From the Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, Missouri 65212
Received for publication, August 7, 2002, and in revised form, October 28, 2002
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ABSTRACT |
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The budding yeast Glc7 serine/threonine protein
phosphatase-1 is regulated by Glc8, the yeast ortholog of mammalian
phosphatase inhibitor-2. In this work, we demonstrated that similarly
to inhibitor-2, Glc8 function is regulated by phosphorylation. The
cyclin-dependent protein kinase, Pho85, in conjunction with
the related cyclins Pcl6 and Pcl7 comprise the major Glc8 kinase
in vivo and in vitro. Several glc7
mutations are dependent on the presence of Glc8 for viability. For
example, glc7 alleles R121K, R142H,
and R198D are lethal in combination with a glc8
deletion. We found that glc7-R121K is lethal in combination
with a pho85 deletion. This finding indicates that Pho85 is
the sole Glc8 kinase in vivo. Furthermore,
glc7-R121K is also lethal when combined with deletions of
pcl6, plc7, pcl8, and
pcl10, indicating that these related cyclins redundantly
activate Pho85 for Glc8 phosphorylation in vivo. In
vitro kinase assays and genetic results indicate that Pho85
cyclins Pcl6 and Pcl7 comprise the predominant Glc8 kinase.
A major fraction of mammalian protein phosphatase-1 exists in a
complex with inhibitor-2
(I-2)1 (1, 2). I-2 appears to
change the conformation of the protein phosphatase-1 catalytic subunit
(PP1c). When I-2 is phosphorylated in an inactive
PP1c·I-2 complex on Thr-72, the PP1c
becomes activated. This activation is thought to occur by the
transference of a phosphorylation-dependent I-2
conformational change to PP1c. This idea is supported by
data showing that phosphorylated I-2 mixed with inactive
PP1c cannot activate it (3, 4). Autodephosphorylation of
phospho-I-2 within the active PP1c·I-2 complex
deactivates PP1c, even though PP1c can be
removed from the complex in an active state. Therefore, I-2 can both
inactivate and activate PP1c by changing its conformation in a phosphorylation-dependent fashion. I-2 interaction
with denatured recombinant PP1c and subsequent
phosphorylation yields an active enzyme, which has inspired the notion
that I-2 is a PP1c molecular chaperone (5).
Several identified protein kinases phosphorylate I-2. Glycogen synthase
kinase-3 (GSK3), extracellular signal-regulated kinase-2, and
cyclin-dependent kinase-5 (CDK5) phosphorylate I-2 Thr-72 in vitro (6-9). Casein kinase-2 (CK2) phosphorylates I-2 on
three residues, Ser-86, Ser-120, and Ser-120, but does not change its activity. CK2 phosphorylation at these sites cooperatively enhances GSK3 phosphorylation at Thr-72 (10). Whether any of these kinases actually phosphorylate I-2 in vivo is unknown.
Glc8 is the budding yeast ortholog of I-2. Mutations in glc8
were discovered because they reduce yeast glycogen accumulation (11).
Glc8 inhibits mammalian PP1c and yeast PP1c,
Glc7, in vitro (12). I-2 has several
PP1c-binding sites in addition to the inhibitory region
(13). Alignments of I-2 and Glc8 sequences indicate that Glc8 may lack
some PP1c-binding sites found in I-2. In contrast to
PP1c in mammalian cells, the majority of Glc7 in yeast is
bound to Sds22 (14, 15). In comparison with other yeast Glc7-binding
proteins, the affinity for Glc8 is weak (16).
Glc7, like mammalian PP1c, regulates many physiological
processes: glycogen metabolism, transcription, translation initiation, membrane fusion, sporulation, and mitosis (17). In regard to its
mitotic function, evidence indicates that Glc7 dephosphorylates proteins phosphorylated by the Ipl1 protein kinase, which regulates chromosome bi-orientation on the spindle (18, 19). Indeed, overexpression or deletion of GLC8 can suppress
ipl1 mutations (12). These data suggest that Glc8 can
activate or inhibit Glc7 activity in vivo.
The regulatory Thr-72 in I-2 corresponds to Glc8 Thr-118. Previous work
indicated that this site must be a phosphorylatable residue for
in vivo Glc8 function (12). In this study, we identify the
Glc8 kinase as the cyclin-dependent kinase, Pho85, which is orthologous with mammalian CDK5. We also demonstrate that Pho85 phosphorylates Glc8 in vivo and that is the sole Glc8 kinase
in budding yeast. Moreover, four redundant Pho85 cyclins are required for in vivo Glc8 phosphorylation.
