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Proc.
Nail.
Aead.
Sci.
USA
Vol.
89,
pp.
28-32,
January
1992
Genetics
Yeast
casein
kinase
I
homologues:
An
essential
gene
pair
(Saccharomyces
cerevisiae/protein
kinase/snf
mutations/salt
tolerance)
Lucy
C.
ROBINSON*,
E.
JANE
ALBERT
HUBBARDt,
PAUL
R.
GRAVESt,
ANNA
A.
DEPAOLI-ROACHt,
PETER
J.
ROACHt,
CHING
KUNG*,
DAVID
W.
HAAS§,
CURT
H.
HAGEDORN§,
MARK
GOEBL,
MICHAEL
R.
CULBERTSON*,
AND
MARIAN
CARLSONt
*Molecular
Biology
and
Genetics,
University
of
Wisconsin,
1525
Linden
Drive,
Madison,
WI
53706;
tDepartment
of
Genetics
and
Development,
Columbia
University,
701
West
168th
Street,
New
York,
NY
10032;
tDepartment
of
Biochemistry
and
Molecular
Biology,
Indiana
University
School
of
Medicine,
635
Barnhill
Drive,
Indianapolis,
IN
46223;
and
§Departments
of
Medicine
and
Cell
Biology,
Vanderbilt
University
School
of
Medicine
and
the
Veterans
Administration
Medical
Center,
Nashville,
TN
37232-2279
Communicated
by
Michael
H.
Wigler,
October
10,
1991
(received
for
review
August
2,
1991)
ABSTRACT
We
report
the
isolation
of
an
essential
pair
of
Saccharomyces
cerevisiae
genes
that
encode
protein
kinase
homologues.
The
two
genes
were
independently
isolated
as
dosage-dependent
suppressors.
Increased
dosage
of
YCKI
sup-
pressed
defects
caused
by
reduced
SNF1
protein
kinase
activ-
ity,
and
increased
dosage
of
YCK2
relieved
sensitivity
of
wild-type
cells
to
salt
stress.
The
two
genes
function
identically
in
the
two
growth
assays,
and
loss
of
function
of
either
gene
alone
has
no
discernible
effect
on
growth.
However,
loss
of
function
of
both
genes
results
in
inviability.
The
two
predicted
protein
products
share
77%
overall
amino
acid
identity
and
contain
sequence
elements
conserved
among
protein
kinases.
Partial
sequence
obtained
for
rabbit
casein
kinase
I
shares
64%
identity
with
the
two
yeast
gene
products.
Moreover,
an
increase
in
casein
kinase
I
activity
is
observed
in
extracts
from
cells
overexpressing
YCK2.
Thus
YCKI
and
YCK2
appear
to
encode
casein
kinase
I
homologues.
Casein
kinase
I
denotes
one
of
two
classes
of widely
distrib-
uted
mammalian
cAMP-
and
Ca2+-independent
protein
ki-
nases
that
were
originally
defined
by
their
ability
to
phos-
phorylate
acidic
substrates,
notably
caseins
(reviewed
in
ref.
1).
Casein
kinase
I
activities
have
been
detected
in
most
eukaryotic
cells
and
can
be
membrane-bound,
cytosolic,
or
nuclear.
A
wide
variety
of
in
vitro
substrates
have
been
identified
for
mammalian
casein
kinase
I.
Among
these
are
the
simian
virus
40
large
tumor
antigen
(1);
the
cytoskeletal
proteins
myosin
and
troponin
(1,
2);
membrane
components
such
as
spectrin
(1)
and
neural
cell
adhesion
molecule
(N-
CAM;
ref.
3);
nonhistone
nuclear
proteins
including
RNA
polymerases
I
and
II
(1);
components
of
the
translation
apparatus
including
initiation
factors
4B,
4E,
and
5
and
tRNA
synthetases
(1,
4,
5);
and
metabolic
enzymes,
notably
gly-
cogen
synthase
(1).
The
site
preference
of
casein
kinase
I
has
been
defined
as
acidic
residues
amino-terminal
to
the
modified
residue
(1,
6).
However,
the
sequence
motif
-Ser(P)-Xaa-Xaa-Ser-
has
re-
cently
been
demonstrated
to
specify
casein
kinase
I
action,
suggesting
a
model
for
casein
kinase
I
biological
activity
termed
hierarchical
protein
phosphorylation
(7).
