From the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229
Received for publication, July 3, 2002, and in revised form, November 8, 2002
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ABSTRACT |
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We report here the identification of
CDC37, which encodes a putative Hsp90 co-chaperone, as a
multicopy suppressor of a temperature-sensitive allele
(cka2-13ts) of the CKA2 gene
encoding the Protein kinase CKII is an essential, ubiquitous
serine/threonine/tyrosine protein kinase of unknown function. From most
sources, the enzyme is composed of two polypeptide subunits, Cdc37 was initially isolated in a genetic screen for mutants defective
in progression through Start (6). Cdc37 function is required for the
proper association of Cdc28 (yeast CDK1) with both G1 and
mitotic cyclins, thus demonstrating a role for Cdc37 in
G2/M progression as well (7). Additional cdc37
mutants generated by Dey et al. (8) arrest in both
G1 and G2/M when shifted to restrictive
temperature. CDC37 interacts genetically with several different protein kinases that are involved in diverse cellular roles.
The list of genetic interactors of CDC37 include
CDC28 (7), KIN28 (9), mammalian v-Src when
expressed in yeast (8), MPS1 (10), STE11 (11),
and CAK1 (12).
Consistent with the genetic results in yeast, mammalian
CDC37 was found to encode the p50 subunit of an Hsp90
molecular chaperone complex that exhibits specificity for protein
kinases (13). Although Hsp90 recognizes a diverse collection of client
proteins, including steroid receptors, protein kinases, and others,
Cdc37/Hsp90 complexes appear to interact exclusively with the catalytic
subunit of protein kinases, with one notable exception (14, 15).
Binding sites for the protein kinase client and for Hsp90 have been
mapped to the N- and C-terminal regions of Cdc37, respectively (13, 15,
16), providing further support for the notion that Cdc37 functions as a
kinase-targeting co-chaperone of Hsp90. Cdc37 has also been
demonstrated to possess chaperone activity on its own, independent of
Hsp90 (17).
We show here that the CKII/Cdc37 connection is unique among the known
interactions between Cdc37 and protein kinases because the highly
conserved N terminus of Cdc37 contains an evolutionarily conserved CKII
consensus site that is phosphorylated by CKII in vivo.
Further, CKII and Cdc37 constitute a positive feedback loop. Although
Hsp90 inhibition reduces CKII function in vivo, Cdc37 and
not Hsp90 is the limiting factor in maintaining CKII function. We also
present genetic evidence that CKII-mediated phosphorylation of Cdc37 is
essential for the ability of the latter to maintain the activity of
multiple protein kinases involved in diverse cellular functions. This
reveals a previously unsuspected role for CKII in regulating the
activity of diverse protein kinases (via Cdc37 phosphorylation).
Strains and Growth Media--
S. cerevisiae strains
used in this study are listed in Table I. Yeast strains were grown in
rich glucose medium (YPD: 1% yeast extract, 2% peptone, and 2%
glucose) or in supplemented minimal medium (18) at different
temperatures as indicated in figure legends. Eviction of
URA3-marked plasmids was accomplished by plating cells on
supplemented minimal medium containing 0.75 mg/ml 5-fluoroorotic acid
(Diagnostic Chemicals Ltd.). Sporulation was carried out in liquid
sporulation medium (1% potassium acetate, 0.1% yeast extract, and
0.5% glucose). Geldanamycin
(GA),1 purchased from Sigma,
was dissolved in dimethyl sulfoxide and added to warm medium soon after
autoclaving. Escherichia coli strain DH5 Multicopy Suppression Screen--
The temperature-sensitive
strain YDH13 (Table I) was transformed via the spheroplast method (18)
with 10-20 µg of an S. cerevisiae genomic library in
multicopy vector YEp24 (19) and plated on CM medium lacking uracil and
leucine. Plates were incubated at 23 °C for 24 h to allow
accumulation of gene products expressed from YEp24 and then placed at
35 °C (2 °C above the maximum permissive temperature of YDH13).
YEp24-based suppressor plasmids were isolated by transforming E. coli strain SK1108 to ampicillin resistance with DNA-containing
extracts prepared from each suppressor strain (20). That suppression
was plasmid-linked was confirmed by retransformation of YDH13, and the
relevant gene was identified by similarly testing various subclones.
Among the genes identified in the screen was CDC37. The
correct assignment in this case was confirmed by constructing a
frameshift mutation (an additional GATC sequence) at the
BglII site in the CDC37 open reading frame. This
construct is predicted to encode a severely truncated product of 120 residues and fails to suppress YDH13.
Construction of CDC37 Phosphorylation Site
Mutants--
Site-directed mutations in CDC37 were prepared
by the general method of Deng and Nickoloff (21). A 3.3-kb
BamHI/KpnI fragment containing the gene was
subcloned into pUC19 to serve as a template. cdc37-S14,17A
was prepared using the Transformer site-directed mutagenesis kit
(Clontech) according to instructions from the manufacturer; cdc37-S14A, cdc37-S17A, and
cdc37-S14,17E were prepared using the equivalent Chameleon
site-directed mutagenesis kit (Stratagene). The four mutagenic
oligonucleotides were: cdc37-S14,17A,
5'-GGATAAAATTGAACTAGCAGATGATGCTGATGTCGAGG-3'; cdc37-S14A, 5'-GGATAAAATTGAACTAGCAGATGATTCTGATGTCG-3';
cdc37-S17A, 5'-GAACTATCAGATGATGCTGATGTCGAGGTGC-3';
cdc37-S14,17E,
5'-GGATAAAA- TTGAACTAGAAGATGATGAAGATGTCGAGGTGCA-3'. The nucleotide changes for each mutation are underlined. The entire coding region of each mutant was sequenced to confirm that only the
desired mutation was introduced. For expression in yeast, the wild-type
and each mutant insert (BamHI/KpnI fragment) were subcloned into BamHI/KpnI-digested single-copy
vectors pRS314 (TRP1) and pRS316 (URA3) (22) and
multicopy vector pRS426 (URA3) (23).
