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Molecular and Cellular Biology, August 2000, p. 5858-5864, Vol. 20, No. 16
Division of Biology, California Institute of
Technology, Pasadena, California 91125
Received 17 March 2000/Returned for modification 26 April
2000/Accepted 19 May 2000
p13suc1 (Cks) proteins have been implicated in the
regulation of cyclin-dependent kinase (CDK) activity. However, the
mechanism by which Cks influences the function of cyclin-CDK complexes
has remained elusive. We show here that Cks1 is required for the
protein kinase activity of budding yeast G1 cyclin-CDK
complexes. Cln2 and Cdc28 subunits coexpressed in baculovirus-infected
insect cells fail to exhibit protein kinase activity towards multiple substrates in the absence of Cks1. Cks1 can both stabilize Cln2-Cdc28 complexes and activate intact complexes in vitro, suggesting that it
plays multiple roles in the biogenesis of active G1
cyclin-CDK complexes. In contrast, Cdc28 forms stable, active complexes
with the B-type cyclins Clb4 and Clb5 regardless of whether Cks1 is present. The levels of Cln2-Cdc28 and Cln3-Cdc28 protein kinase activity are severely reduced in cks1-38 cell extracts.
Moreover, phosphorylation of G1 cyclins, which depends on
Cdc28 activity, is reduced in cks1-38 cells. The role of
Cks1 in promoting G1 cyclin-CDK protein kinase activity
both in vitro and in vivo provides a simple molecular rationale for the
essential role of CKS1 in progression through
G1 phase in budding yeast.
Cyclin-dependent kinases (CDKs)
trigger a variety of events in the division cycle of eukaryotic cells
(28). Multiple regulatory mechanisms conspire to ensure that
CDKs are switched on at the right time and in the right place as cells
proceed through the cell division program. At least four distinct
posttranslational mechanisms positively regulate CDK function,
including binding of cyclins, binding of p13suc1 (Cks)
proteins, phosphorylation of a threonine within the T-loop of CDKs by
the CDK-activating kinase (CAK), and dephosphorylation of threonine and
tyrosine residues within the ATP-binding site by Cdc25 (27).
Of these mechanisms, the least well understood is how binding of Cks
proteins promote the function of CDKs.
The gene encoding p13suc1 was originally identified as a
multicopy suppressor of cdc2ts mutations in
Schizosaccharomyces pombe (15) and was
subsequently isolated as a multicopy suppressor (named CKS1)
of cdc28ts mutations in Saccharomyces
cerevisiae (14). Characterization of budding yeast
cks1 thermosensitive mutants revealed that Cks1 function is
required at two points in the cell cycle: prior to start in
G1 and at some point in mitosis (42). As was
observed in p13suc1-depleted fission yeast (26),
G2-arrested cks1ts mutants
accumulate high levels of CDK activity.
The role of Cks proteins in progression through mitosis has been the
subject of numerous recent studies. Xenopus extracts depleted of the Cks1 homolog p9 fail to enter mitosis due to
accumulation of inhibitory phosphate on the Y15 residue of
p34cdc2 (29), perhaps because p9 promotes
CDK-dependent phosphorylation of the Y15 kinase Wee1 and the Y15
phosphatase Cdc25 (31). p9-depleted cycling extracts
programmed with a p34cdc2 mutant that cannot be
phosphorylated on Y15 no longer arrest at the G2/M
transition but instead fail to initiate cyclin B destruction and
consequently remain arrested in mitosis (29). These results suggest that p9 also promotes activation of cyclin B proteolysis by the
anaphase-promoting complex-cyclosome (APC/C)-26S proteasome pathways
(22). p9 is thought to activate cyclin B proteolysis by
directly facilitating the phosphorylation of components of the APC/C,
including Cdc27, by mitotic CDK (30, 36). However, there is
some controversy on this point, since budding yeast Cks1 is dispensable
for cyclin B ubiquitination but appears instead to promote degradation
of ubiquitinated cyclin B by the 26S proteasome (19).