For
exam-
ple,
phosphorylation
of
N-CAM
by
casein
kinase
I
in
vitro
is
abolished
by
prior
treatment
with
phosphatase
(3).
Also,
phosphorylation
of
glycogen
synthase
by
cAMP-dependent
protein
kinase
(cAPK)
potentiates
that
by
casein
kinase
1
(6).
Replacement
of
phosphoserine
by
a
block
of
three
or
four
acidic
residues
in
synthetic
peptide
substrates
results
in
poor
but
relatively
specific
casein
kinase
I
substrates
(8).
Casein
kinase
I
activity
has
been
inferred
to
be
central
to
cellular
function
from
its
wide
distribution,
numerous
in
vitro
substrates,
and
multiple
subcellular
localizations.
However,
the
importance
of phosphorylation
by
the
enzyme
in
vivo
has
been
demonstrated
in
few
cases,
notably
glycogen
synthase.
Phosphorylation
of
glycogen
synthase
by
casein
kinase
I
in
vitro
results
in
inactivation
of
the
enzyme
(7)
and
phosphor-
ylation
of
one
site,
Ser10,
occurs
in
rabbit
muscle
in
vivo
in
response
to
stimulation
by
epinephrine
(9).
Other
physiolog-
ically
important
phosphorylations
by
casein
kinase
I
have
been
suggested
but
its
overall
physiological
roles
remain
unclear.
The
study
of
casein
kinase
I
function
would
be
greatly
facilitated
by
the
approaches
that
are
possible
with
the
genetically
tractable
budding
yeast
Saccharomyces
cere-
visiae.
Enzymological
studies
have
revealed
multiple
forms
of
casein
kinase
in
yeast
that
share
properties
with
mamma-
lian
casein
kinase
I
(10-12),
but
no
biological
function
has
been
assigned
to
these
activities.
We
describe
here
the
isolation
of
two
structural
homo-
logues
of
casein
kinase
I
in
S.
cerevisiae.
The
YCKI
and
YCK2
(yeast
casein
kinase
I
homologue)
genes
were
identi-
fied
independently
based
on
the
effects
of
increased
gene
dosage.
YCKJ
was
isolated
as
a
suppressor
of
the
require-
ment
for
SNF4
function.
The
SNF4
protein
is
a
positive
effector
of
the
SNF1
protein
kinase
(13),
which
is
required
for
carbon
catabolite
derepression
(14).
Increased
dosage
of
YCK2
allows
wild-type
cells
to
withstand
extreme
salinity
stress
without
prior
adaptation.
We
report
that
YCKJ
and
YCK2
show
a
high
degree
of
sequence
similarity
to
each
other
and
to
partial
sequence
of
rabbit
casein
kinase
I.
We
find
that
increased
gene
dosage
of
YCK2
is
correlated
with
increased
casein
kinase
I
activity
in
cell
extracts.
Finally,
we
demon-
strate
that
the
two
genes
comprise
an
essential
gene
family.l
MATERIALS
AND
METHODS
Genetic
Methods.
S.
cerevisiae
strains
were
derived
from
JC482
(MATa
his4
leu2
ura3;
ref.
15),
LRB188
(MATa/a
his3/his3
leu2/leu2
ura3/ura3;
ref.
16),
MCY1093
(MATa
his4
lys2
ura3
SUC2),
MCY1094 (MATa
ade2
ura3
SUC2),
or
MCY2372
(MATa
his3
leu2
ura3
SUC2).
The
snf
strains
were
MCY1853
(MA
Ta
snf4-A2
his4
lys2
ura3
SUC2)
and
MCY1845
(MATa
snfl-A10
ade2
ura3
SUC2).
Genetic
anal-
ysis
and
transformation
were
by
standard
methods
(17,
18).
Nucleic
Acid
Methods.
Standard
methods
for
DNA
manip-
ulations
were
used
(19,
20).
DNA
sequence
was
determined
for
both
strands
(21)
using
the
Sequenase
(United
States
Abbreviation:
cAPK,
cAMP-dependent
protein
kinase.
$The
sequences
reported
in
this
paper
have
been
deposited
in
the
GenBank
data
base
[accession
nos.