Strains carrying the above mutations as the only CDC37
allele were constructed as follows. CDC37 (in pRS316) was
introduced into a diploid yeast strain (gift from David Morgan)
carrying a LEU2-marked deletion in one copy of the
CDC37 gene (7). The resultant strain was sporulated, and
haploid progeny that were auxotrophic for leucine and uracil
(i.e. cdc37 null and rescued by wild-type
CDC37 on pRS316) were selected for further manipulation. After selection of a haploid strain with the a mating type, cells were transformed with CDC37, cdc37-S14,17A,
cdc37-S14A, cdc37-S17A, and
cdc37-S14,17E (all on pRS314). Transformants were plated
twice in succession on minimal medium containing 5-fluoroorotic acid to
evict the URA3-marked plasmid, yielding strains YSB11 through YSB15, respectively (Table I). All strains were tested for
growth at a range of temperatures from 23 to 38 °C.
Overexpression of CKII and Hsc/p82--
The Western Blotting--
Five A600 units of
cells were pelleted by centrifugation, resuspended to a total volume of
100 µl by addition of sterile deionized water, and stored at
Metabolic Labeling and Immunoprecipitation--
Strains were
grown in YPD to mid-log phase at 26 °C, and aliquots containing 5 A600 units of cells were removed, labeled with [32P]orthophosphate (500 µCi/sample) for 2.5 h at
26 or 37 °C, harvested, and washed in water. Protein extracts were
prepared as above, except that extracts were adjusted to 1% Nonidet
P-40, 0.5% deoxycholate (to trap SDS in mixed micelles) prior to
incubation with 1 µl of affinity-purified rabbit polyclonal antibody
against Cdc37 (gift of David Morgan, University of California, San
Francisco, CA) (7). Immunoprecipitation was performed essentially as
described by Cardenas et al. (26). Immunoprecipitated
proteins were resolved by electrophoresis in an SDS-polyacrylamide gel
(10%), and labeled Cdc37 was visualized with a Molecular
Dynamics PhosphorImager.
Morphological Examinations--
Strains were grown to mid-log
phase at 23 °C and fixed by addition of formaldehyde to a final
concentration of 3.7%. Cell morphology was visualized using Nomarski
optics. For generation of growth curves, saturated cultures of the
relevant strains were inoculated into fresh YPD at a starting
concentration of 3 × 105 cells/ml. Cultures were
grown at 23 °C with vigorous shaking at 450 rpm. To determine cell
number, aliquots were removed at the indicated times, fixed with 3.7%
formaldehyde, and counted in a hemacytometer.
In Vivo Assay for CKII Activity--
Steady-state Tyr
phosphorylation of Fpr3 in the absence of Ptp1 function was used as an
assay for CKII activity in vivo (5). Deletion of
PTP1 was carried out by the PCR method of Wach et al. (27). Two primers were used to amplify the kanamycin
resistance gene from pFA-6A (gift of Peter Philippsen, Biozentrum,
Switzerland) together with flanking sequences derived from the
immediately upstream and downstream regions of PTP1. The
forward primer used was
5'-CAAGAAAAGCGTTTGTTAGTAGTAGTTTGCGACAGTGGAGCGTACGCTGCAGGTCGAC-3', and the reverse primer used was
5'-TATTGAAAAAATGCACAATTAGGAACTTTTATATAGCTGTATCGATGAATTCGAGCTCG-3' (PTP1 flanking sequences underlined). Disruptions were
verified by PCR.
To assay steady-state Tyr phosphorylation of Fpr3, isogenic wild-type
and mutant strains were routinely grown in minimal medium to mid-log
phase at 23 °C. For experiments involving GAL-regulated overexpression of Drosophila CKII, strains were grown at
30 °C in 2% raffinose medium lacking uracil. Early log phase
cultures were shifted to 37 °C for 3 h, adjusted to 2%
galactose to induce CKII expression, and incubated for an additional
3 h. To assay Tyr phosphorylation of Fpr3 at different cell cycle
stages, cultures of YSB48 (Table I) were first grown in YPD to mid-log
phase at 25 °C. Cultures were then adjusted to 10 µg/ml Isolation of Cdc37 as a Multicopy Suppressor of cka2-13
Mutants--
To identify genes which interact genetically with CKII in
S. cerevisiae, we utilized strain YDH13 (Table
I) to screen for multicopy suppressors of
the temperature-sensitive cka2 allele, cka2-13ts (28). The screen was carried out at
the minimum restrictive temperature of 35 °C, and one of the
suppressor plasmids identified encoded the protein kinase-specific
(co-)chaperone, Cdc37 (Fig. 1A). Multicopy
CDC37 also suppressed the slow growth rate at permissive temperature and flocculation displayed by YDH13 (data not shown). CDC37 suppressed four other
cka2ts alleles (data not shown), as well as two
alleles of the CKA1 gene, encoding the
Because Cdc37 has been shown to act as a chaperone either by itself
(17) or in concert with Hsp90 (13), we assessed the effect(s) of GA, an
Hsp90-specific inhibitor, on cka2-13 mutants. GA is a member
of the benzoquinoid ansamycin family of antibiotics that binds Hsp90
within a conserved pocket that constitutes the nucleotide binding site
of Hsp90, and inhibits Hsp90 ATPase activity (29-31). GA has been
shown to indirectly inhibit a variety of oncogenic tyrosine kinases and
other substrates of Hsp90 including mineralocorticoid receptor and
glucocorticoid receptor (32-36). Refolding of denatured firefly
luciferase by the Hsp90 chaperone complex is also inhibited by
treatment with ansamycins (37). GA has been shown to inhibit Hsp90
function in vivo in yeast as well (25). In contrast to human
cell lines, which are sensitive to GA at nanomolar concentrations, the
growth of wild-type yeast is unimpaired even at concentrations of 2 mM GA (25). We have also found similar results with strains wild-type for CKII activity (data not shown).
