49).
The Saccharomyces cerevisiae ortholog of MO15, Kin28p
(56), is a subunit of yeast TFIIH but does not display CAK
activity (7). Originally identified because of its homology
with Cdc28p, Kin28p is essential (56), and mutants show
diminished CTD phosphorylation and impaired RNA Pol II transcription
(7, 66). In addition to Kin28p (20), the yeast
orthologs of cyclin H and MAT1, Ccl1p (67) and Tfb3p
(19) respectively, are both subunits of yeast TFIIH.
Consistent with these observations, kin28 (7,
66), ccl1 (65), and tfb3
(17) mutant strains fail to display cell cycle arrest
morphologies, and are all severely deficient in RNA Pol II transcription.
Biochemical purification and characterization of Cak1p, the CAK enzyme
from yeast (15, 32, 64), showed that it is active as a
monomer, inactive toward the CTD of RNA Pol II (31), the physiological CAK of Cdc28p (32, 64), and not a subunit of TFIIH. In contrast with kin28 mutants, cak1
mutants are blocked in cell cycle progression (32, 64). The
genetically simpler yeast, therefore, has two separate gene products
for CAK and TFIIH functions, whereas higher eukaryotes use MO15 and
cyclin H for both.
Like MO15, Kin28p contains a potential site of activating
phosphorylation. In order to characterize the function and regulation of Kin28p, we examined the properties of kin28 strains
lacking this activating threonine in vivo. Kin28pT162A has
significantly reduced kinase activity and displays a very strong
phenotype in a tfb3-ts strain background, thus supporting the dual regulation of MO15 by activating phosphorylation and MAT1
binding suggested by experiments in vitro. In contrast to a recent
report (16), activating phosphorylation of Kin28p is not
essential for Kin28p function or cell viability in the presence of
wild-type TFB3. Finally, we provide evidence that Cak1p
regulates the activating phosphorylation of Kin28p in vivo,
phosphorylates Kin28p on this site in vitro, and thereby activates
Kin28p. Together, these experiments link the functions of Cak1p and Kin28p.
Plasmids and strains.
Unless otherwise noted, all yeast
strains were derived from YMW1 (MAT
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Activating Phosphorylation of the Kin28p Subunit of
Yeast TFIIH by Cak1p
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
ade2-1 ade3-22 his3-11,15
leu2-3,112 trp1-1 ura3-1 can1-100). Specific plasmids and
genotypes of strains in this study are listed in Tables
1 and 2.
Temperature-sensitive tfb3 strains (rig2-ts
[17]) were crossed with YJK1744, transformed with
pGK13 or pGK36, and grown on 5-fluoro-orotic acid (FOA) to derive
YJK1844, YJK1845, YJK1848, and YJK1849.
TABLE 1.
Plasmids
TABLE 2.
Strains
-galactosidase reporter plasmid contained lacZ after
a GAL1/GAL10 promoter. The cloning strategy for the
GAL1/GAL10 promoter-containing vector was described
previously (34); lacZ was cloned by using BamHI and HindIII cloning sites in the vector.
Recombinant baculoviruses expressing FLAG-Kin28p and FLAG-Ccl1p were
prepared and purified via the FLAG tags as described previously
(28). FLAG-Kin28pT162A was produced by
site-directed in vitro mutagenesis with oligonucleotide CCACATGAGATACTGGCAAGTAACGTCGTAACA. The mutation
was verified by DNA sequencing. Staining of protein gels indicated that
the preparations of FLAG-Kin28p and FLAG-Ccl1p were homogeneous
(28) and that the preparations of monomeric FLAG-Kin28p were
~1 to 5% pure. As indicated below, despite these differences in
purity, approximately equal amounts of Kin28p were used as substrates
for Cak1p.
Buffers.
The 1× protease inhibitor mix (PI) contained 10 µg each of leupeptin, chymostatin, and pepstatin (Chemicon) per ml.
EB is 80 mM -glycerophosphate (pH 7.3)-20 mM EGTA-15 mM
MgCl2-10 mM dithiothreitol (DTT)-1 mg of ovalbumin per
ml-1× PI; 5× sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) sample buffer is 0.5 M Tris (pH 8.0)-10%
SDS-20 mM EDTA-250 mM DTT-50% glycerol. Plates and media were made
as described previously (5).