M74552
(YCKI)
and
M74453
(YCK2)].
28
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertisement"
in
accordance
with
18
U.S.C.
§1734
solely
to
indicate
this
fact.
Proc.
Natl.
Acad.
Sci.
USA
89
(1992)
29
Biochemical)
and
Amplitaq
(Cetus)
enzymes,
in
part
using
the
Applied
Biosystems
373A
DNA
sequencer.
Gene
Disruption.
A
deletion
of
YCKI
(yckl-AJ::
URA3)
was
constructed
by
replacing
the
Bgl
II
fragment
with
URA3
(Fig.
1A).
yck2::HIS3
was
constructed
by
TnS
mutagenesis
(22)
of
pLS2.3
followed
by
replacement
of
a
portion
of
TnS
with
HIS3
(Fig.
1B).
The
yckl-Al::URA3
and
yck2::HIS3
alleles
were
introduced
(23)
into
the
wild-type
diploid
strains
MCY1093
x
MCY1094
and
LRB188
to
generate
strains
heterozygous
for
each
disruption
mutation.
The
diploid
strains
LRB237
and
LRB238
(yckl/+
yck2/+)
were
gener-
ated
by
introduction
of
yckl
-Al::
URA3
into
a
diploid
strain
heterozygous
for
yck2::HIS3.
LRB263
(MATa
his3
leu2
ura3
yck2)
and
LRB266
(MATa
his3
leu2
ura3
yckl)
are
meiotic
progeny
of
LRB237.
Strains
LRB271
(MATa/MA
Ta
his3/
his3
leu2/leu2
ura3/ura3
yckl/yckl
yck2/+)
and
LRB272
(MATa/MATa
his3/his3
leu2/leu2
ura3/ura3
yckl/+
yck2/
yck2)
were
derived
from
meiotic
progeny
of
LRB237.
Diploid
strains
MCY2394
and
MCY2395
were
generated
by
introduc-
tion
of
yck2::HIS3
into
MCY2393
(MATa/MATa
ade2/+
his3/his3
+
/Ieu2
+
/Ilys2
ura3/ura3
yckl/yckl).
Replacement
of
wild-type
sequences
with
the
disruption
alleles
was
veri-
fied
by
Southern
blot
analysis.
Characterization
of
an
Amplifled
DNA
Fragment
Encoding
Rabbit
Casein
Kinase
I.
Casein
kinase
I
used
for
protein
sequencing
was
purified
from
rabbit
reticulocyte
lysate
(4).
This
enzyme
was
identical
to
casein
kinase
I
purified
from
rabbit
skeletal
muscle
with
respect
to
molecular
mass
and
tryptic
phosphopeptide
map
(4,
6).
Phosphopeptide
maps
of
initiation
factor
4E
phosphorylated
by
reticulocyte
and
skel-
etal
muscle
casein
kinase
I
were
identical,
and
the
specificity
of
the
reticulocyte
enzyme
for
other
substrates
was
consis-
tent
with
that
of
casein
kinase
I
(4).
Reticulocyte
casein
kinase
1
(30
,ug)
was
subjected
to
SDS/PAGE
and
transferred
to
nitrocellulose
(4,
24).
The
polypeptide
of
35
kDa
was
excised
and
digested
with
trypsin
(trypsin/protein
ratio,
1:20)
at
37°C
for
24
hr.
Tryptic
peptides
were
separated
on
a
reverse-phase
HPLC
C18
cartridge
(OD-300,
Applied
Biosys-
tems)
with
a
gradient
of
acetonitrile
in
0.1%
trifluoroacetic
acid.
The
purified
peptides
were
subjected
to
automated
A.
YCKJ
pB65
-
pJH21
p
pB65R7
pJH2O
pUJ721
(YEp352)
pJH79
2
(pUC19)
B.
YCK2
pIS2
pI]S2.3
pLS2.33
(YEp351)
pLS2.6
pIS2.3T
EX
N
H
II
Tn5:iIIS3
+
1
kb
FIG.
1.
YCKI
and
YCK2
plasmids.
Only
yeast
DNA
sequences
are
shown.
Arrows
indicate
the
position
and
5'
3'
direction
of
the
open
reading
frames,
starting
at
the
first
ATG.