Strains and General Methods--
Rich (YEPD) and omission (SC)
media used for growth of Saccharomyces cerevisiae and LB
broth have been previously described (24, 25). Low adenine medium
contained 0.6% adenine (w/v). Glycogen accumulation was assayed
qualitatively by exposure of yeast colonies grown on YEPD plates to
iodine crystals for 3 min at room temperature (11). Wild-type strains
stain brown, and glycogen-deficient strains stain yellow.
Most strains of S. cerevisiae and Escherichia
coli used in this study are listed in Table
I. Additional strains included BY4743
diploids with kanMX4-marked deletions (20). Strains in Table
I with these deletions were made congenic to JC746-9D by six or more
backcrosses. The pcl8::HIS3 mutation
was made by transformation with a PCR fragment containing
HIS3 flanked by 40 bases from the 5' and 3' ends of
PCL8. This fragment was made by amplification of
HIS3 from pRS303 (21) with primers 466 (5'-AAGATTTTGT
TTGAGGTTTT GCAGATAAAC AAGAAGCAGT CTGTGCGGTATTTCACACCG-3') and 467 (5'-TATAGCAAAA CTCATGTGTT TCCTAATGCC TGTCTTGTAT
AGATTGTACTGAGAGTGCAC-3'). The pcl10::mTnURA3 was transferred from
plasmid V82B4 (22). The glc7-R121K mutation was scored by a
DraI restriction site polymorphism. The DraI site
present in the mutant allele was tested in a 1476-bp GLC7
fragment amplified by colony PCR (23) using primers 462 (5'-ATGACGAGTG
ATGATTGCATC-3') and 463 (5'-AGAAGCCCCA ATTAAAGTATGT-3'). URA3 was inserted 7 kb 5' from the glc7-R121K
allele using plasmid p1736 (11).
Plasmids--
All of the plasmids used in this study
are listed in Table II with their
relevant genotypes. Plasmid p1841 contains an
EcoRI-SalI GLC8 fragment from plasmid
p1814 (11) cloned into YEp352 (26). The SalI site in p1841
comes from its YCp50 vector sequences. Plasmid p2087 was made by
ligation of the SacI-SalI fragment from p1841,
containing GLC8, into pTSV30A, a 2µ ADE3 LEU2
plasmid from John Pringle (University of North Carolina, Chapel Hill,
NC). To make the GLC8 expression plasmid pPM1539, a
NdeI site was created at the GLC8 initiating
methionine codon by mutagenesis of single-stranded DNA from p1945 with
primer 86 (5'- CATCAGACATATGGGAGG-3', the initiating
methionine codon is underlined) (27). The
NdeI-HindIII GLC8 fragment was ligated
into pT7-7 (28), placing GLC8 behind the T7 promoter- and
ribosome-binding sites. Plasmid pYT114 contains a T118A
mutation in GLC8 made by overlapping PCR mutagenesis (29, 30). Primer pair 243 (5'-ATTAACCCTCACTAAAGGGA-3') and 379 (5'-GCACCTTGGTAGGGGGCCTTG-3') and primer pair 386 (5'-AATTCTCATGTTTGACAGCTT-3') and 378 (5'-GCCCAAGGCCCCCTACCAAGG-3') generated two overlapping fragments from pPM1539. These fragments, along with primers 386 and 243, were used to generate the complete T7p-GLC8-T118A-containing fragment in a subsequent PCR. A
ClaI-HindIII fragment from this final PCR product
was cloned into pT7-7.
The pYEX4T-1 derivatives that express GST fusions in EJ758
transformants (32) were from Elizabeth Grayhack and Eric Phizicky (University of Rochester) or ResGen. GST fusion plasmids from original
EJ758 transformant pools were retrieved by E. coli
transformation. Recombinational cloning generated these plasmids and
the yeast transformants contained a mixture of plasmid species (32).
Therefore, we chose an individual, isolated plasmid from the mixture
for the studies in this paper. We determined the DNA sequence of the entire open reading frame for each chosen plasmid. By comparison with
yeast DNA (33), mutations were found in many of the fusion genes (Table
III). We assume that these mutations do
not adversely affect the biochemistry of the fusion proteins. For the
Pcl1 and Pho81 pools, an authentic GST fusion plasmid could not be
found.