Preparation of lysates.
Nondenaturing lysates were prepared
by using a modified protocol previously applied for extracting TFIIH
from yeast (18). Exponentially growing cultures were
harvested by filtration, washed with deionized water, and transferred
into 1.5-ml Microfuge tubes (0.2 to 0.4 g of pellets per tube),
with addition of 0.8 g of acid-washed glass beads (0.5-mm
diameter; Sigma) and lysis buffer [150 mM Tris acetate (pH 7.5), 300 mM (NH4)2SO4, 1 mM EDTA, 1 mM
spermidine, 1 mM DTT, 10% glycerol, and 1× PI] to fill the remaining
volume. Samples were lysed by seven 1-min pulses on a bead beater
(Mini-Beadbeater-8; Biospec Products) followed by 1-min incubations in
a 
10°C ice-NaCl slurry. Glass beads and debris were removed by
centrifugation at 15,000 rpm in a Microfuge at 4°C for 10 min. The
supernatant was clarified by centrifugation at 70,000 rpm for 30 min at
4°C in the TLA 100.2 rotor in a Beckman Optima ultracentrifuge. Crude
extracts were aliquoted, frozen in liquid nitrogen, and stored at
80°C. Typical extract concentrations were 4 to 7 mg/ml.
Immunoblotting. SDS-12.5% polyacrylamide gels were transferred to polyvinylidene difluoride membranes (Millipore) by using either a tank or semidry blotting transfer system. Membranes were blocked for at least 1 h with TBST Blotto (10 mM Tris [pH 8.0], 150 mM NaCl, 0.1% Tween, 5% milk powder), incubated with primary antibodies for at least 1 h, washed with TBST (TBST Blotto without milk powder) five times for 5 min each, incubated for 1 to 2 h with secondary antibodies, washed with TBST five times for 5 min each, washed with TBS (TBST without Tween 20), incubated with chemiluminescence reagents (Pierce), and exposed to film.
One-dimensional (1-D) antihemagglutinin (anti-HA) blots were probed with either 12CA5 antibody (10 µg/ml) or rabbit anti-HA antibodies (80 ng/ml) (Santa Cruz). 2-D blots were probed with rabbit anti-HA antibodies (50 ng/ml). FLAG immunoblotting was conducted with 40 ng of polyclonal rabbit anti-FLAG (Santa Cruz) per ml followed by 160 ng of horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Pierce) per ml.Immunoprecipitation.
Twenty microliters of protein A-agarose
beads (Gibco BRL) was used per immunoprecipitation. The beads were
washed seven times with 800 µl of EB containing 1% Nonidet P-40
(NP-40), incubated for a minimum of 1 h at 4°C with 10 µg of
12CA5 antibody per immunoprecipitation, washed seven times with 800 µl of EB containing 1% NP-40, incubated for at least 1 h with
400 µg of yeast lysate diluted into 4 volumes of EB containing 1%
NP-40, washed three times with 300 µl of EB containing 1% NP-40, and
then washed three times with 300 µl of EB. Final pellets were
resuspended to a total volume of 100 µl and either used in
experiments or stored at 80°C following freezing in liquid nitrogen.
Phosphatase treatments.
Kin28p immunoprecipitates were
prepared as described above, with EB replaced by HB (EB containing 20 mM HEPES [pH 7.3] instead of -glycerophosphate).
Immunoprecipitates were incubated at 37°C for 45 min in 1× lambda
phosphatase buffer (New England Biolabs)-1× PI-1 mM
phenylmethylsulfonyl fluoride with various combinations of 800 U of
lambda phosphatase (New England Biolabs) and 20 mM sodium pyrophosphate.
2-D electrophoresis.