As shown in Fig. 1B, cka2-13 mutants were
sensitive to 35 µM GA at 23 °C (permissive
temperature). Moreover, this sensitivity was suppressible by
CDC37 overexpression. As might be expected, overexpression
of the yeast homologs of Hsp90, HSC82 or HSP82 (which are the targets of GA in yeast; Refs. 25 and 31), also suppressed the GA sensitivity of cka2-13 mutants. A mutant
allele of the yeast heat shock factor, hsf1-583, was
marginally sensitive to GA at this temperature (used as a positive
control) (25), whereas yeast that are wild-type for CKII were
unaffected. CDC37 overexpression suppressed the GA
sensitivity of cka2-13 mutants at temperatures from 21 to
34 °C (lowest and highest temperatures tested; data not shown). The
minimum restrictive temperature for cka2-13 mutants is
35 °C, and the fact that they do not grow even at 21 °C in the
presence of GA emphasizes the importance of Hsp90 function for the
survival of cka2-13 mutants. Consistent with the reported
effects of GA on protein kinases in mammalian cells, treatment of
cka2-13 cells with GA was found to cause decreased steady-state levels of CKII-dependent phosphorylation of
Fpr3 (Fig. 1C; see below for a discussion of this assay). We
therefore conclude that, upon prolonged incubation of
cka2-13 cells with GA, CKII activity drops below a critical
threshold causing these cells to die.
Cdc37 and Not Hsp90 Is the Limiting Factor in Maintenance of CKII
Function--
CDC37 overexpression has been shown to
suppress the temperature-sensitivity of hsp90 mutants in an
allele-specific manner (17), raising the possibility that Cdc37
enhances Hsp90 function in vivo in S. cerevisiae.
Alternatively or in addition, Cdc37 might function independently of
Hsp90 to protect one or more critical targets from denaturation in an
hsp90 mutant background. Given the GA sensitivity of
cka2-13 mutants, we sought to determine whether suppression
of cka2-13 by CDC37 was mediated via Hsp90. To
explore the general relationship between Cdc37 and Hsp90 in yeast, we
made use of the previously reported observation that hsf1-583 cells are sensitive to GA because of reduced Hsp90
expression (25, 38). If Cdc37 enhances Hsp90 function, CDC37
overexpression might be expected to suppress the GA sensitivity of an
hsf1-583 mutant. As shown in Fig.
2A, overexpression of
CDC37 did not enhance the ability of hsf1-583
mutants to grow in the presence of GA at either 25 or 30 °C. This
result suggests that CDC37 overexpression does not enhance
Hsp90 function (or expression) in vivo. To determine whether
the ability of CDC37 to suppress the GA sensitivity of cka2-13 mutants might be mediated via an increase in Hsp90
function, we tested whether direct overexpression of HSC82
or HSP82 could rescue the temperature sensitivity of a
cka2-13 strain. As shown in Fig. 2B, neither gene
is able to suppress this mutant under conditions where CDC37
is an effective suppressor.
The above results make it unlikely that CDC37 overexpression
suppresses the GA sensitivity of a cka2-13 strain by
enhancing Hsp90 function. One possibility is that Cdc37 is itself an
independent chaperone of CKII, a conclusion consistent with the ability
of purified Cdc37 to protect CKII activity in vitro (17).
However, this interpretation does not explain the fact that
cka2-13 mutants are sensitive to GA (Fig. 1B),
which implies that Hsp90 function is required for CKII activity. An
alternative possibility is thus that Hsp90 and Cdc37 are both required
for CKII activity, possibly as a functional complex, but that Cdc37 is
the limiting component (overexpression of Cdc37 but not Hsp90
suppresses the temperature sensitivity of a cka2-13 strain).
A similar conclusion has been reached regarding the nature of the
Cdc37/Hsp90 relationship with a temperature-sensitive mutant of the Hck
kinase in human cells (39). Because our results were consistent with
the possibility that Cdc37 is the limiting factor in the maintenance of
CKII function, we decided to characterize further the nature of the
CKII/Cdc37 relationship.
Cdc37 Is a Physiological Substrate of CKII--
Alignment of Cdc37
sequences from diverse organisms revealed an evolutionarily conserved
CKII phosphorylation motif near the N terminus of the protein (see Fig.
4A). All available Cdc37 sequences conserve a potential CKII
phosphorylation site at Ser-14 (S. cerevisiae numbering),
and the fungal sequences share a second potential site at Ser-17.