Glc8 Preparation--
E. coli strain BL21(DE3) (34)
transformed with either pPM1539, pYT114, or pYT116 was grown to early
exponential phase in LB broth containing 100 µg/ml ampicillin at
37 °C. Isopropyl- In Vitro Glc8 Kinase Assays--
Yeast crude extracts were
prepared by vortexing exponential phase cells 4 × 30 s at
4 °C in the presence of glass beads and an equal volume of
homogenization buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, and 10 mM NaF, 1× complete protease
inhibitor mixture (Roche Molecular Biochemicals)) followed by a 10-min
centrifugation at 4 °C. To remove contaminating endogenous ATP, the
extracts were further purified through P25 spin columns (Bio-Rad). The eluants were collected, and the protein concentrations were quantitated using bovine serum albumin as a standard (35). Glc8 kinase assay reactions were performed by mixing 10 µg of crude yeast extract or
the indicated amounts of purified GST fusion protein, 2.5 µg of Glc8,
5 µl of ATP mixture (1 mM ATP, 25 mM
MgCl2, 1200 cpm/pmol [ Isolation of Glc8-dependent Mutants--
The strain
JC945-8C/p2087 was mutagenized with ethyl methanesulfonate to 10%
survival and plated on YEPD (37). A colony sectoring assay was used to
recognize and isolate GLC8-dependent mutants
(38-40). Putative GLC8-dependent mutants were
initially selected by their inability to sector. True
GLC8-dependent mutants regained their ability to
sector when transformed with p1945. By this method we isolated 11 independent GLC8-dependent mutants after
screening 2 × 105 colonies. All of the mutations were
complemented by CEN GLC7 plasmids and were genetically
linked to the GLC7 locus.
We retrieved the GLC7 locus from one of the mutant strains,
JC968-1, by plasmid gap repair (11, 41). Transformation of JC968-1
with p1855 deleted for a GLC7 BglII-SalI fragment
yielded sectoring and nonsectoring transformants. The sectoring
transformants had repaired the p1855 gap to wild-type GLC7,
whereas the nonsectoring ones did not contain wild-type
GLC7. Sequencing of DNA of the retrieved GLC7
locus from two independent nonsectoring transformants revealed a G to A
transition in codon 121, which converted the wild-type Glc7 Arg-121 to
Lys (glc7-R121K). The Glc8 dependence of previously isolated
glc7 mutations (16) was tested by complementation of the
glc7-R121K sectoring trait and by lethality in the absence of GLC8.
Isolation of GST Fusion Proteins from
Yeast--
EJ758 transformants were grown at 30 °C to exponential
phase in SC In Vitro Phosphorylation of Glc8 on Thr-118--
The homology
between Glc8 and mammalian I-2 is greatest surrounding I-2 Thr-72, the
residue that is phosphorylated by GSK3, extracellular signal-regulated
kinase-2, and CDK5 (6-9, 11). The corresponding Thr-118 in Glc8 was
previously shown to be important for Glc8 function in vivo
(12). Consequently, we examined whether Glc8 Thr-118 was actually
phosphorylated. Indeed, yeast crude extracts efficiently phosphorylated
Glc8 in vitro (Fig.
1A). In contrast, a mutant
Glc8 in which alanine is substituted for Thr-118 (Glc8-T118A) was not
phosphorylated (Fig. 1A, lane 4). I-2 is phosphorylated at additional sites by CK2 (7, 44). Although yeast
contains CK2 and our reaction conditions were favorable for its
activity, Glc8-T118A was not phosphorylated in vitro. This
is consistent with the fact that sites that correspond to I-2 CK2
phosphorylation are not found in Glc8. Therefore, Thr-118 is the major
site of Glc8 phosphorylation under the conditions of our in
vitro reactions.
Phosphorylation Is Required for Glc8 in Vivo
Function--
Presently, two methods exist for assaying Glc8 in
vivo function. The first, reduction of glycogen accumulation was
the trait that initially identified glc8 loss of function
mutations (11). Subsequently, high copy GLC8 was found to
suppress ipl1 mutations caused by the antagonistic
relationship of Ipl1 protein kinase and Glc7 phosphatase (12). However,
the latter method drastically altered in vivo Glc8
concentration. Thus, we hunted for mutants that were dependent on Glc8
for viability. We anticipated that mutations lethal in combination with
loss of Glc8 could reveal additional functions of Glc8. Such mutants
would provide a way of assessing Glc8 function in vivo at
normal Glc8 concentrations.