Bg,
Bgl
II;
E,
EcoRI;
H,
HindIII;
N,
Nco
I;
P,
Pst
1;
S,
Sal
I;
Sm,
Sma
I;
X,
Xba
I;
Xh,
Xho
I.
(A)
The
vector
is
YEp24
unless
otherwise
indicated.
The
ability
of
each
multicopy
plasmid
to
suppress
the
growth
defect
of
an
snf4
mutant
on
raffinose
at
30'C
is
indicated
at
right.
*,
pLJ721
was
tested
only
for
salt
tolerance.
HindIll
and
Bgl
II
sites
were
mapped
only
to
the
left
of
the
Sma
I
site.
(B)
The
vector
is
YEp352
unless
otherwise
indicated.
The
ability
of
each
multicopy
plasmid
to
confer
salt
tolerance
is
indicated
at
right.
kb,
Kilobase.
Edman
degradation
on
a
Porton
Instruments
(Tarzana,
CA)
model
2090
integrated
microsequencing
system
equipped
with
an
on-line
HPLC
for
detection
of
phenylthiohydantoin
derivatives.
Amino
acid
sequences
were
obtained
from
four
peptides.
Degenerate
oligonucleotide
primers
for
polymerase
chain
reaction
(PCR)
DNA
amplification
were
designed
from
the
peptide
sequences
DNFLMG
(sense
strand)
and
MYFNLQ
(antisense
strand).
A
BamHI
site
and
Sma
I
site
were
added
at
the
5'
ends
of
the
sense
and
antisense
primers,
respectively.
PCR
was
carried
out
in
two
separate
phases
(25),
using
cDNA
synthesized
from
total
rabbit
testis
RNA.
The
first
phase
was
10
cycles
with
10
pmol
of
primers
and
the
second
phase
was
40
cycles
with
50
pmol
of
primers.
The
reaction
yielded
a
discrete
DNA
product
of
230
base
pairs
(bp)
that
was
subcloned
into
phage
vector
M13
by
using
the
BamHI
and
Sma
I
sites
and
subjected
to
sequence
analysis.
Measurement
of
Yeast
Casein
Kinase
I
Activity.
Cells
from
overnight
cultures
were
disrupted
by
shaking
with
glass
beads
in
25
mM
Hepes/2
mM
EDTA/2
mM
EGTA,
pH
7.5.
The
extract
was
centrifuged
at
11,000
x
g
and
the
supernatant
was
assayed
for
casein
kinase
I
activity
(8).
32p
incorporation
from
[y-32P]ATP
(310
cpm/pmol)
into
the
synthetic
peptide
DDDDVASLPGLRRR
(1
mM)
was
measured
using
a
P81
paper
(Whatman)
filter
binding
assay
(26).
Values
from
control
reactions
lacking
peptide
were
subtracted.
RESULTS
Isolation
of
the
YCK1
and
YCK2
Genes.
YCKI
was
isolated
as
a
multicopy
suppressor
of
growth
defects
due
to
reduced
SNF1
protein
kinase
activity.
The
SNF1
protein
kinase
is
required
for
expression
of
many
genes
in
response
to
glucose
deprivation
(14).
The
SNF4
protein
is
physically
associated
with
SNF1
and
required
for
maximal
SNF1
protein
kinase
activity
(13,
27).
To
recover
genes
that
are
functionally
related
to
SNFI
and
SNF4,
we
isolated
multicopy
(2
am)
plasmids
that
suppress
the
raffinose
growth
defect
of
snf4
mutants.
The
snf4
mutant
strain
MCY1853
was
transformed
with
a
YEp24
yeast
genomic
library
(28),
and
transformants
able
to
grow
on
raffinose
at
30°C
were
isolated
as
described
(29).
Among
the
plasmids
recovered
was
pB65,
which
was
a
weak
suppressor
recovered
from
three
transformants.
To
localize
the
functional
region
within
pB65,
we
con-
structed
subclones
and
tested
their
ability
to
allow
growth
of
the
snf4-A2
mutant
on
raffinose.
The
functional
region
lies
across
the
Sal
I
site
(Fig.
1A).
A
1.8-kb
RNA
hybridized
to
a
probe
derived
from
this
region
(data
not
shown).
YCK2 was
isolated
by
its
ability
to
act
as
a
suppressor
of
high
salinity
intolerance.