Residue 17 represents the important +3 determinant of the Ser-14 site,
and this is potentially satisfied by a phospho-Ser residue in the
fungal species or by a glutamate in the others. Consistent with these
facts, yeast Cdc37 has been shown to be a substrate of mammalian CKII
in vitro (17).
The above data suggest that Cdc37 may be an in vivo
substrate of CKII. To test this possibility, we examined Cdc37
phosphorylation in vivo via metabolic labeling and
immunoprecipitation in wild-type versus CKII-deficient
strains. Western blotting revealed that these strains had comparable
Cdc37 levels (data not shown). As shown in Fig.
3 (lanes 1-4), Cdc37 is
radiolabeled in vivo in wild-type cells and also in a
cka1
To confirm the importance of the CKII phosphorylation site motif on
Cdc37, we compared the in vivo phosphorylation of wild-type Cdc37 with that of a Cdc37-S14,17A mutation (see below). As shown in
Fig. 3 (lanes 7 and 8), in vivo
labeling of the mutant is dramatically reduced compared with that of
wild-type Cdc37. Although we cannot formally exclude the possibility of
a conformational change that affects CKII phosphorylation elsewhere on
the molecule, these results strongly suggest that Ser-14 and/or Ser-17
constitute a major site of Cdc37 phosphorylation in
vivo.
CKII Phosphorylation Site Mutants of CDC37--
To probe the
functional significance of Cdc37 phosphorylation by CKII, mutant
alleles encoding non-phosphorylatable (cdc37-S14,17A), semi-phosphorylatable (cdc37-S14A and
cdc37-S17A), and quasi-phosphorylated (cdc37-S14,17E) derivatives of CDC37 were
constructed (Fig. 4A) and
expressed from their native promoters in a CDC37 null
background. Western blotting with anti-Cdc37 antibody revealed that all
of the mutants are expressed at levels comparable to that of wild-type (data not shown). The cdc37-S14,17A strain was the most
severely affected, displaying an extremely slow growth rate (doubling
time of 10 h versus 90 min for the isogenic wild-type
control) and an elongated, enlarged morphology, even at 23 °C (Fig.
4, B and C). The cdc37-S14A strain was
more severely affected for both growth and morphology than the
cdc37-S17A mutant (Fig. 4, B and C),
supporting the idea that the evolutionarily conserved CKII site at
Ser-14 is more important for Cdc37 function than Ser-17. The
cdc37-S14,17A and cdc37-S14A strains were found
to be temperature-sensitive for growth at 30 and 37 °C,
respectively, whereas cdc37-S17A was not sensitive for
growth at any of the temperatures tested (data not shown). A
cdc37-1 allele, which encodes a C-terminal truncation of the
last third of the protein (7), had an essentially wild-type morphology
under the same conditions (Fig. 4C), underscoring the importance of the CKII phosphorylation sites in Cdc37.
When compared with cdc37-S14,17A, the
cdc37-S14,17E strain displayed a faster growth rate
(doubling time of 4 versus 10 h) and less severe
morphological defects (Fig. 4, B and C).
Furthermore, the minimum restrictive temperature of
cdc37-S14,17E was 35 °C compared with 30 °C for
cdc37-S14,17A (data not shown). The ability of the double
glutamate replacement to partially rescue the phenotype associated with
the double alanine replacement is consistent with Ser-14 and Ser-17
serving as phosphorylation sites. The inability of
cdc37-S14,17E to provide wild-type function might reflect
the difference between a phospho-Ser versus a glutamate
residue and/or a requirement for phosphoryl group turnover.
All of the CKII phosphorylation site alleles were recessive to
wild-type (data not shown), indicating that they represent loss-of-function mutations.
Because of the known relationship between Cdc37 and Hsp90, we also
examined the growth of CKII phosphorylation site mutants of Cdc37 in
the presence of the Hsp90 inhibitor GA. As shown in Fig.
5, a cdc37-S14A mutant is
sensitive to GA (Fig. 5, vector), and this sensitivity is
suppressed by overexpression of wild-type CDC37 as well as
by cdc37-S17A. This mutant is also weakly suppressed by
overexpression of cdc37-S14A itself, indicating dosage
sensitivity of this mutant allele. In contrast, overexpression of
cdc37-S14,17A not only fails to rescue the GA sensitivity of
the cdc37-S14A mutant but results in a dominant-negative
effect. These data confirm the importance of the CKII sites for Cdc37
function in vivo and support a potential interaction between
Cdc37 and Hsp90 in yeast. They also corroborate the conclusions that
Ser-14 is the more important of the two sites but that both are
required for optimal function.
CDC37 and CKII Constitute a Positive Feedback Loop--
The
ability of CDC37 overexpression to suppress the temperature
and GA sensitivity of a cka2-13 mutant (Fig. 1, A
and B) prompted us to examine CKII activity in a strain with
impaired Cdc37 function using the phosphorylation state of Fpr3, a well
characterized substrate of CKII in S. cerevisiae, as an
indicator of CKII activity in vivo (5). Fpr3 is the only
known substrate of CKII phosphorylated on tyrosine (in addition to
serine and probably threonine), and phosphorylation of Fpr3 (at
Tyr-184) by CKII can thus be conveniently monitored by Western blotting
of whole cell extracts with anti-phosphotyrosine antibody.
Phosphorylation of Fpr3 at Tyr-184 is completely dependent upon
functional CKII in vivo, but accumulation of
Tyr-phosphorylated Fpr3 requires prior deletion of PTP1,
which encodes a tyrosine phosphatase active against this site (5).