We used the synthetic lethal screening method (38-40) to isolate
mutants that became dependent on Glc8 for viability. The 11 GLC8-dependent mutants isolated all contained
mutations in GLC7. The GLC7 locus from one of
these mutants contained a glc7-R121K mutation
("Experimental Procedures"). In a previous study, 18 glc7 point mutations were isolated based on their effects on
glycogen metabolism (16). Analysis of these 18 mutants revealed that glc7 alleles R121K, R142H, and
R198D are strictly dependent on Glc8 for growth and that
some other glc7 alleles show intermediate growth traits with
glc8.
To test whether phosphorylation is necessary for Glc8 function in
vivo, we tested the ability of glc8-T118A to complement two traits: the glc7-R121K Glc8-dependent trait
and the glc8 glycogen deficiency trait. As shown in Fig.
2, plasmid pYT115 (glc8-T118A) was incapable of complementing both of these traits. Therefore, glc8-T118A does not encode a functional Glc8 in
vivo. Because Glc8-T118A protein is not phosphorylated in
vitro, we conclude that Glc8 function requires Glc8
phosphorylation.
Pho85/Pcl7 Is the Major Glc8 Kinase in Vitro--
To
determine which yeast protein kinase phosphorylates Glc8, we used a
collection of yeast strains that each express a GST fusion to a yeast
protein kinase (32). Initially, GST fusion proteins were purified from
pools of cells. The GST protein kinase fusions were purified from 12 pools of 10 strains each and used for in vitro Glc8 kinase
assays. Protein from pool 6 had the greatest Glc8 kinase activity (Fig.
1B). Next, GST protein kinases were purified and assayed
from the ten component strains in pool 6. GST-Pho85 had the greatest
Glc8 kinase activity (Fig. 1C). Many protein kinases could
phosphorylate Glc8 in these in vitro reactions. However, we
assumed that the in vivo Glc8 kinase would demonstrate the
best phosphorylation kinetics and yield the greatest Glc8 phosphorylation in these assays. Therefore, we disregarded protein kinases such as Hal5, which phosphorylate Glc8 greater than the average protein kinase.
The strain containing GST-Pho85 may contain a mixture of plasmids
because of the way the GST fusions were constructed (32). Therefore,
yeast DNA was purified from the GST-Pho85 containing yeast strain and
retrieved by E. coli transformation, and individually isolated DNAs were used to transform EJ758. Of the eight DNAs analyzed,
seven had a restriction map expected for a GST-PHO85 plasmid. The DNA sequence from one of these isolates was identical to
PHO85 with the exception of an A to T transition in codon
61, which converts a lysine to an isoleucine (Table III). This
GST-PHO85 plasmid expressed high Glc8 kinase activity when
the GST-Pho85 was purified from EJ758 transformants. Together, these
data identify Pho85 as an in vitro Glc8 kinase. Although
there were other protein kinases that phosphorylate Glc8 in
vitro, Pho85 was most active, and as our genetic analyses
demonstrate below, it is the only in vivo Glc8 kinase.
Pho85 is a cyclin-dependent kinase that is regulated by 10 alternative cyclins (45). The purified GST-Pho85 fusion presumably had
one or more of these cyclins associated with it. Two biochemical experiments delineated which Pho85 cyclins comprise the active Glc8
kinase. In the first experiment, GST-Pho85 was purified from BY4743
diploids with homozygous deletions of Pho85 cyclins (pho80, clg1, pcl1, pcl2, pcl6,
pcl7, pcl8, and pcl9), and the Glc8
kinase activity was assayed. The Glc8 kinase activity of GST-Pho85 was uniquely diminished when it was purified from the pcl6
deletion strain (Fig. 1D).
In the second experiment, GST fusions to each of the Pho85 cyclins were
purified from EJ758 yeast. Pho85 binds to each of these cyclins and
hence should co-purify with them (45). Affinity purification of
GST-Pcl7 from yeast consistently (n = 4) yielded the
greatest Glc8 kinase activity (Fig. 3).
Although equivalent masses of GST-cyclin/Pho85 were used in these
assays, the relative affinity of Pho85 to these GST-cyclin fusions will
temper the activity we measured. Therefore, we only used the above data
to indicate that the Pho85 cyclins Pcl6 and Pcl7 contribute to the Glc8
kinase in vitro. Genetic analyses (see below) were employed to confirm whether this contention was true in vivo. Based
on amino acid sequence comparison, Pcl6 and Pcl7 are closely related and belong to a family that includes Pcl8 and Pcl10 (45, 46).