Nondenaturing yeast lysates were made
by using yeast extract buffer (100 mM NaCl, 20 mM Tris [pH 7.5], 10 mM EDTA, 2 mM EGTA, 5% glycerol, 1× PI) according to the procedure
described above. Fifty microliters of nondenaturing yeast lysate was
treated with 10 U of protease-free DNase and 25 µg of protease-free
RNase A (both from Worthington Biochemical Corp.) and 5 mM
MgCl2 on ice for 30 min, followed by incubation at 4°C
for 15 min. Proteins were precipitated overnight with 9 volumes of
acetone at 20°C, pelleted at 8,000 rpm for 15 min in a Microfuge,
and air dried to translucence. The dried pellets were dissolved in 100 µl of IEF sample buffer (9 M urea [American Bioanalytical], 65 mM
CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, 65 mM DTT, 5% Resolyte 4-8 [BDH Biochemicals]), and 10 µl was loaded
into 8-mm tube gels containing 5% acrylamide premix (derived from a
37.5:1 acrylamide-bisacrylamide mix), 9.25 M deionized urea solution,
27 mM CHAPS, 2.8% Resolyte 4-8, and 2.8% Resolyte 5-7 (BDH
Biochemicals). Gels were focused for 4.5 h at 400 V with 20 mM
NaOH catholyte and 10 mM phosphoric acid anolyte. The gels were
extruded into equilibration buffer (77 mM Tris buffer [pH 6.8], 3.1%
SDS, 32 mM DTT), incubated for 5 min, and subjected to SDS-PAGE and
immunoblotting as described above.
Kinase assays. (i) CTD kinase assay.
CTD kinase assays were
performed essentially as described previously (7). Briefly,
10 µl of immunoprecipitate was incubated in the presence of 3.0 µCi
of [
-32P]ATP, 0.375 µM ATP, and 4 µg of CTD
peptide [(YSPTSPS)4] in a total volume of 16 µl (filled
with EB) at room temperature. Assays were terminated after 15 min by
adding 4 µl of 5× sample buffer, and the mixtures were loaded onto
SDS-12.5% polyacrylamide gels. The gels were Coomassie blue stained,
dried, and visualized by using phosphorimaging (Molecular Imager
GS-250; Bio-Rad) and autoradiography.
(ii) Phosphorylation of CDKs.
Purified glutathione
S-transferase-Cak1p (7.5 ng) was incubated with 10 ng of
Cdk2, ~30 ng of FLAG-Kin28p-Ccl1p, or ~50 ng of
FLAG-Kin28pT162A in the presence of 5 µCi of
[-32P]ATP, 10 µM ATP, and 20 mM MgCl2 in
EB (final volume, 16 µl; EB was as described above except that 10×
PI mix was used). The reactions were terminated after 30 min at room
temperature by adding 7 µl of 5× SDS-PAGE sample buffer, and the
products were run on SDS-10% PAGE and analyzed by phosphorimaging and autoradiography.
Expression assays. Northern blotting was conducted as follows. Cultures were grown to exponential phase at 30°C in yeast extract-peptone-dextrose (YPD) and reinoculated into YPD containing 0.9 M NaCl. RNA was extracted from intact yeast cells by a hot-phenol-chloroform protocol (5), and duplicate samples were run in formaldehyde-containing agarose (5) and transferred to a GeneScreen membrane (Dupont). RNA was UV-cross-linked to the membrane at 1,200 mJ/cm2. Labeled probe was prepared from a PstI/BglII fragment of GPD1 (the pUCGPD1 plasmid was a gift from Michael Gustin, Rice University [4]). Results were visualized and quantitated by phosphorimager analysis.
Galactose promoter induction assays were conducted as follows. Cells were grown to exponential phase in CM-Trp-Ura (5) with raffinose at 30°C, and expression of the GAL1/GAL10 promoter-linked lacZ reporter was induced by addition of 30% galactose to 2%. One-milliliter samples were harvested by centrifugation in Microfuge tubes. Cells were permeabilized by resuspending them in 500 µl of ZF1 buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 38 mM 2-mercaptoethanol, pH 7.0), adding 10 µl of 0.1% SDS and 20 µl of chloroform, vortexing, and incubating at 32°C for 5 min. Assays were conducted by adding 100 µl of o-nitrophenyl--D-galactopyranoside (4 mg/ml), incubating at 32°C for various intervals, stopping the
reaction by adding 500 µl of Na2CO3,
pelleting the cells, and measuring the optical density at 420 nm
(OD420) of the supernatant. Because the assays were only
approximately linear with respect to substrate incubation times and OD,
the substrate incubation times were equal within each time point.