Despite these recent advances in our understanding of how Cks proteins
promote mitosis, it remains unclear why Cks1 is required for
progression through Start.
Structural insights into the function of Cks proteins have emerged from
X-ray crystallography of human Cks1-Cdk2 complexes (2). The
binding of Cks1 does not cause significant conformational changes in
either the active site or the cyclin-binding interface of Cdk2. A
comparison of the Cks1-Cdk2 and cyclin A-Cdk2 (17) structures reveals that Cks1 and cyclin A bind to opposite sides of
Cdk2 flanking the active site (2). The structure of the ternary complex suggests that Cks1 may help position substrates for
phosphorylation by Cdk2.
In this report, we provide evidence that Cks1 is required both in vitro
and in vivo for the protein kinase activity of G1 cyclin-CDK complexes from budding yeast. These findings suggest a
simple explanation for why Cks1 activity is required for budding yeast
cells to traverse Start.
Preparation of cell lysates from baculovirus-infected cells.
Sf9 or Hi-5 cells were grown in suspension in supplemented Grace's
insect media. For infection, 107 cells were plated per
10-cm-diameter dish and overlayed with working stocks of viruses at a
multiplicity of infection of ~10. After incubation for 40 to 44 h, cells were harvested in a clinical centrifuge at 4°C, washed once
in 1× phosphate-buffered saline, and resuspended in ice-cold lysis
buffer (10 mM HEPES-KOH [pH 7.4], 150 mM NaCl, 0.2% Triton X-100, 1 mM Activation of Cln2-Cdc28HA, immunoprecipitations,
protein kinase assays, and immunoblots.
Clarified lysates from
baculovirus-infected Sf9 cells were incubated in a reaction mixture
consisting of activation buffer (1/10 volume of 40 mM magnesium
acetate, 5 mM PMSF, 50 mM benzamidine, 10 mM dithiothreitol (DTT), 50 µg of pepstatin and 50 µg of leupeptin/ml), with or without yeast
extract, Cks1, and ATP-regenerating system (1/10 volume of 500-µg/ml
creatine phosphokinase, 10 mM ATP, 20 mM HEPES [pH 7.2], 10 mM
magnesium acetate, 300 mM creatine phosphate). Activation mixtures were
incubated for 15 min at 24°C and then chilled on ice.
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Cks1 Is Required for G1
Cyclin-Cyclin-Dependent Kinase Activity in Budding Yeast
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
-mercaptoethanol, 5 µg of pepstatin and 5 µg of leupeptin/ml,
2 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM benzamidine, 50 mM
-glycerophosphate, 50 mM NaF, and 0.2 mM sodium orthovanadate).
After a 10-min incubation on ice, lysates were sonicated three times
for 10-s intervals at a setting of 4 (Branson sonifier 450) and then
clarified by centrifugation at 4°C (35,000 rpm for 10 min; Sorvall
RP100AT4 rotor). Supernatant fractions (typically 4 to 10 mg of
protein/ml) were frozen in liquid N2 and stored at
80°C.
-glycerophosphate [pH 7.5],
100 mM NaCl, 50 mM NaF, 5 mM EDTA, 2 mM sodium orthovanadate, 0.2%
Triton X-100, 1 mM DTT, 0.5 mM PMSF, 5 µg of pepstatin and 5 µg of
leupeptin/ml) and supplemented with 0.75 µl of 12CA5
antihemagglutinin (anti-HA) ascites fluid. After a 45-min incubation on
ice, 20 µl of a 50% slurry of protein A-Sepharose beads was added,
and tubes were incubated on a rotating wheel for 1 h at 4°C.
Immune complexes were washed twice with 1 ml of IPB (minus protease
inhibitors, NaF, and sodium orthovanadate). All samples were then
washed twice with 1 ml of kinase assay buffer (KAB; 10 mM HEPES [pH
7.2], 10 mM MgCl2, 50 mM NaCl, 2 mM EDTA, 1 mM DTT, 0.02%
Triton X-100) and supplemented with 10 µl of KAB adjusted to 150 µg
of histone H1/ml, 11 µM ATP, and 1.5 µCi of
[
-32P]ATP (4,500 Ci/mmol)/µl. Following a 15-min
incubation at room temperature, kinase reactions were terminated by
addition of 7 µl of 3× sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) loading buffer and heated for 3 min at
95°C. Boiled samples were subjected to SDS-PAGE, and the
32P radiolabel was quantitated by imaging dried gels with a
Molecular Dynamics PhosphorImager.