Pho85 affinity with its substrates Pho4 and Gsy2 is sufficient to
detect the interaction by co-precipitation or two-hybrid methods (46,
47). In our analysis, we found that GST-Glc8 purified from yeast
co-purified Glc8 kinase activity (Fig. 3). Glc8 affinity for Pho85 was
independently reported while this work was in progress (48). Our
results show that the Pho85 associated with Glc8 is catalytically
active. GST-Glc8 was also phosphorylated somewhat in the reaction,
which indicates that GST-Glc8 isolated from yeast was partially dephosphorylated.
Pho85/Pcl6 Is the Major in Vivo Glc8 Kinase--
The
above experiments show that Pho85, in association with certain cyclins,
can phosphorylate Glc8 in vitro. We wished to test whether
Pho85 is the sole Glc8 kinase in vivo. The
glc7-R121K mutation is lethal in combination with
glc8 null mutations. Because Glc8 function requires Thr-118
phosphorylation, any mutation that compromises in vivo Glc8
phosphorylation should be lethal in combination with
glc7-R121K. Therefore, we generated strains with a
pho85 glc7-R121K genotype to test their viability. Congenic
strains were used for these analyses to eliminate the influence of
cryptic background mutations. Control crosses with either
pho85 or glc7-R121K strains showed good spore
viability. However, pho85 by glc7-R121K crosses
yielded inviable double mutant spores. Typical tetratype tetrads from
these crosses are shown in Fig.
4A, in which the pho85 slow growth and missing pho85 glc7-R121K
spores were apparent. The pho85 glc7-R121K spores germinated
but stopped dividing at the ~16 cell stage. Therefore, this result
demonstrates that Pho85 is the sole Glc8 kinase in vivo.
To determine which Pho85 cyclin promotes Glc8 in vivo
phosphorylation, additional crosses were performed. We focused on the role of the related Pcl6, Pcl7, Pcl8, and Pcl10 cyclins because they
were implicated by the in vitro analyses above. No
individual pcl mutation was lethal in combination with
glc7-R121K. However, strains in which pcl6 pcl7
double mutations were combined with glc7-R121K showed slow
spore germination (data not shown) and temperature sensitivity (Fig.
5). The pcl6 pcl7 glc7-R121K
cells arrested as large budded cells with replicated DNA at the
nonpermissive temperature. When PCL6 was the sole wild-type
gene in this set, glc7-R121K did not inhibit growth at
37 °C (JC1349-17C; Fig. 5). In contrast, when PCL7 was
the sole wild-type gene in this set, glc7-R121K caused a
detectable temperature sensitivity (JC1347-20B; Fig. 5). Strains with
pcl6 and pcl7 combined with glc7-R121K
showed great temperature sensitivity, which was further compromised by elimination of pcl8. These results are consistent with
redundant functions of Pcl6, Pcl7, and Pcl8 for Glc8 kinase activity
with the strength of function increasing in the order: Pcl8, Pcl7, and
Pcl6. Although the above results did not address the contribution of
Pcl10, spores with a pcl6 pcl7 pcl8 pcl10 glc7-R121K
genotype were uniquely inviable (Fig. 4B). Therefore,
temperature sensitivity results from the significant reductions of Glc8
phosphorylation in a pcl6 pcl7 pcl8 glc7-R121K strain, but
elimination of the four related Pcl6, Pcl7, Pcl8, and Pcl10 prevented
growth of glc7-R121K strains at any temperature. In
conclusion, Pcl6, Pcl7, Pcl8, and Pcl10 function redundantly as Pho85
cyclins for Glc8 phosphorylation in vivo with Pho85/Pcl6
providing the majority of the Glc8 kinase activity.
From the primary sequence alignment of Glc8 with its homologs,
Thr-118 was anticipated to be phosphorylated (11, 12). Indeed, Tung
et al. (12) showed that a high copy Glc8-T118A mutant
protein was defective in ipl1 suppression (12). Mammalian protein kinases GSK3, extracellular signal-regulated kinase-2, or CDK5
phosphorylate the homologous I-2 residue in vitro (6-9). Budding yeast has at least one homolog to each of these protein kinases
(49). Using a collection of GST fusions to yeast protein kinases, we
discovered that Pho85 was the prevailing Glc8 kinase in
vitro (Fig. 1C). However, this collection of gene
fusions is known to be incomplete (50), so there was the possibility
that other protein kinases could also phosphorylate Glc8.