Cell synchronization.
A MATa bar1
strain (YJK1773) was grown in CM-Trp (5) at 30°C. When the
OD600 reached ~0.6, three 50-ml cultures were harvested.
One was saved for the asynchronous sample; the other two were
reinoculated into YPD containing 50 µg of benomyl (Dupont) per ml.
Cultures were arrested for 2.5 h in YPD containing 100 ng of
-factor per ml. Cultures were harvested by filtration, washed twice
with at least 100 ml of CM-Trp, and resuspended in prewarmed CM-Trp.
Samples (50 ml) were harvested at 15-min intervals following release
from -factor. Benomyl-arrested cells were collected after 3.75 and
4.75 h of incubation. Each harvested sample was washed once with
deionized water, frozen in liquid nitrogen, and stored at 80°C
until lysis.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Kin28p is phosphorylated on threonine 162. In the course of our studies, we noticed that Kin28p resolved into two to four species on SDS-PAGE (unless stated otherwise, all Kin28p used in this work contains a C-terminal influenza virus HA epitope tag [7]). To test whether Kin28p, like most other CDKs, is a phosphoprotein, we treated Kin28p immunoprecipitates with lambda phosphatase and monitored the effect on electrophoretic mobility by immunoblotting. Treatment with phosphatase converted the immunoprecipitated Kin28p to a slower-migrating species (Fig. 1A, compare lane 4 with lane 1). Inclusion of a phosphatase inhibitor partially blocked this effect (lane 3), indicating that the higher-mobility form of Kin28p is due to phosphorylation.
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Biochemical activity of Kin28p point mutants. As part of our characterization of the biochemical properties of Kin28p, we tested whether any of the Kin28p point mutants affected kinase activity, expecting that the Kin28pT162A mutants would be severely compromised, as are analogous mutants of Cdc2 and Cdk2. We performed CTD kinase assays and anti-HA immunoblotting on immunoprecipitates of wild-type Kin28p and Kin28p point mutants expressed from multicopy plasmids (Fig. 3). Wild-type Kin28p showed strong CTD kinase activity (upper panel, lane 1), as did the Kin28pAF mutant (lane 3). In contrast, Kin28pT162A mutants had significantly lower kinase activity (lanes 4 and 5). Full kinase activity was present in mutants containing substituted serine (T162S) (lane 6) or acidic residues (T162D/T162E) (lanes 8 and 9) designed to mimic a constitutively phosphorylated Thr-162. Immunoprecipitates from a strain containing untagged Kin28p displayed no activity (lane 2), as did immunoprecipitates of a mutant designed to lack catalytic activity (D147N) (lane 7). Corresponding immunoblots demonstrated comparable loadings and indicated that the Kin28pT162E and Kin28pT162D mutants ran with the faster mobility observed for phosphorylated Kin28p (Fig. 3, lower panel, lanes 8 and 9). Subsequent experiments in which Kin28p was expressed from low-copy-number plasmids produced essentially identical results (data not shown), and phosphorimager quantification normalized to protein loading indicated that Kin28pAF and Kin28pT162S had the same kinase activities as wild-type Kin28p; Kin28pT162A, however, was only 20 to 25% as active. These results indicate that phosphorylation of Thr-162, although not essential for kinase activity, substantially increases Kin28p activity.
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Activating phosphorylation of Thr-162 is not essential in vivo. The substantial reduction of kinase activity observed in the T162A mutant, combined with the fact that KIN28 is an essential gene, led us to expect that strains containing only the T162A allele would be inviable. To test this prediction, we disrupted KIN28 in a diploid strain and attempted to isolate haploid progeny that were rescued by KIN28 or kin28T162A expressed from a low-copy-number plasmid. To our surprise, kin28T162A fully rescued the kin28 disruption, as did the AF, AF/T162A, T162S, T162E, and T162D mutants (data not shown); kin28D147N strains were inviable (see Fig. 5A). All of the viable strains containing the indicated KIN28 alleles and lacking a chromosomal copy of KIN28 were isolated at the expected frequencies.