Preparation of yeast extracts.
Yeast extracts used as a
source of Cln2-Cdc28 activator were prepared as described previously
(5) from RJD875 (trp1 ura3 his2 ade1 bar1 cln1
cln2 cln3
leu2::GAL-CLN3::LEU2 cdc28-4 pep4::URA3 MAT
) cells arrested in
G1 by growth in yeast extract-peptone-dextrose (YPD) medium
for 5.5 h at 24°C.
-glycerophosphate, 0.2 mM sodium orthovanadate, 1 mM DTT, 2 mM
PMSF, 2 mM benzamidine, 5 µg of pepstatin and 5 µg of
leupeptin/ml). The cell suspension was vortexed with 2 ml of glass
beads (0.5 mm in diameter), and the resulting lysate was collected from
the beads and centrifuged at 4°C for 10 min in a microcentrifuge,
followed by 10 min at 40,000 rpm in a Sorvall microultracentrifuge
(RP100AT4 rotor). Clarified lysates were used for immunoprecipitation
and immunoblotting of Cln23×HA as described in the legend
to Fig. 5. Cln3-Cdc28 activities in untagged (RJD539; ade1 his2
leu2 trp1 ura3 MATa), CKS1 CLN33×HA
(RJD1280;
cln3::LEU2::GAL1::CLN33×HA
cks1::LEU2 ade1 leu2 his2 trp1 ura3
MATa pSE271-CKS1), and cks1-38
CLN33×HA (RJD1279;
cln3::LEU2::GAL1::CLN33×HA
cks1::LEU2 ade1 leu2 his2 trp1 ura3
MATa pSE271-cks1-38) cells were evaluated as
described previously (18). RJD539, -1279, and -1280 are
isogenic to each other and to BF264-15d.
Cell extracts for the -factor block-release experiment depicted in
Fig. 5C and D were prepared as follows. Yeast strains RJD1091 and
RJD1093 were grown in 500 ml of YPD at 24°C to an OD600
of ~0.5, supplemented with 5 µg of -factor/ml, and incubated an
additional 3 h at 24°C, followed by 1.5 h at 38.5°C.
G1-arrested cells were collected by filtration, washed with
500 ml of yeast extract-peptone, and resuspended in 500 ml of YPD
prewarmed to 38.5°C. At 15-min intervals following release from
-factor, 45-ml aliquots of the culture were harvested by
centrifugation, and cell pellets were washed with 10 ml of 100 mM NaCl
and frozen in liquid nitrogen. Once aliquots at all time points were
collected, cell pellets were thawed, resuspended in 1 ml of GBLB, and
lysed by vortexing with 1 ml of glass beads (0.5 mm in diameter). Cell lysates were clarified by centrifugation in a Sorvall
microultracentrifuge (10 min at 35,000 rpm; RP100AT4 rotor) and used
for immunoprecipitation and immunoblotting of Cln23×HA as
described in the legend of Fig. 5.
Recombinant proteins. (i) Cks1.