Wild-type
cells
require
a
period
of
adaptation
or
conditioning
at
0.4-0.6
M
NaCl
to
enable
growth
on
medium
containing
>1
M
NaCl.
During
this
period
the
cells
accumulate
high
levels
of
the
protective
osmolyte
glycerol
(30,
31).
The
multicopy
plasmid
pLS2
was
recovered
from
four
transformants
of
the
strain
JC482
with
a
YEp352
genomic
library
(provided
by
D.
Conklin,
University
of
Wisconsin,
Madison).
These
transformants
no
longer
re-
quired
conditioning
to
grow
at
high
salinity.
The
functional
region
of
pLS2
was
determined
by
subclone
analysis
(Fig.
1B).
A
probe
derived
from
the
HindIII-Xba
I
fragment
hybridized
to
a
1.9-kb
RNA
(data
not
shown).
YCK1
and
YCK2
Show
Sequence
Similarity
to
Each
Other
and
to
Protein
Kinases.
The
nucleotide
sequence
of
the
pB65
functional
region
contains
an
open
reading
frame
of
1.6
kb.
The
538
codons
between
the
first
ATG
in
the
insert
and
the
first
stop
codon
encode
a
61.7-kDa
protein.
The
nucleotide
sequence
of
the
pLS2
functional
region
contains
a
547-codon
open
reading
frame
that
could
encode
a
protein
of
62
kDa.
Both
predicted
amino
acid
sequences
were
compared
with
sequences
in
the
GenBank
and
EMBL
data
bases
(32)
(Feb-
ruary
1991)
and
with
protein
sequences
in
the
data
base
Genetics:
Robinson
et
al.
SB
B
H
Sm
II,
f
O-
Proc.
Natl.
Acad.
Sci.
USA
89
(1992)
maintained
by
M.G.
The
YCK1
and
YCK2
proteins
are
most
similar
to
each
other
(Fig.
2),
with
overall
amino
acid
identity
of
77%.
The
N-terminal
two-thirds
of
each
predicted
product
contains
sequence
elements
that
are
conserved
among
pro-
tein
kinases
(33).
Within
this
region,
the
YCK
products
are
96%
identical.
The
C
termini
of
the
YCK
proteins
show
less
similarity
to
each
other,
but
both
are
rich
in
glutamine
residues
and
terminate
with
the
sequence
Gly-Cys-Cys.
YCK1
and
YCK2
Resemble
Rabbit
Casein
Kinase
I.
Both
YCK1
and
YCK2
are
closely
related
to
partial
sequences
obtained
for rabbit
casein
kinase
I.
Degenerate
oligonucleo-
tide
primers
corresponding
to
peptide
sequence
obtained
for
rabbit
reticulocyte
casein
kinase
I
were
used
in
a
PCR
with
rabbit
testis
cDNA,
and
the
sequence
of
the
product
was
determined.
The
predicted
amino
acid
sequence
(Fig.
2)
contained
the
sequences
of
the
peptides
used
to
design
the
primers
as
well
as the
sequences
of
two
other
tryptic
pep-
tides.
There
were
three
conservative
substitutions
(see
leg-
end
to
Fig.
2)
that
could
reflect
either
PCR
artifacts
or
the
presence
of
distinct
casein
kinase
I
isoforms
in
rabbit
testis
and
reticulocytes.
When
the
sequence
predicted
for
rabbit
testis
casein
kinase
I
was
compared
with
sequences
in
a
data
base
maintained
by
M.G.,
the
highest
similarity
was
to
YCK1
and
YCK2
(Fig.
2),
with
64%
amino
acid
identity
over
the
77
residues.
Con-
versely,
YCK1
and
YCK2
showed
greater
similarity
to
the
rabbit
casein
kinase
I
sequence
than
to
any
yeast
sequence.
All
three
sequences
were
much
more
distantly
related
to
other
protein
kinases,
showing
<26%
identity
to
enzymes
in
the
Ca2+/calmodulin-dependent,
cdc2,
and
cAMP-depen-
dent
protein
kinase
families.
Casein
kinases
I
and
II
are
among
the
few
known
kinases
with
substrate
specificity
for
acidic
sites,
but
in
terms
of
primary
sequence,
the casein
kinase
II
enzymes
are
quite
distinct
from
these
putative
casein
kinase
I
sequences.