Independently, we know that none of the four yeast GSK3 homologs
contribute Glc8 kinase activity because extracts from the quadruple
mds1 mck1 mrk1 yol128c mutant strain have the same Glc8
kinase specific activity as wild-type strains (data not shown). We used
a glc7 mutant that requires Glc8 to grow to test whether
Pho85 phosphorylates Glc8 in vivo. The inviability of
glc7-R121K pho85 double mutants not only demonstrated that
Pho85 phosphorylates Glc8 in vivo, but it also illustrates
that Pho85 must be the only Glc8 kinase. This result is consistent with
a single Glc8 kinase resolved by sedimentation velocity centrifugation
and by several chromatographic steps (data not shown). Note that the
in vivo phosphorylation of I-2 by the mammalian kinases
has not always been demonstrated.
While this work was in progress, mammalian CDK5, the ortholog of Pho85,
was reported to phosphorylate I-2 in vitro (9). Furthermore,
CDK5 binds directly to PP1c (9). This binding suggests that
I-2 in the PP1c·I-2 complex would be predominantly active
because of its phosphorylation by CDK5 and hence exhibit PP1c activity. The CDK5 region that binds to
PP1c, RVRLDDDD, weakly corresponds to the Pho85 sequence
EKKLDSEE, matching only two of eight residues. In particular the Pho85
sequence does not have the canonical (R/K)(V/I) X(F/Y)
sequence that many PP1c-binding proteins possess (51).
Therefore, Pho85 probably does not bind directly to Glc7. However,
others and we found that Pho85 binds to Glc8 (Fig. 3A and
Ref. 49). Our data additionally show that the Pho85 when bound to Glc8
is active for transphosphorylation. In Fig. 3 the added Glc8 was
predominantly phosphorylated; however, there was a small amount of
phospho-GST-Glc8 detectable. Therefore, Glc8 would be highly
phosphorylated in vivo as long as the redundant Pcl6, Pcl7,
Pcl8, and Pcl10 levels were high.
Pho85 cyclins are functionally partitioned; subsets of them promote
Pho85 phosphorylation of substrates involved in specific processes. For
example, the related Pcl1 and Pcl2 cyclins direct Pho85 to
phosphorylate the Clb-Cdc28 kinase inhibitor, Sic1, and thus provide
additional Cdc28 in regulation of the G1 to S phase transition (52-54). In this work we found that in vivo Glc8
kinase activity was predominantly derived from Pcl6- and
Pcl7-associated Pho85. These cyclins are members of the "metabolic
regulation" group of Pho85 cyclins because they regulate glycogen
metabolism and prevent growth on alternative carbon sources (47, 55). The redundant Pcl6 and Pcl7 influence both glycogen phosphorylase and
glycogen synthase activity, whereas Pcl8 and Pcl10 only regulate glycogen synthase. Glc8, through its regulation of Glc7, was previously recognized as a regulator of glycogen metabolism. Therefore, the metabolic group of Pho85 cyclins influences glycogen metabolism on
several different levels.
Although glycogen synthesis is not essential for yeast viability,
glc8 glc7-R121K cells are inviable. This indicates that Glc8
may modulate the functions of Glc7 essential for viability. An
essential function of Glc7 seems to be modulation of the kinetochore during mitosis (56). Because activity of Pho85/Pcl7 is maximized during
the S phase of the cell cycle, Pcl7 may also modulate cell cycle
functions (46). Two-hybrid results connect Pcl6 to the kinetochore-associated protein Spc24 via YLR190W (58-60). Therefore, Glc8 phosphorylation may change during the cell cycle, perhaps close to
the kinetochore, and thereby regulate Glc7 phosphatase activity at the kinetochore.
The temporal expression of Pcl7 can explain the discrepancy in the
determination of the Pho85 cyclin in the in vitro Glc8 kinase. The panel of assays in Fig. 1D clearly shows a
reduction in the Glc8 kinase activity from the pcl6 mutant
strain. We interpret this to show that Pcl6 is the major Pho85 cyclin
for Glc8 kinase activity. Because we used asynchronous cells to prepare
the extracts for these assays, few cells would be in the S phase, and
thus little Pcl7 would be provided. In contrast, when GST-Pcl7/Pho8 was
purified for the assays shown in Fig. 3, it was no longer subject to
the normal PCL7 transcriptional control and perhaps could
overcome post-translational influences on Pcl7 levels because of its
great expression. The comparatively low Glc8 kinase activity mustered
by GST-Pcl6/Pho85 in this assay probably reflects poor affinity of
Pho85 for the GST-Pcl6 fusion. However, note that we did not rely on
these in vitro assays entirely in our assignment of the Glc8
kinase Pho85 cyclin. We used the dependence of glc7-R121K for Glc8 function, which in turn required in vivo Glc8 phosphorylation.