A 4-ml overnight culture of
BL21(DE3) plus pLysS transformed with the plasmid pCKS1-1, which
expressed Cks1 from the T7 promoter, was inoculated into 1 liter of
Luria broth-100 µg of ampicillin/ml and grown at 37°C to an
OD600 of 0.5. The culture was then cooled to 24°C in an
ice water bath, and IPTG
(isopropyl--D-thiogalactopyranoside) was added to 0.3 mM
to induce synthesis of Cks1. After a 4-h induction at 24°C, cells
were harvested (Sorvall GSA rotor; 7,000 rpm for 7 min), washed once
with 250 ml of 50 mM Tris (pH 7.6)-100 mM NaCl, and resuspended in 10 ml of 1× phosphate-buffered saline. To prepare a lysate, the cell
suspension was frozen in liquid nitrogen, thawed on ice, and sonicated
for 30 s at setting 5 (Branson sonifier 450). The resulting lysate
was clarified by centrifugation in an IEC clinical centrifuge, and the
supernatant was incubated in a boiling-water bath for 5 min and then
centrifuged for 10 min at 40,000 rpm (Beckman 60Ti rotor). The
supernatant fraction was supplemented with ammonium sulfate (1 g per
6.1 ml of supernatant), incubated for 30 min at 4°C, and centrifuged
at 40,000 rpm for 10 min (Beckman 60Ti rotor). Precipitated proteins
were redissolved in 10 ml of 50 mM Tris (pH 7.6)-100 mM NaCl-2 mM
EDTA and dialyzed against three changes of 50 mM Tris (pH 7.6)-100 mM
NaCl. The dialysate was chromatographed on a Sepharose CL-6B column
(107 cm by 16-mm diameter), and fractions containing Cks1 were pooled and concentrated in a 15-ml Millipore centrifugal concentrator (nominal
molecular mass cutoff = 10 kDa).
(ii) Cdc28HA. A recombinant baculovirus that expresses Cdc28HA was generated by cloning a blunted, CDC28HA-containing BstBI/XbaI fragment from pSF19 (39a) into the SmaI site of pVL1392.
(iii) Cln2mycHis6. Epitope-tagged Cln2 was generated by PCR amplification of CLN2 from a pAS101 template (provided by A. Sil) using the oligonucleotides RDO53 (GGGGGATCCCATATGGCTAGTGCTGAACC) and RDO54 (GGGCTGCAGCTATATTACTTGCGGCCGCTGGGTATTGCCCATACC). The resulting PCR fragment, which contains a unique NotI site at the 3' end of the CLN2 coding sequence, was digested with BamHI plus PstI and subcloned into the corresponding sites of pUC119 to generate pUC119-Cln2(NotI). Complementary oligonucleotides (RDO59: GGCCTCTAGAGGAGCAGAAATTAATCAGCGAAGAGGACCTCCTCAGGCATCATCACCATCATCACG; RDO60: GGCCCGTGATGATGGTGATGATGCCTGAGGAGGTCCTCTTCGCTGATTAATTTCTGCTCCTCTAGA) encoding the bipartite mycHis6 epitope were hybridized, kinased, and ligated into the unique NotI site of pUC119-CLN2(NotI) to yield pUC119-CLN2mycHis6. Finally, the BamHI/PstI fragment of pUC119-CLN2mycHis6 was cloned into the BamHI/PstI sites of pVL1393. All PCR-amplified coding sequences were validated by DNA sequencing.
(iv) MBP-Clb2.
CLB2 was amplified by PCR from a pC2408
template (provided by K. Nasmyth) using oligonucleotides RDO70
(GAACGGTCGACTCAGAATTCTTCATGCAAGGTCATTATATCATAGCC) and RDO71
(GAACGGCTAGCATATGTCGCGGCCGCTATCCAACCCAATAGAAAACAC). The resulting fragment was digested with SalI
plus NheI and cloned into the
XbaI/SalI sites of pRS306 (37), which
had been previously modified by filling in the unique NotI
site (pRS306NotI). All PCR-amplified coding sequences were validated
by DNA sequencing. CLB2 coding sequences were excised from
this plasmid by digestion with NdeI (filled in with Klenow
fragment) and SalI and inserted into the EcoRI
(filled in with Klenow fragment) and SalI sites of pMAL-c1
(New England BioLabs). Maltose-binding protein (MBP)-Clb2 was
expressed and purified on amylose resin by standard methods (New
England BioLabs).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Activation of baculovirus-expressed Cln2-Cdc28 complexes requires a
factor from yeast extract.