Although
yeast
and
Drosophila
casein
kinase
II
sequences
share
64%
amino
acid
identity
(1),
they
show
<20%
identity
to
the
catalytic
domains
of
the
YCK
products
and
to
the
partial
rabbit
casein
kinase
I
sequence.
The
crystal
structure
of
cAPK
with
substrate-analogue
inhibitory
peptide
bound
(34,
35)
has
allowed
definition
of
side
chains
involved
in
substrate
recognition,
some
of
which
lie
within
the
conserved
kinase
domain.
For
example,
Glu170
in
cAPK
contacts
a
substrate
Arg
residue.
This
Glu
is
replaced
by
His
in
casein
kinase
II
and
by
Arg
in
some
receptor
tyrosine
kinases
that
also
prefer
acidic
substrates
(33).
Thus,
a
basic
residue
in this
position
might
correlate
with
recognition
of
acidic
substrates.
However,
the
corre-
sponding
residue
in
casein
kinase
I
and
the
YCK
products
is
Asp.
Three
residues
that
are
important
for
substrate
recog-
nition
by
cAPK-Leu1",
Pro202,
and
Leu205-are
replaced
in
casein
kinase
II
by
Arg, Arg,
and
Lys,
respectively,
consis-
tent
with
a
preference
for
acidic
substrates.
However,
in
the
casein
kinase
I
family,
the
residues
are
Leu,
Ala,
and
Met/Ala,
respectively.
Thus,
casein
kinase
I
is
distinct
from
casein
kinase
II
as
judged
by
overall
sequence
as
well
as
these
key
residues
potentially
involved
in
substrate
recognition.
Another
characteristic
of
the
casein
kinase
I
family
is
the
absence
of
the
highly
conserved
Ala-Pro-Glu
sequence
of
subdomain
VIII
(33),
and
it
is
not
possible
to
assign
unequiv-
ocally
the
residue
corresponding
to
the
invariant
Glu
(cAPK
Glu21).
In
summary,
it
appears
that
these
gene
products
define
a
novel
subfamily
of
protein
kinases
with
distinctive
sequence
features.
YCKI
and
YCK2
Are
Functionally
Homologous.
The
high
degree
of
sequence
similarity
between
YCK1
and
YCK2
prompted
us
to
assess
their
functional
similarity.
Plasmid
pB65
weakly
suppressed
the
raffinose
and
sucrose
growth
defects
of
snf4-A2
and
snfl-AJO
mutants
MCY1853
and
MCY1845.
Transformation
of
these
strains
with
pLS2
showed
that
increased
dosage
of
YCK2
also
weakly
sup-
pressed
the
growth
defects.
Because
invertase
catalyzes
the
hydrolysis
of
these
sugars,
we
assayed
invertase
activity
(36)
in
snf4
mutants
carrying
pB65,
pB65R7,
or
pLS2.
None
of
these
plasmids
restored
significant
invertase
expression
after
a
shift
to
low
glucose
(2-3.5
units
for
YCKJ
and
YCK2
plasmids;
1.5-2
units
for
vectors;
160
units
for
SNF4
plas-
mid).
In
the
reciprocal
experiment,
a
multicopy
subclone
of
YCKI
(pLJ721;
Fig.
1A)
was
introduced
into
JC482
and
transformants
were
tested
for
the
ability
to
grow
on
medium
containing
1.2
M
NaCl.
pLJ721
was
as
effective
as
pLS2
in
this
assay,
while
cells
carrying
the
vector
were
unable
to
grow
(data
not
shown).
Loss
of
YCK
Function
Is
Lethal.
The
phenotype
caused
by
loss
of
YCKJ
function
was
examined
by
constructing
a
diploid
strain
heterozygous
for
the
yckl-AJ::URA3
allele.
The
URA3
marker
segregated
in
a
Mendelian
fashion
among
tetrads
derived
from
this
strain,
and
none
of
the
Ura+
1
.
MSMPIASTTLAVNNLTNINGNANFNVQANKQLHHQAVDSPAR.
.SSMTATTAANSNSNS
...
SRDDSTIVGLHYKIGKKIDET.SF
.VLFEGT
YCK1
:
11111
1
1:
:
:
1:
1111111111111111
1
MSQVQSPLTATNSGLAVNNNT
...