We therefore sought to determine whether T162A strains showed gross phenotypes that could be uncovered by simple selective conditions or assays. We found no differences in the survival of KIN28 or kin28T162A strains upon growth at various temperatures, heat shock, osmotic shock, UV exposure, cadmium exposure, or carbon starvation (data not shown). To test for subtle growth defects, a coculture experiment in which genetically marked KIN28 and kin28T162A strains were mixed and maintained in exponential phase for over 2 weeks was performed. We were unable to distinguish survival of the KIN28 and kin28T162A strains under these conditions (data not shown). We next determined whether Thr-162 phosphorylation was necessary for high-level transcriptional induction. We first compared the induction kinetics of GPD1, a gene involved in survival following osmotic stress (4). GPD1 encodes an enzyme involved in glycerol synthesis; following hyperosmotic stress, yeast accumulates glycerol to counteract extracellular osmotic pressure (4, 39). Cultures of KIN28 and kin28T162A strains were transferred into medium containing 0.9 M NaCl and grown. GPD1 transcripts were quantitated by Northern blot analysis. KIN28 and kin28T162A cells showed very similar GPD1 induction kinetics (Fig. 4A). Comparable kinetics were also seen for HSP104, a heat shock gene also induced during osmotic stress (data not shown). We next compared the induction kinetics of a lacZ reporter gene from a galactose-inducible promoter following the transition from raffinose- to galactose-containing medium. Expression of-galactosidase activity was virtually
identical for the two strains (Fig. 4B). These findings were
surprising, since they indicated that fine tuning of TFIIH activity via
Kin28p probably plays little role during the transcriptional cycle.
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Role of Thr-162 phosphorylation in vivo. Our observations that Kin28p kinase activity is substantially reduced in T162A mutants led us to explore whether Thr-162 phosphorylation might be essential when Kin28p is otherwise compromised or limiting.
We first tested whether a temperature-sensitive allele, kin28-ts16 (7), would have increased thermosensitivity if we introduced a T162A point mutation (kin28-tsT162A). We constructed a kin28 strain containing both a URA3-marked KIN28 and a TRP1-marked
kin28-tsT162A plasmid. Cells were plated on FOA
to select against the wild-type plasmid. Although cells containing a
TRP1-marked KIN28 gene survived on FOA at any
temperature, both catalytically inactive
(kin28D147N) and
kin28-tsT162A plasmids were unable to rescue
viability (Fig. 5A), indicating that both
mutants are nonfunctional. To corroborate this result, we introduced
kin28-tsT162A into a diploid strain in which one
allele of KIN28 was disrupted with LEU2. No
leucine prototrophs were recovered at 23°C; in contrast, Leu
cells contained the
kin28-tsT162A plasmid, indicating that the
inviability of kin28 disruptants was not due to plasmid loss
or toxicity during sporulation (data not shown).
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strains
containing low-copy KIN28 or
kin28T162A combined with high- or low-copy
kin28D147N. We observed that
kin28T162A strains were impaired for growth in a
dose-dependent manner when Kin28pD147N was simultaneously
expressed (Fig. 5B) and were inviable when Kin28pD147N was
expressed from a high-copy-number plasmid at 37°C (Fig. 5B). In
contrast, strains containing the wild-type allele were not sensitive to
overexpression of kin28D147N at any temperature.
The higher eukaryotic Kin28p ortholog, MO15, can be activated in a
phosphorylation-independent manner by binding MAT1 (13, 22,
63), an assembly factor that is also a subunit of TFIIH. The
yeast ortholog of MAT1 is TFB3, an essential gene
whose product is a subunit of TFIIH (17, 19). We tested
whether kin28T162A could enhance the effects of
a tfb3-ts mutation. We reasoned that if Kin28p-Ccl1p
complexes can be activated either by Thr-162 phosphorylation or by
association with Tfb3p, then the function of the
Kin28pT162A mutant might be severely attenuated in a
tfb3-ts strain. We constructed tfb3-ts strains
containing either KIN28 or
kin28T162A. Two different tfb3-ts
alleles (tfb3-ts14 and tfb3-ts23) (17) were barely temperature sensitive in our strain background (Fig. 6A). However, when the tfb3-ts
alleles were combined with kin28T162A, these
strains were critically impaired for growth at 34°C (Fig. 6A).