Cyclin-CDK subunits that have been
expressed from recombinant baculoviruses typically assemble into active
protein kinase complexes (4, 23, 25). Thus, to characterize
the budding yeast Cln2-Cdc28 complex, we constructed recombinant
baculoviruses to express Cln2 and Cdc28 either singly or in combination
in insect Sf9 cells. The Cln2 and Cdc28 subunits were tagged at their C termini with a dual myc-hexahistidine epitope (Cln2MH6) and
an HA epitope (Cdc28HA), respectively, to facilitate the
recovery of Cln2-Cdc28 complexes from cell lysates. Following
immunoprecipitation with the anti-HA monoclonal antibody 12CA5, immune
complexes were assayed for histone H1 protein kinase activity and
evaluated by SDS-PAGE (see Materials and Methods). Unexpectedly, Sf9
cells coinfected with recombinant baculoviruses encoding
Cln2MH6 and Cdc28HA failed to express histone
H1 protein kinase activity (Fig. 1, lane
5), even though both subunits were produced as judged by immunoblotting
(data not shown).
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The yeast extract Cln2-Cdc28 activator is Cks1.
In preliminary
attempts to characterize the Cln2MH6-Cdc28HA
activator in yeast extract, we tested its sensitivity to heat.
Surprisingly, boiled yeast extract fully retained its ability to
activate baculovirus-expressed Cln2MH6-Cdc28HA
(Fig. 2A). Eukaryotic cells express a
small protein that binds tightly to the CDK subunit of cyclin-CDK
complexes and that is known by a variety of names including p13, suc1,
and Cks (3, 14, 33). Depending on the species,
p13suc1 or Cks1 ranges in size from 9 to 19 kDa. Given that
the S. pombe p13suc1 protein is heat stable
(39), we tested whether the
Cln2MH6-Cdc28HA activator might correspond to
Cks1, which is the budding yeast homolog of p13suc1
(14). Untreated or boiled E. coli extract that
contained Cks1 could substitute for yeast extract to activate
Cln2MH6-Cdc28HA complexes in insect cell lysate
(Fig. 2A), whereas control E. coli extract had no effect
(not shown). Half-maximal activation of
Cln2MH6-Cdc28HA complexes in crude Sf9 cell
extracts (Fig. 2C) was obtained with ~100 nM purified Cks1 (Fig. 2B).
Based on these observations, we conclude that the heat-stable
Cln2MH6-Cdc28HA activator present in yeast
extract is Cks1.
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Cks1 assembles with and activates the ability of Cln2-Cdc28 to
phosphorylate multiple substrates.
Cks proteins are well known for
binding tightly to cyclin B-CDK complexes. To test if Cks1 likewise
assembles with Cln2-Cdc28, all three proteins were expressed from
baculovirus vectors in insect cells. Coomassie blue staining and
immunoblot analysis confirmed that Cln2-Cdc28 complexes bound to Cks1
beads (data not shown) and that both Cln2 and Cks1His6 were
recovered along with GST-Cdc28HA on glutathione resin (Fig.
3A; these complexes exhibited high histone H1 kinase activity [data not shown]). These results confirm a
previous report that Cdc28 and Cks1 copurify with Cln2 from yeast cells
(46).
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Cks1-dependent activation of Cdc28 is Cln specific.
Since
expression of many different cyclin-CDK combinations in Sf9 cells has
yielded active protein kinase complexes in the absence of added Cks
proteins, we examined whether the Cks1-dependent activation of
Cln2MH6-Cdc28HA was a peculiarity of the
budding yeast CDK or of the G1 cyclin. Whereas coinfection
of Sf9 cells with baculoviruses encoding Cln2MH6 and
Cdc28HA yielded little precipitable protein kinase activity
unless Cks1 was added prior to immunoprecipitation with anti-HA
antibodies, Sf9 lysates containing Clb4 plus Cdc28HA or
Clb5 plus Cdc28HA yielded substantial histone H1 kinase
activity even in the absence of added Cks1 (Fig.
4). Besides Cln2-Cdc28 complexes,
Cln1-Cdc28 complexes expressed in insect cells require Cks1 for protein
kinase activity (38; J. W. Harper, personal communication).