PTP1 deletions had no effect on the growth, morphology, or
temperature sensitivity of the strains used to monitor CKII activity
(data not shown).
As shown in Fig. 6A,
Tyr-184-phosphorylated Fpr3 represented a major band in a Western blot
of a whole-cell extract prepared from a CDC37 ptp1 strain.
An increase in the intensity of this band was observed upon
overexpression of Drosophila CKII, as described previously
(5). Relative to the CDC37 strain, extracts made from
cdc37-S14A or cdc37-S17A cells had a lower steady
state level of phosphorylated Fpr3, either with or without
overexpression of Drosophila CKII (the comparable behavior
displayed by the S14A and S17A mutants in this experiment may reflect
the stringent conditions employed, such that both mutants are severely
impaired). The observed decrease in Fpr3 Tyr phosphorylation in the
cdc37 mutant strains indicated that normal Cdc37 function is
required to sustain CKII activity in vivo. Consistent with
this conclusion, overexpression of cdc37-S14,17A or
cdc37-S14A (unlike wild-type CDC37
overexpression) failed to suppress the GA sensitivity (Fig. 6B) or temperature sensitivity of a cka2-13
strain (data not shown). These results also implicate the CKII
phosphorylation site(s) in the ability of Cdc37 to maintain CKII
activity.
The genetic and biochemical results described here support the idea
that CKII and Cdc37 exist in a positive feedback loop wherein CKII
activates Cdc37 by phosphorylation, which then can activate/maintain
CKII. Because Cdc37 is required at the G1 and G2/M phases of the cell cycle (7, 8), we asked whether CKII activity might peak during these same stages. To monitor CKII activity
during the cell cycle, we arrested a strain that is wild-type for CKII
in the G1, S, and G2/M phases of the cell cycle
by treatment with The CKII/CDC37 Feedback Loop Helps Maintain the Activity
of Multiple Protein Kinases--
The CKII phosphorylation sites on
Cdc37 are important for its ability to activate/maintain CKII activity
(Fig. 6B). Because Cdc37 functions as a chaperone for a
number of protein kinase catalytic subunits, we wished to determine
whether phosphorylation of Cdc37 by CKII was important for these
substrates as well. Several potential Cdc37 clients have been
identified by genetic studies in S. cerevisiae, including
Kin28 (9), Mps1 (10), and Cdc28 (7). Because Hsp90 and Cdc37 often
share similar kinase substrates (17), we reasoned that
temperature-sensitive mutants of KIN28, MPS1, and
CDC28 might be sensitive to GA in a fashion similar to the
cka2-13 strain (Fig. 1B).
The normal restrictive temperature for mps1-1,
kin28-ts3, and cdc28-109 is reported to be
30 °C, 35 °C, and 37 °C, respectively. All three mutants were
found to be GA-sensitive at temperatures lower than their restrictive
temperature (see Fig. 7), whereas their
isogenic wild-type controls were insensitive (data not shown). As shown
in Fig. 7, the GA sensitivity of kin28-ts3 and
mps1-1 mutants was suppressed by overexpression of
CDC37 but not by cdc37-S14,17A or
cdc37-S14A. This is consistent with the idea that the CKII phosphorylation sites on Cdc37 are essential for its ability to protect
Kin28 and Mps1 in addition to CKII and that these kinases are reduced
in function/activity in both cdc37-S14,17A and
cdc37-S14A.
The GA sensitivity of cdc28-109 was not suppressed by
CDC37 overexpression (Fig. 7) at a variety of temperatures
tested (nor was its temperature sensitivity suppressible by
CDC37 overexpression; data not shown). However, we consider
it unlikely that the CKII phosphorylation site mutants of
CDC37 have adequate Cdc28 function because these mutants are
partially suppressed by Cdc28 overexpression as well as by deletion of
SWE1, which inhibits the mitotic form of
Cdc28.3 Moreover,
cdc37-1 mutants have been shown to be limiting for Cdc28
function previously (7, 12), and cdc37-1 mutants are synthetically lethal when combined with the cdc28-109
mutation (41). Cdc37 function thus is clearly required for Cdc28.
However, in contrast to the other kinase mutants tested, Cdc37 function might not be limiting in cdc28-109 mutants.
We have presented biochemical and genetic evidence for a positive
feedback loop between CKII and Cdc37. According to our model (see Fig.
8), CKII phosphorylates and activates
Cdc37, which in turn promotes/maintains the activity of CKII. This
model is supported by the following observations: 1) CDC37
functions as a multicopy suppressor of temperature-sensitive CKII
alleles; 2) loss-of-function mutations in CDC37 result in
reduced CKII activity toward a known physiological substrate, Fpr3,
in vivo; 3) CKII phosphorylates Cdc37 in vivo at
Ser-14 and/or Ser-17 (or at a site affected by mutation of these
residues); 4) replacement of Ser-14 and Ser-17 with alanine results in
severe phenotypic deficits that are partially reversed in the
corresponding glutamate mutant; and 5) CKII phosphorylation site
mutants of Cdc37 fail to suppress the GA sensitivity of a cka2-13 strain. Additionally, CKII activity peaks at the
same stages of the cell cycle that Cdc37 function has been shown to be
essential by several groups.