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Kin28p phosphorylation and activity are invariant throughout the cell cycle. The activity of MO15 in mammalian TFIIH was recently shown to be reduced during mitosis as part of a pathway in which transcription is repressed during mitosis (3, 38). This effect is due to both an increase in Ser-164 phosphorylation (which inhibits phosphorylation of the CTD by MO15) and a decrease in Thr-170 phosphorylation (3). We therefore determined whether Kin28p activity or its phosphorylation on Thr-162 varies during the cell cycle. (Kin28p lacks a phosphorylatable residue corresponding to Ser-164 in MO15.) Kin28p was immunoprecipitated from extracts derived from synchronized cells, assayed for CTD kinase activity, and immunoblotted. Neither Thr-162 phosphorylation nor kinase activity varied significantly during the cell cycle (Fig. 7A) or in extracts from asynchronous or mitotically arrested cells (Fig. 7A, lanes 1, 13, and 14). We conclude that Kin28p activity and phosphorylation are not regulated in a cell cycle-dependent manner.
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Kin28p is phosphorylated by Cak1p in vivo and in vitro. Cak1p phosphorylates Cdc28p in vivo on a site (Thr-169) equivalent to Thr-162 in Kin28p (32, 64). To investigate the relationship between Cak1p and Kin28p, we isolated cak1-22 kin28-ts16 double mutants and examined them for synthetic interactions. We found that cak1-22 kin28-ts16 strains had increased temperature sensitivity compared with strains containing the single mutations and grew poorly even at 26.5°C (Fig. 8A). Presumably, the already-compromised protein made by kin28-ts16 is rendered nonfunctional in the absence of phosphorylation by Cak1p, similar to the inviability of strains containing kin28-tsT162A (Fig. 5A).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Phosphorylation of Kin28p. Our goals in these experiments were to characterize the in vivo functions of Kin28p posttranslational regulation, to establish whether the modes of regulation of MO15 activity apply to Kin28p, and to clarify the relationship between Cak1p and Kin28p. We have shown that Kin28p, like many other CDKs, is phosphorylated in vivo on an activating residue (Thr-162) within its T loop. A Kin28pT162A mutant had significantly reduced activity in vitro and reduced function in vivo. In contrast to the situation in mammalian cells (38), both the extent of this phosphorylation and Kin28p activity were constant during the cell cycle, suggesting that the CTD kinase in yeast TFIIH is not a target for the regulation of transcription during mitosis.
Dual regulation of Kin28p. Like most CDKs (27, 30, 33, 53, 60), Kin28p requires phosphorylation for full activity. Unlike Cdc28p (7) in S. cerevisiae and Cdc2 in Schizosaccharomyces pombe (14, 26), where analogous activating phosphorylation site mutants are nonfunctional, kin28T162A is functional in vivo. Like MO15, Kin28p is positively regulated both by phosphorylation and by binding to an assembly factor. In the case of human MO15, the MO15-cyclin H complex can be activated by phosphorylation on Thr-170. In contrast, the trimeric MO15-cyclin H-MAT1 complex is active whether or not Thr-170 is phosphorylated. Our results indicate that this model, which is based on experiments in vitro, also applies in vivo. A strain carrying kin28T162A or a temperature-sensitive allele of TFB3 grows well under normal conditions. However, a kin28T162A tfb3-ts strain is severely compromised at all temperatures. This genetic interaction was confirmed at the biochemical level: Kin28p activity in the double-mutant strains was greatly reduced compared to that in a wild-type strain or in a strain containing either single mutation.