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Cks1 stabilizes Cln2-Cdc28 complexes and can activate preexisting complexes. To determine how Cks1 influences the activity of Cln2MH6-Cdc28HA, we first examined whether Cks1 influences the assembly of the complex. Immunoblotting revealed that greater amounts of Cln2MH6 consistently coimmunoprecipitated with Cdc28HA from insect cell lysates that were supplemented with Cks1 (Fig. 3B, bottom, lane 5 versus lane 6; Fig. 3C, bottom, lane 3 versus lane 4). Although Cks1 enhanced the stability of the Cln2MH6-Cdc28HA complex, there was substantial Cln2MH6 antigen in Cdc28HA precipitates prepared in the absence of Cks1, even though these precipitates had little or no protein kinase activity (Fig. 3B and C). The addition of Cks1 to Cln2MH6-Cdc28HA immunoprecipitates prepared in the absence of Cks1 led to the appearance of Cln2MH6-dependent protein kinase activity towards multiple substrates (Fig. 3C, lanes 5 to 8; note that although the level of protein kinase activity is higher in lane 4 than in lane 8, there is significantly less Cln2MH6 in the latter samples). Thus, activation of Cln2MH6-Cdc28HA by Cks1 can proceed in the absence of insect cell lysate proteins and ATP. Taken together, these data suggest that Cks1 has two functions: it can promote the assembly of Cln2MH6 with Cdc28HA and it can activate preassembled Cln2MH6-Cdc28HA complexes.
Cks1 is required for Cln2-Cdc28 activity in vivo.
Temperature-sensitive mutant cks1-38 cells arrest cell
division in both G1 and M phases at the nonpermissive
temperature (42). To determine whether Cks1 function is
required for the appearance of active Cln2-Cdc28 complexes in vivo, we
examined the level of histone H1 kinase activity associated with
epitope-tagged Cln2 (Cln23×HA) in asynchronous and
G1-synchronized populations of CKS1 and cks1-38 cells (Fig. 5). Both
histone H1 kinase activity and Cdc28 antigen were recovered in anti-HA
immunoprecipitates prepared from asynchronous populations of CKS1
CLN2HA cells (Fig. 5A, lanes 5 and 7) but not from
CKS1 cells that did not express tagged Cln23×HA
(lanes 1 and 3). In contrast, little or no histone H1 kinase activity
and Cdc28 antigen were detected in anti-HA precipitates prepared from
asynchronous cultures of cks1-38 cells maintained at the
permissive temperature (24°C; lane 9) or shifted to the restrictive
temperature (38.5°C; lane 11). Immunoblot analysis confirmed that
these cks1-38 cultures contained normal levels of Cdc28 and
Cln23×HA antigens (data not shown). Moreover, recovery of
both Cln23×HA-associated protein kinase activity and Cdc28
antigen was restored by the addition of purified Cks1 to the
cks1-38 lysate prior to the immunoprecipitation step (Fig.
5A, lanes 10 and 12), indicating that the Cdc28 and
Cln23×HA subunits present in cks1-38 cell
extracts were competent to form an active kinase complex.
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Cks1 is required for Cln3-Cdc28 activity in vivo.
Cks1 is
important for activation of recombinant Cln1-Cdc28 and Cln2-Cdc28
complexes, and cks1ts cells fail to transit from
G1 to S phase. Given that either Cln1, Cln2, or Cln3 is
able to sustain progression through G1 phase in budding
yeast cells (34), we reasoned that Cks1 might also be
important for Cln3-Cdc28 activity. To test this, we examined the
formation of active Cln3-Cdc28 complexes in cks1-38 strains. GAL-CLN33×HA CKS1 and
GAL-CLN33×HA cks1-38 cells were grown in
galactose medium at 25°C and one-half of each culture was shifted to
38.5°C for 3 h. Cln33×HA-Cdc28 activity and
Cln33×HA plus Cdc28 antigens were evaluated by protein
kinase assay and immunoblotting of anti-HA immunoprecipitates.