' catalytic subunit of protein kinase CKII. Unlike
wild-type cells, cka2-13 cells were sensitive to the
Hsp90-specific inhibitor geldanamycin, and this sensitivity was
suppressed by overexpression of either Hsp90 or Cdc37. However, only
CDC37 was capable of suppressing the temperature
sensitivity of a cka2-13 strain, implying that Cdc37 is the
limiting component. Immunoprecipitation of metabolically labeled Cdc37
from wild-type versus cka2-13 strains revealed
that Cdc37 is a physiological substrate of CKII, and Ser-14 and/or
Ser-17 were identified as the most likely sites of CKII phosphorylation
in vivo. A cdc37-S14,17A strain lacking these
phosphorylation sites exhibited severe growth and morphological defects
that were partially reversed in a cdc37-S14,17E strain.
Reduced CKII activity was observed in both cdc37-S14A and
cdc37-S17A mutants at 37 °C, and cdc37-S14A
or cdc37-S14,17A overexpression was incapable of protecting
cka2-13 mutants on media containing geldanamycin.
Additionally, CKII activity was elevated in cells arrested at the
G1 and G2/M phases of the cell cycle, the same
phases during which Cdc37 function is essential. Collectively, these
data define a positive feedback loop between CKII and Cdc37.
Additional genetic assays demonstrate that this CKII/Cdc37
interaction positively regulates the activity of multiple protein
kinases in addition to CKII.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(35-44 kDa) and
(24-28 kDa), which combine to form an
2
2 tetramer (for review, see Refs. 1-4).
In Saccharomyces cerevisiae, the enzyme consists of two
distinct catalytic subunits,
and
' (encoded by the genes
CKA1 and CKA2, respectively), and two regulatory subunits,
and
' (encoded by the genes CKB1 and
CKB2, respectively). CKII recognizes Ser/Thr (or in
exceptional cases Tyr) (5) in an acidic environment (2). CKII
phosphorylates a broad spectrum of endogenous substrates involved in
transcription, translation, signal transduction, and other functions.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Clontech) was grown in Luria broth containing 50 µg/ml ampicillin.
and
subunits of Drosophila melanogaster CKII were expressed
from the single-copy vector pBM272 under control of the GAL1,10 promoter, as described previously (24).
Overexpression of Hsc82 or Hsp82 was achieved with multicopy YEp24
plasmids (gift of Kevin Morano) expressing each gene under control of
its own promoter (25).
80 °C. Thawed cell suspensions were mixed rapidly with 100 µl of
boiling 2× SDS sample buffer, vortexed, and incubated at 100 °C for
5 min. After heat treatment, the samples were centrifuged at
11,000 × g for 10 min, and the clarified supernatants were stored at
20 °C. Protein concentration was determined with the BCA protein assay (Pierce) using bovine serum albumin as a standard, and equal amounts of protein were electrophoresed in a 10%
SDS-polyacrylamide gel and transferred to polyvinylidene difluoride
membrane (Immobilon). Immunodetection was performed using the Amplified
Alkaline Phosphatase Immun-Blot kit (Bio-Rad). Mouse monoclonal
anti-Cdc37 (gift from Avrom Caplan, Mt. Sinai School of Medicine, New
York, NY) was used at 1:1500 dilution, rabbit polyclonal anti-Cdc37 at
1:3000 dilution (gift of Steve Reed, The Scripps Research Institute, La
Jolla, CA), mouse anti-phosphotyrosine antibody 4G10 (Upstate
Biotechnology, Inc.) at 1:1000 dilution, and rabbit anti-Fpr3 (gift
from Jeremy Thorner, University of California, Berkeley, CA) at 1:1500 dilution.
-factor
(4 h), 0.1 M hydroxyurea (5 h), or 30 µg/ml benomyl (4 h)
to promote arrest in the G1, S, or G2/M phases
of the cell cycle, respectively. Tyr-phosphorylated Fpr3 was detected
by Western blotting as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit of
S. cerevisiae
CKII.2
S. cerevisiae strains
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Fig. 1.
Cdc37 and Hsp90 augment CKII function.
A, multicopy suppression of cka2-13 by
CDC37. YDH6 (cka1 CKA2; row
1), YDH13 (cka1
cka2-13ts;
row 2), and YDH13 carrying multicopy CDC37
(row 3) were spotted in triplicate on YPD and grown at the
indicated temperatures. B, suppression of GA sensitivity of
cka2-13 mutant by CDC37 or
HSC/P82 overexpression. Empty vector (YEp24) or
multicopy plasmids expressing CDC37, HSC82, or
HSP82 were transformed into YDH13, spotted in duplicate on
YPD or YPD supplemented with 35 µM GA, and grown at
23 °C. NSY-B (hsf1-583) and YDH6 were used as controls
(bottom row). All cells were spotted at 5000 cells/spot.
C, GA-mediated inhibition of CKII activity in a
cka2-13 strain. YSB48 (cka1
CKA2
ptp1) and YSB49 (cka1
cka2-13ts ptp1) were grown in minimal
medium at 23 °C to mid-log phase, transferred to YPD with or without
100 µM GA, and incubated for 3 h. Immunoblots of
cell lysates were probed with anti-Tyr(P) antibody to assay for levels
of phosphorylated Fpr3 as a measure of CKII activity in
vivo. Blots were stripped and reprobed with anti-Fpr3.
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Fig. 2.