The reason glc7-R121K and some other glc7 alleles
confer a dependence on Glc8 for viability is unknown. Reduction of Glc7 activity causes cell cycle arrest at the spindle assembly checkpoint (61, 62). Depletion of Glc8 in glc7-R121K cells appears to cause the same block in the cell cycle because they arrest as large
budded cells with replicated chromosomes. The hyperactivity of
Pho85/Pcl7 in S phase phosphorylating Glc8 would activate Glc7 for
mitotic activity. If Glc8 mimics the I-2
phosphorylation-dependent chaperone function (5), then
Glc8-dependent glc7 alleles may encode proteins
that fold poorly or fail to adopt a conformation suitable to
dephosphorylate critical substrates without Glc8. We know that the
Glc8-dependent glc7 alleles neither encode
enzymes with uniformly low protein phosphatase activity nor exhibit a trend in Glc8 affinity (16, 63). Furthermore, they affect residues
unnecessary for catalytic activity.
Mammalian I-2 is found in two complexes that contain PP1 protein
phosphatase and a protein kinase. The first is the CDK5 complex described above, which is involved in neuronal development (9). The
second is a Nek2 protein kinase complex, which regulates the duplication and separation of centrosomes (57). I-2 promotes centrosome
separation by its inhibition of PP1 in this complex with concomitant
increase of Nek2 activity (57). Spindle pole body (equivalent to
centrosome) duplication occurs in yeast in late G1, but
separation continues through the S phase. The arrest of pcl6 pcl7
glc7-R121K cells in metaphase at the nonpermissive temperature
that we found is inconsistent with phospho-Glc8 promoting spindle pole
dynamics, but it is consistent with kinetochore activity required in
the metaphase to anaphase transition (56). Therefore, I-2 orthologs are
found in kinase-phosphatase complexes in these two eukaryotic species
and play roles in cell cycle regulation, but the specific processes
regulated are different.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Strain list
Plasmid list
Mutations in GST fusion plasmids
-D-thiogalactopyranoside was added
to a 6 mM final concentration, and the culture was grown an
additional 5 h. The harvested cell pellets were resuspended in
1/20th culture volume of sonication buffer (50 mM
Tris-HCl, pH 8.0, 10 mM EDTA, 100 mM NaCl, 1.0 mM phenylmethylsulfonyl fluoride) and sonicated three times
for 1-min intervals on ice with a 250 watt sonicator at 50% maximum
power. The cell debris was removed by centrifugation at 15,000 × g for 15 min, and the supernatant proteins were heated at
95 °C for 15 min. The denatured proteins were removed by
centrifugation. Contaminating high molecular weight substances were
removed by passing supernatant through a 300,000 nominal molecular
weight limit cellulose membrane (Millipore). The final
supernatant containing Glc8 was stored at
20 °C.
-32P]ATP), and
homogenization buffer without protease inhibitor, in a final volume of
25 µl. The reactions were incubated for 30 min at 30 °C. Adding
equal volumes of 2× SDS loading buffer (36) stopped reactions. The
samples were then heated for 3 min at 95 °C and resolved with
SDS-PAGE (36). The gels were dried and exposed to Kodak XAR5
film or to a Kodak phosphorimaging screen. The data on phosphorimaging
screens were read on Molecular Imager FX and analyzed with Quantity One
software (Bio-Rad).
Ura (A600 = 0.8) and induced with
500 µM CuSO4 for 2 h. Note that
transformants with purified Pho85 cyclin fusions could not grow on SC
Ura
Leu, which would elevate the plasmid copy number. The cells
were glass bead extracted in 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 4 mM MgCl2, 5 mM dithiothreitol, 10% (v/v) glycerol, 1 M
NaCl, 1× complete protease inhibitor mixture. Binding to
glutathione-agarose (Sigma), washing, and dialysis were as described
(32). The fusion proteins were dialyzed against 20 mM
Tris-HCl, pH 7.4, 2 mM EDTA, 4 mM
MgCl2, 1 mM dithiothreitol, 55 mM
NaCl, 50% (v/v) glycerol and then stored at
20 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Phosphorylation of Glc8 in
vitro. A, Glc8 was purified as described
under "Experimental Procedures." 10-µg amounts of protein from
E. coli crude extract (lane 1) or soluble protein
after heat treatment (lane 2) were separated by SDS-PAGE and
Coomassie-stained. Lanes 3-5 show detection of
phosphorylated Glc8 by Glc8 kinase assays. In these lanes, 10 µg of
JC746-9D yeast protein extract was incubated with 1 µg Glc8
(lane 3), Glc8-T118A (lane 4), or buffer
(lane 5) and [ -32P]ATP (1200 cpm/pmol) for
20 min, then separated by SDS-PAGE, and exposed to film. The
closed arrow indicates the position of full-length Glc8.