Cln33×HA-associated histone H1 kinase activity was
substantially diminished in cks1-38 cells grown at either
the permissive or restrictive temperature (Fig.
6A). As was observed for Cln2, little
Cdc28 was recovered in Cln3HA precipitates prepared from
cks1-38 cells incubated at the nonpermissive temperature
(Fig. 6B). Interestingly, Cdc28 was recovered in association with Cln3
from cks1-38 cells grown at the permissive temperature (Fig.
6B), even though these complexes had little protein kinase activity
(Fig. 6A). Taken together with the data shown in Fig. 3C and 5A, this
observation suggests that Cks1 stimulates both the assembly of Cln
proteins with Cdc28 and the activity of preformed Cln-Cdc28 complexes.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cks1 activates budding yeast G1 cyclin-CDK complexes. We provide evidence that Cks1 is required for the protein kinase activity of Cln2-Cdc28 and Cln3-Cdc28 protein kinase complexes. Cks1 potently activates the ability of immunoisolated Cln2-Cdc28 complexes to phosphorylate multiple substrates but has only a modest effect on the activity of Clb-Cdc28 complexes. Immunoprecipitation experiments suggest that Cks1 promotes the assembly of active Cln2-Cdc28 complexes in vivo and in vitro. However, intact Cln2-Cdc28 complexes recovered from insect cells in the absence of yeast Cks1 are nonetheless activated by added Cks1, suggesting that Cks1 may promote Cln2-Cdc28 protein kinase activity by an additional mechanism besides complex formation.
A previous analysis of the phenotype of cks1-38 mutants failed to reveal the defect in G1 cyclin-Cdc28 protein kinase activity reported here (42). This discrepancy is probably due to the fact that, in the prior work, Cdc28 protein kinase activity was monitored using an antibody directed against Cdc28. G1 cyclins account for only a small fraction of total Cdc28 protein kinase activity (47), and Cks1 is not required for the protein kinase activity of the more abundant Clb-Cdc28 complexes (Fig. 4 and 5B; the small contribution of Cks1 to Clb-Cdc28 protein kinase activity is probably obscured by the accumulation of Clb2 in cks1-38 cells). Thus, since anti-Cdc28 antibodies are expected to retrieve both Cln-Cdc28 and Clb-Cdc28 complexes, it is not surprising that Tang and Reed (42) failed to detect the effect reported here.Mechanism of Cln2-Cdc28 activation by Cks1. How does Cks1 promote Cln2-Cdc28 protein kinase activity? First, Cks1 appears to enhance the interaction between Cln2 and Cdc28. It is difficult to envision how Cks1 stabilizes the interaction of Cln2 with Cdc28 since Cks1 and cyclin A bind to opposite sides of Cdk2 and since the binding of Cks1 to the C-terminal lobe of Cdk2 has little effect on the conformation of the cyclin-binding interface (2). However, the binding of cyclin B to p34cdc2 enhances the binding of human Cks2 (7), suggesting that cyclin and Cks proteins may interact cooperatively with CDKs. Another possibility is that the long C-terminal tail following the cyclin box of Cln2 may form stabilizing contacts with Cks1 bound to the carboxy-terminal lobe of Cdc28.