Cdc37 and not Hsp90 is the
limiting factor in the maintenance of CKII function. A,
overexpression of Cdc37 does not enhance Hsp90 function. NSY-B
(hsf1-583) cells were transformed with empty plasmid
(pRS426) or CDC37 on a multicopy plasmid. The resulting
strains were spotted in duplicate on YPD without or with 35 µM GA at 5000 and 500 cells/spot and incubated at 25 or
30 °C for 3 days. B, Cdc37 but not Hsp90 suppresses a
CKII temperature-sensitive mutant. YDH13 (cka1
cka2-13) cells were transformed with empty vector (YEp24) or
multicopy plasmids encoding CDC37, HSP82, or
HSC82. The resulting strains were streaked on YPD and
incubated at 35 °C for 3 days.
CKA2 strain, at both permissive and
non-permissive temperature. In contrast, incorporation into Cdc37 is
strongly diminished in a cka1
cka2ts strain at permissive temperature, and
essentially abolished at non-permissive temperature (Fig. 3,
lanes 5 and 6). The simplest interpretation of
these data is that Cdc37 is a direct substrate of CKII in
vivo.
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Fig. 3.
Cdc37 is a physiological substrate of
CKII. Cells were grown at 26 °C and then labeled with
[32P]orthophosphate for 2.5 h at either 26 °C
(lanes 1, 3, 5, 7, and
8) or 37 °C (lanes 2, 4, and
6). Total cell extracts were immunoprecipitated with a
polyclonal antibody against Cdc37, and immunoprecipitated material was
analyzed by gel electrophoresis and phosphorimaging. Lanes 1 and 2, YPH499 (CKA1 CKA2 CDC37); lanes
3 and 4, YDH6 (cka1 CKA2
CDC37); lanes 5 and 6, YDH13
(cka1
cka2-13 CDC37); lanes 7 and
8, YRM 14.0 (CKA1 CKA2 cdc37-1) transformed with
a single-copy plasmid expressing CDC37 or
cdc37-S14,17A, respectively. The cdc37-1 allele
contains a premature termination codon and does not produce a 70-kDa
Cdc37 signal (7).
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Fig. 4.
CKII phosphorylation site mutants of
CDC37. A, sequence alignment of the
conserved N terminus of Cdc37 from various species (human, chicken,
D. melanogaster, Caenorhabditis elegans,
Schizosaccharomyces pombe, Candida albicans, and
S. cerevisiae) plus S. cerevisiae Cdc37
phosphorylation site mutations. The conserved CKII motif is shown
boxed, with the potentially modified Ser residues in
bold. B, growth rates of CDC37 mutant
strains. Strains (YSB11-YSB15) harboring the indicated
CDC37 alleles were inoculated into YPD at a starting
concentration of 3 × 105 cells/ml and grown at
23 °C. Cell number was monitored as described under "Experimental
Procedures." C, morphology of CDC37 mutant
strains. Strains harboring the indicated CDC37 alleles were
grown at 23 °C to mid-log phase, and photographs of cells were taken
on a Zeiss IM 35 epifluorescence microscope fitted with Nomarski
optics. SR672-1, carrying the C-terminal truncation allele
cdc37-1, is included for comparison.
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Fig. 5.
GA sensitivity of cdc37
phosphorylation site mutants. YSB13 (cdc37-S14A)
was transformed with empty vector (pRS426) or a multicopy plasmid
carrying CDC37, cdc37-S14,17A,
cdc37-S14A, or cdc37-S17A. Resulting strains were
spotted at 5000 cells/spot on YPD or YPD supplemented with 35 µM GA and incubated at 28 °C for 2 days.
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Fig. 6.
CKII phosphorylation sites on Cdc37 are
essential for the ability of Cdc37 to maintain CKII function.
A, cdc37-S14A and -S17A mutants
exhibit reduced CKII activity. YSB41 (CDC37 ptp1), YSB40
(cdc37-S14A ptp1), or YSB72 (cdc37-S17A ptp1)
transformed with empty vector (pBM272) or a plasmid expressing both
subunits of D. melanogaster CKII were grown to mid-log phase
in raffinose medium at 30 °C, shifted to 37 °C for 3 h, and
then incubated in the presence of galactose for 3 h. Cell extracts
were immunoblotted with anti-Tyr(P) antibody 4G10. A parallel blot was
probed with anti-Fpr3. B, CKII phosphorylation sites on
Cdc37 are essential for the latter to support CKII function. YDH13
(cka2-13) transformed with empty multicopy plasmid (pRS426)
or multicopy plasmid expressing CDC37,
cdc37-S14,17A, or cdc37-S14A were spotted at 5000 cells/spot on YPD or YPD supplemented with 35 µM GA and
incubated at 21 °C for 3 days. C, CKII activity is
elevated in cells arrested at G1 or G2/M.
Extracts prepared from asynchronous cultures of YSB48 (ptp1)
or YSB48 cultures arrested in G1, S, or G2/M
were probed with anti-P-Tyr antibody to assay steady state levels of
phosphorylated Fpr3. Blots were stripped and reprobed with
anti-Fpr3.
-factor, hydroxyurea, and benomyl, respectively.
As shown in Fig. 6C, CKII activity toward Fpr3 is high in
G1 and G2/M phase-arrested cells and low in S
phase-arrested cells. A requirement for CKII in the G1 and
G2/M phases of the cell cycle has been documented previously (28), and another group has previously reported that CKII
activity is most likely inhibited during S phase in human cells (using
a substrate other than Fpr3) (40). Failure to phosphorylate Cdc37 could
therefore contribute to the previously reported G1 and
G2/M arrest of temperature-sensitive CKII mutants (28).
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Fig. 7.