Kin28pT162A appears to be destabilized in strains defective for TFB3. Both tfb3-ts strains had reduced levels of Kin28pT162A at the restrictive temperature, and the tfb3-14 strain had much less Kin28pT162A than a wild-type strain even at the permissive temperature. In contrast, the amount of wild-type Kin28p was unaffected by the status of TFB3, suggesting that either Thr-162 phosphorylation or association with Tfb3p can stabilize Kin28p. The stability of Kin28p may depend on its association with its cyclin partner, Ccl1p, resulting in the rapid elimination of free Kin28p. Activating phosphorylations of CDKs, in addition to increasing kinase activity, may also stabilize the CDK-cyclin complex (12). Thr-170 phosphorylation of MO15, for example, stabilizes MO15-cyclin H complexes (41, 43). Furthermore, MAT1 (the homolog of Tfb3p) stabilizes the MO15-cyclin H complex in the absence of an activating phosphorylation (13, 43). The instability of Kin28p can be further inferred from the observation that temperature-sensitive alleles of CDC37 are synthetically lethal with kin28-ts alleles (reference 66 and our unpublished results). A number of newly translated CDKs, including Cdc28p and Cdk4, require stabilization by the Cdc37p chaperone (24, 61).Relationship between Cak1p and Kin28p. The data presented here establish Kin28p as the second physiological substrate for the yeast CAK, Cak1p. Cak1p can phosphorylate activating sites in a number of CDKs, including Cdc28p and a variety of mammalian CDKs (15, 31, 32, 64), and was a logical candidate for the activating kinase acting on the equivalent site in Kin28p. We showed that Cak1p could phosphorylate wild-type Kin28p but not Kin28pT162A in vitro and that Thr-162 phosphorylation of Kin28p was eliminated upon inactivation of Cak1p in vivo. In contrast, inactivation of Kin28p does not affect Cak1p activity (data not shown). In addition, we and others (66) have shown that mutations in KIN28 and CAK1 display synthetic lethal interactions.
The phosphorylation of Kin28p by Cak1p is highly specific. Cak1p was unable to phosphorylate MO15 (a Kin28p ortholog), and MO15 (another CAK) was unable to phosphorylate Kin28p (data not shown). Although MO15-cyclin H is a substrate for Cdk2-cyclin A (22, 43), Kin28p was not (data not shown). Furthermore, we observed no genetic interaction between kin28-ts16 and cdc28-1 alleles (data not shown) or any changes in Kin28p phosphorylation in a cdc28-ts strain under restrictive growth conditions (data not shown). A highly mutated allele of CDC28 that is functional but whose protein product cannot be phosphorylated by Cak1p has been isolated (8). A strain carrying this allele can grow in the absence of CAK1, but not as well as when CAK1 is present. These findings revealed a nonessential role for Cak1p in addition to its essential function as an activating kinase for Cdc28p. A plausible nonessential function is the activating phosphorylation of Kin28p. That our work should bring together Cak1p and Kin28p is ironic, since these two proteins reflect the evolutionary separation of the CAK and TFIIH functions performed by MO15 in other species. Although Cak1p is a very different CAK from MO15, the close connection of each to both the cell cycle and the basal transcription apparatus is intriguing (Fig. 10). Both control the cell cycle via phosphorylation of the major CDKs in their respective species. MO15 directly controls transcription as a subunit of TFIIH by phosphorylating the CTD of RNA Pol II, whereas Cak1p modulates TFIIH activity by phosphorylating Kin28p. Both yeast and mammalian cells can therefore coordinately affect both the cell cycle and CTD phosphorylation through a single CAK.
|
strain (45), and Csk1 can phosphorylate
and activate Mcs6 in vitro and in vivo (29). Whether
organisms such as humans also contain a Cak1p-like or Csk1-like CAK
capable of phosphorylating MO15 remains an open question.