Although Cks1 strongly stabilizes the interaction between Cln2 and Cdc28 in yeast extract, it has a far less dramatic effect on the assembly of stable Cln2-Cdc28 complexes in insect cell extract. We do not understand the reason for this discrepancy. Perhaps the far greater concentration of Cln2 and Cdc28 subunits expressed in insect cells favors complex assembly even if Cks1 is absent. Alternatively, insect cells may contain a factor (insect Cks1-like protein?) that stabilizes Cln2-Cdc28 association but that is insufficient to sustain protein kinase activity. Although it is unclear how the binding of Cks1 activates preformed Cln2-Cdc28 complexes isolated from insect cells, it seems unlikely that this effect is brought about through conformational changes propagated from the Cks1 binding site to the active site of Cdc28, because binding of Cks1 does not evoke substantial rearrangements within the catalytic site of Cdk2 (2) and because Cks1 has little effect on the activity of Clb-Cdc28 complexes. We suggest four alternative hypotheses to explain how Cks1 selectively activates Cln2-Cdc28 complexes. First, Cln2 may exert both positive and negative effects on Cdc28. Positive regulation would presumably be accomplished in the same manner as is observed for cyclin A: binding of Cln2 rearranges key residues in the active site of Cdc28, favoring a geometry that permits transfer of the phosphate of ATP to bound
substrate (17). In contrast, other domains within Cln2
(e.g., the C-terminal tail) might act negatively on Cdc28 unless
displaced via binding of Cks1. A second possibility is that Cln
proteins may bind Cdc28 more weakly than do Clb proteins and that Cks1
may be required to stabilize the Cln-Cdc28 complex. Third, Cks1 may
provide substrate interaction sites that are normally provided by the
cyclin in Clb-Cdc28 complexes (21) but that are otherwise
absent from Cln-Cdc28 complexes. Last, Cks1 may promote Cln2-Cdc28
activity by acting directly on Cln2. Although it seems likely that the
target of action of Cks1 is the 5 helix-L14 loop within the
C-terminal lobe of Cdc28, this last possibility is suggested by the
observation that a Cdc28 mutant (Cdc28-1N) that binds Cks1 poorly
(2) nonetheless proceeds through Start and arrests at
G2/M instead (32, 41).
Cdc37 has also been reported to promote interactions between Cln2 and
Cdc28 (13). Unlike cdc37-1 mutants,
cks1-38 cells contain normal levels of Cdc28 and accumulate
high levels of Clb-Cdc28 activity. Cdc37 is thought to promote the
assembly of cyclin-CDK complexes by promoting conformational
maturation of the CDK subunit (10, 40). Thus, Cdc37 and Cks1
probably promote Cln2-Cdc28 activity by different means.
Is Cks1 required for the activity of other cyclin-CDK complexes? To date, all cyclin-CDK complexes that have been expressed in insect cells (including cyclin A-Cdc2, cyclin B-Cdc2, cyclin D-Cdk4, and cyclin E-Cdk2) are active in the absence of any added proteins (4, 23, 25). Thus, the role of Cks1 in Cln2-Cdc28 activation may be unique to this particular combination. However, because mammalian cyclin-Cdk complexes may recruit endogenous Cks1-like proteins in insect cells, it is possible that mammalian G1 cyclins also require Cks proteins to activate their cognate CDKs. It is unclear whether insect cell Cks1-like proteins bind to Cln2-Cdc28, but if they do they are not sufficient to sustain protein kinase activity in vitro.
There is precedent for assembly factors contributing to the formation of active cyclin-CDK complexes in animal cells. Besides the Cdc37 example noted above, Mat1 is required for the assembly of cyclin H-Cdk7 (CAK) complexes (6, 12, 43). Further work will be required to test the generality of the observations we report here and to deduce the exact mechanism by which Cks1 promotes the activation of Cln2-Cdc28 complexes.| |
ACKNOWLEDGMENTS |
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We acknowledge M. Weinreich and B. Stillman for providing recombinant baculoviruses encoding Clb4 and Clb5. We also thank M. Olson for MBP-Clb2 and anti-Clb2 serum, R. Booher for Cks1 plasmids, S. Reed for cks1-38, F. Cross for the Far1 fragment and Cln33×HA strains, Wade Harper for baculoviruses encoding Cks1 and GST-Cdc28HA, members of W. Dunphy's laboratory for advice on baculovirus expression, and W. Dunphy, R. Feldman, G. Turner, R. Verma, and D. Patra for discussions and comments on the manuscript.
R.J.D. is a Searle and Markey Scholar, and this work was supported by the Searle Program/The Chicago Community Trust, The Lucille P. Markey Charitable Trust, and the National Institutes of Health (NIH RO1 GM52466).
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Biology, 156-29, California Institute of Technology, Pasadena, CA 91125. Phone: (626) 395-3162. Fax: (626) 449-0756. E-mail: deshaies{at}its.caltech.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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