CKII phosphorylation sites on Cdc37 are
essential for its ability to protect Kin28 and Mps1. GF2092
(kin28-ts3), WX241-17a (mps1-1), and CMY677
(cdc28-109) were transformed with empty vector (pRS426) or a
multicopy plasmid expressing CDC37,
cdc37-S14,17A, or cdc37-S14A. Resulting strains
were spotted on YPD or YPD supplemented with 35 µM GA and
incubated at either 23 °C (WX241-17a and CMY677) or 28 °C
(GF2092) for 3 days.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
A positive feedback loop between CKII and
Cdc37 positively regulates multiple protein kinases. Cdc37
promotes the activity of diverse protein kinase clients including CKII,
which in turn phosphorylates and activates Cdc37. P'ase
indicates a potential protein phosphatase active against CKII-modified
Cdc37.
In addition to augmenting the activity of Cdc37 and CKII, the positive
feedback loop between these two proteins also promotes the activity of
additional protein kinases including, Mps1 (required for spindle pole
duplication as well as the spindle checkpoint) (10), Kin28 (a
C-terminal domain kinase of RNA polymerase II that mediates the
interaction of polymerase II with capping enzymes during transcription
of genes) (9, 42), and Cdc28 (the yeast cell cycle engine) (7). At
least two other kinases also represent substrates of the positive
feedback loop, because the cdc37-34 allele (encoding a
Ser-14 to leucine replacement) (14) has been shown to be defective in
the maturation/activation of Ste11 (a MAP kinase involved in -factor
signaling in yeast) (11) as well as the mammalian oncoprotein v-Src
(when expressed in yeast) (8). Additional Cdc37 clients such as Cak1
(12) may be dependent upon CKII-mediated phosphorylation of Cdc37 as
well, but have not been tested explicitly. By regulating Cdc37
phosphorylation, CKII plays an important role in promoting the activity
of multiple cellular kinases involved in diverse functions.
Because CKII, Cdc37, and the CKII phosphorylation site on Cdc37 are evolutionarily conserved, a similar mechanism that positively regulates multiple kinases (including possible orthologs of yeast kinase clients of the CKII/Cdc37 feedback loop shown in Fig. 8) may be in place in higher organisms as well. Such a model also gives us a deeper appreciation of the molecular basis for the pleiotropic nature of CKII since, by phosphorylating and activating Cdc37 (which represents only one of the many CKII substrates identified so far), it can play a role in diverse signal cascades regulated by Cdc37 clients.
Cdc37 overexpression has been shown to induce tumors in mice (43, 44). Although Cdc37 seems to cooperate with c-Myc and cyclin D1 in producing tumors, the biochemical mechanisms underlying its action most likely include multiple kinase substrates of Cdc37 that work in concert to promote proliferation. Proto-oncogenic kinases that Cdc37 has been shown to interact with include v-Src (8), Raf-1 (16), and CDK4 (13). CKII overexpression also produces tumors in mice (45, 46), and the mechanism for this might involve its ability to activate Cdc37, which can then activate a host of other oncogenic kinases. It would be interesting to see whether mice overexpressing non-phosphorylatable Cdc37 would remain able to cooperate with c-Myc or cyclin D1 in promoting oncogenesis.
How does phosphorylation of Cdc37 by CKII regulate its function? At least two possibilities exist. Phosphorylation might regulate the interaction of Cdc37 with Hsp90 and/or other co-chaperones. Alternatively, it might affect the interaction(s) between Cdc37 and its protein kinase clients or other substrates. We have been unable to detect protein-protein interactions between yeast Cdc37 and any other protein kinase using a two-hybrid system, and others have had trouble isolating proteins that interact with yeast Cdc37 as well (12). However, a physical interaction between Cdc37 and Ste11 has been reported in yeast (11), and the highly conserved N-terminal half of Cdc37 (which contains the conserved CKII phosphorylation site) has been shown to interact with at least one protein kinase, Raf-1, in mammalian cells (16). Based on the latter observation, we suspect that the CKII phosphorylation-deficient mutants of CDC37 might be impaired in interactions with target protein kinases. Consistent with such a possibility, overexpression of the N-terminal half of yeast CDC37 has a dominant negative effect in a cdc37-S14A but not a CDC37 strain.3
Because a feedback loop between CKII/Cdc37 regulates several other
proteins as well and because CKII activity (against Fpr3) shows a cell
cycle dependence, we suspect that there might also exist regulators
(activators/inhibitors) of the feedback loop. Such gene(s) might also
be responsible for modulation of CKII activity during the cell cycle.
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ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge Avrom Caplan, David Morgan, Steve Reed, and Jeremy Thorner for antibodies and Carl Mann, Gerard Faye, Kevin Morano, Mark Winey, and Peter Phillipsen for strains, plasmids, and reagents.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM54626 (to C. V. C. G.).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.
The first and second authors contributed equally to this work.
§ Present address: Dept. of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208.
¶ Present address: Dept. of Biochemistry, University of Kentucky, Lexington, KY 40506.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, Life Sciences Bldg., University of Georgia, Athens, GA 30602-7229. Tel.: 706-542-1769; Fax:
706-542-1738; E-mail: glover@arches.uga.edu.
Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M206662200
2 A. Rethinaswamy and C. V. C. Glover, unpublished data.
3 S. Bandhakavi and C. V. C. Glover, unpublished observations.
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ABBREVIATIONS |
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The abbreviation used is: GA, geldanamycin.
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REFERENCES |
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