Espinoza et al. (16) reported related results after the
completion of this work. They found that Kin28p is phosphorylated on
Thr-162, that Kin28p activity is reduced in the absence of this
phosphorylation, and that Cak1p is probably the kinase that phosphorylates Kin28p on Thr-162. Our results differ in two notable aspects. First, Espinoza et al. concluded that Kin28pT162A
was nonfunctional in that it could not rescue a temperature-sensitive allele of KIN28 at the nonpermissive temperature. In
contrast, we observed little phenotypic effect of expressing
kin28T162A at wild-type levels as the sole
Kin28p in the cell. Both studies were carried out in the W303 strain
background. We are confident that our strains contained
kin28T162A, since the lack of Thr-162
phosphorylation was detectable by SDS-PAGE. Moreover, sequencing of our
kin28T162A allele indicated the absence of any
unintended mutations. These different conclusions may reflect
differences in experimental design. It is possible, for example, that
the activity of Kin28pT162A was compromised in the
kin28-3 strain used by Espinoza et al., particularly at the
nonpermissive temperature for kin28-3. In fact, we observed
that Kin28pT162A functions very poorly in a strain
overexpressing a catalytically inactive form of Kin28p,
Kin28pD147N (Fig. 5B), a situation somewhat similar to that
employed by Espinoza et al. (This comparison is imperfect, however,
since we expressed Kin28pD147N from the KIN28
promoter on a high-copy-number plasmid, resulting in ~three to
fivefold overexpression of Kin28p compared to that with a
low-copy-number plasmid.) Their observation that the activity of Kin28p
(wild type or mutant) expressed from a heterologous promotor on a
plasmid was surprisingly low (their data not shown) strengthens the
possibility that the presence of inactivated Kin28-3p could compromise
Kin28pT162A, presumably via competition for Ccl1p and/or
Tfb3p. Whatever the explanation for the different conclusions, we feel
that the direct approach taken in this study indicates that
Kin28pT162A is fully functional as the only Kin28p in the
cell. In a separate experiment, Espinoza et al. found that a strain
with CAK1 deleted was viable (see also reference
8), even though Kin28p lacked activating
phosphorylation, supporting our conclusion that Thr-162 phosphorylation
is not essential for Kin28p function. Activating phosphorylation also
appears to be nonessential for the Kin28p homolog in S. pombe, Mcs6 (29).
A second difference between our results and those of Espinoza et al.
(16) concerns the requirements for Kin28p phosphorylation by
Cak1p. We found that Cak1p could phosphorylate Kin28p approximately equally well whether the Kin28p was free or bound to Ccl1p, whereas they observed only weak phosphorylation of monomeric Kin28p, moderate phosphorylation of Kin28p bound to Ccl1p, and strong phosphorylation of
Kin28p in the presence of both Ccl1p and Tfb3p. These results may also
reflect differences in experimental design. Espinoza et al. examined
Kin28p phosphorylation following coinfection of insect cells with
recombinant baculoviruses expressing Kin28p, Ccl1p, Tfb3p, Cak1p, and
Cdc37p, whereas we examined phosphorylation by using isolated proteins.
Free Kin28p may be rapidly dephosphorylated by an insect cell
phosphatase. An intriguing second possibility is that high expression
of the Cdc37p protein kinase chaperone both aids in the proper folding
of Kin28p and interferes with phosphorylation by Cak1p. Further work
will be required to resolve this difference.
| |
ACKNOWLEDGMENTS |
|---|
Many technical aspects of this work depended on the help of our coworkers, including Janet Burton, Beth Egan, Deb Enke, Karen Ross, Zach Pitluk, Joyce Wall, and the other members of the Solomon lab. We are especially grateful for the patience of Jackie Vogel and Kate Long, who helped navigate the flatlands of isoelectric focusing. Critical reagents were graciously provided by Gerard Faye (rig2-ts strains), Mike Gustin (GPD1 plasmids), Ann Sutton (cak1 plasmids and strains), Peter Novick (HSP104 probe), Henrik Dohlman (bar1 disruption plasmid), and Amy Fluegge (pAF21 reporter plasmid). For their sharing of equipment and materials, we thank Ken Williams, Bill Konigsberg, Peter Lengyel, Peter Novick, Sandy Wolin, and the members of their labs. David Stern, Mike Snyder, and David Gonda are gratefully acknowledged for advice and reagents.
This work was supported by a long-term fellowship from the Swiss National Science Foundation (to P.K.), the National Institutes of Health (grants GM34365 to R.A.Y. and GM47830 to M.J.S.), and the Searle Scholars Program/The Chicago Community Trust (M.J.S.). M.J.S. is a Leukemia Society of America Scholar.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Yale University School of Medicine, Department of Molecular Biophysics and Biochemistry, 333 Cedar St., New Haven, CT 06520-8024. Phone: (203) 737-2702. Fax: (203) 785-6404. E-mail: mark.solomon{at}yale.edu.
J.K. dedicates this paper to Sara Laimon.
Present address: Department of Molecular Biology, Princeton
University, Princeton, NJ 08544.
§ Present address: Antigenics LLC, Woburn, MA 01801.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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