B, GST-protein kinase fusions were purified from 120 yeast
strains in 12 pools of 10 fusions each. Kinase reactions with 5 µg of
Glc8, [
-32P]ATP, and 120 ng of GST fusion protein were
separated by SDS-PAGE and autoradiographed. C, the
first ten lanes show Glc8 kinase assays of pool 6 members
(200 ng of GST fusion) or pools 6 and 7 (150 ng of GST fusion).
D, GST-Pho85 was purified from BY4743 derivatives homozygous
for the indicated mutations. Glc8 kinase assays used 100 ng of
GST-Pho85/reaction.
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Fig. 2.
Effects of Glc8-T118A in
vivo. Strain JC968-1 (ade2 ade3 leu2 ura3
glc8::HIS3 glc7-R121K/p2087 [2µ ADE3
LEU2 GLC8]) was transformed with p1945 (CEN URA3 GLC8)
in A or pYT115 (CEN URA3 glc8-T118A) in
B. The resulting transformants were selected on SC Ura and
restreaked onto low adenine SC
Ura and grown until the red pigment
was clearly seen (3-5 days). C, glycogen accumulation was
assayed in strains JC746-9D (GLC8), JC840B
(glc8), JC840B/p1945 (glc8/GLC8), and
JC840B/pYT115 (glc8/glc8-T118A) by iodine
staining. The colonies were spotted onto YEPD grown 24 h and then
exposed to iodine vapor for 3 min before photographing.
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Fig. 3.
Pcl7 is the major Pho85 cyclin in the Glc8
kinase in vitro. A, indicated
GST-protein fusions were purified from EJ758 transformants. Kinase
reactions with 5 µg of Glc8, [ -32P]ATP, and 100 ng
of GST fusion protein were separated by SDS-PAGE and autoradiographed.
The migration of GST-Glc8 and Glc8 is shown on the left, and
the sizes of molecular mass markers are shown on the right.
B, quantitation of the autoradiogram in A.
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Fig. 4.
Pho85 is the in vivo
Glc8 kinase. A, three typical tetratype tetrads
from JC1327-11A (glc7-R121K) × JC1328-5A
(pho85::kanMX4) show two faster growing
spores (PHO85+): one slow growing spore
(pho85 GLC7+) and one inviable spore
(pho85 glc7-R121K). Sister spore clones are in the same
column. The genotypes of spores are indicated on the right.
B, typical tetrads from JC1347-20B (pcl6 pcl8 pcl10
glc7-R121K) × JC1349-17C (pcl7 pcl8 pcl10
glc7-R121K) reveal that spores with a pcl6 pcl7 pcl8 pcl10
glc7-121K genotype fail to grow. Sister spore clones are in the
same column. The PCL6 and PCL7 genotypes of
spores are indicated on the right.
View larger version (32K):
[in a new window]
Fig. 5.
Four redundant Pcls comprise the Glc8 kinase
in vivo. A, strains with the indicated
relevant genotypes were grown overnight in YEPD, and serial 10-fold
dilutions were spotted on YEPD plates, which were incubated at the
indicated temperatures for 2 days.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Joaquim Ariño, Elizabeth Grayhack, Eric Phizicky, and Mike Snyder for plasmids and strains. The excellent technical assistance by Jill Adams and the contribution by Chad Lambert are also appreciated.
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FOOTNOTES |
---|
* This work was supported by the National Science Foundation, the University of Missouri Molecular Biology Program, and the Department of Molecular Microbiology and Immunology.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.
Present address: Div. of Hematology/Oncology, Children's Hospital
Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027.
§ Present address: Gene Tools LLC, 2680 SW 3rd St., Corvallis, OR 97333.
¶ To whom correspondence should be addressed: Dept. of Molecular Microbiology and Immunology, 1 Hospital Dr., University of Missouri, Columbia, MO 65212. Tel.: 573-882-2780; Fax: 573-882-4287; E-mail: CannonJ@missouri.edu.
Published, JBC Papers in Press, October 28, 2002, DOI 10.1074/jbc.M208058200
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ABBREVIATIONS |
---|
The abbreviations used are: I-2, inhibitor-2; PP1c, protein phosphatase-1 catalytic subunit; GSK3, glycogen synthase kinase-3; CDK5, cyclin-dependent kinase-5; CK2, casein kinase-2; GST, glutathione S-transferase.
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