MNSQMPNRSNVRLVNGTLPPSLHVSSNLNHNTGNSSASYSGSQSRDDSTIVGLHYKIGKKIfEcSFcVLFEGT
YCK2
II
88
NMINGVPVAIKFEPRKTEAPQLRDEYKTYKILNGTPNIPYAYYFGQEGLHNILVIDLLGPSLEDLFDWCGRKFSVKTVVQVAVQMITLIEDLHAHDL
YCK1
11111:1111111111111111:111:11111
111
11
111111111ll111111111111111111111:1111111111111111111111111
95
NMINGLPVAIgEPRKTEAPQLKDEYRTYKI
LAGTPGIPQEYYFGQEGLHNILVIDLLGPSLEDLFDWCGRRFSVKTVVQVAVQMITLIEDLHAHDL
YCK2
III
185
IYRDIKPD.fFLIGRPGQPDANNIHLIDEIMAKQYRDPKTKQHIPYREKKSLSGTARYMSINTHLGREQSRRDAMEALcHVFFYFLRGHLPWQGLKAP
YCK1
111111111111111111111
:1111111111111111111111111111111111111111111111111
1111:1111111111:111111111
192
IYRwIKPDEFLIGRPGQPDANKVHLIDEMAKQYRDPKTKQHIPYREKKSLSGTARYMSINTHLGREQSRRDnMEAMcHVFFYFLRGQLPWQGLKAP
YCK2
1111:1
:II:IIII:III
:IIIIIIIIIIIIIIIIIIII1lIIII1lI:1
I11
DIJFLMGLG.
.
KKGNLVYI
IDFlLAKKYRDARTHQHIPYRENKNLTGTARYASINTHLGIEQSRRDDLESLcYVLMYFNL
CK1
VI
VII
Ix
282
NNKQKYEKIGEKKRSTNVYDLAQGLPVQFGRYLEIVRSLSFEECPDYEGYRKLLLSVLDDLGETADGQYDWMKLNDGRGWDLNINKKPNLHGYGHPN
YCK1
289
NNKQKYEKIGEKKRLTNVYDLAQGLP
IQFGRYLEIVRNLSFEETPDYEGYRMLLLSVLDDLGETADGQYDWMKLNGGRGWDLSINKKPNLHGYGHPN
YCK2
379
PPNEKSRKHR
..............
NKQLQMQQ
............
LQMQQLQQQQQQQQYAQKTEADMRNSQYK
.....
PKLDPTSYEAYQHQTQQK
YCK1
111111::11
~~1
111
111111111
1:IIII:I
:
:1:1
111111:11
386
PPNEKSKRHRSKNHQYSSPDHHHHYNQQQQQQQAQAQAQAQAQAKVQQQQLQQAQAQQQ
.
ANRYQLQPDDSHYDEEREASKLDPTSYEAYQQQTQQK
YCK2
444
YLQEQQKRQQQQKLQEQQLQEQQLQQQQQQQQQLRATGQPPSQPQAQTQSQQFGARYQPQQQPSAALRTPEQHPNDDNSSLAASHKGFFQKLGCC*
YCK1
1
1:111
I
11
1:
11
I
I
11
::
1:
11
1111
11111
483
YAQQQQK
.....
.....
QMQQKSKQFANTG
.......
ANGQTNKYPYNAQPTANDEQNAKNAAQDRNSNKSS
.....
KGFFSKLGCC*
YCK2
FIG.
2.
Alignment
of
the
YCK1
and
YCK2
predicted
amino
acid
sequences
and
partial
rabbit
testis
casein
kinase
I
(CK1)
sequence.
The
comparison
was
generated
by
the
GAP
program
(32).
Vertical
lines
denote
identity
and
double
dots
denote
conservative
changes.
Underlined
residues
correspond
to
readily
identifiable
conserved
protein
kinase
subdomains
(ref.
33;
subdomains
are
numbered
below
the
sequence).
The
YCKI
clone
includes
an
additional
54
codons
5'
to
the
first
ATG
(not
shown).
The
sequences
determined
for
four
tryptic
peptides
of
rabbit
reticulocyte
casein
kinase
I
were
XIFPDNFLMGIG,
XDDXESLGYVLMYFNL,
XHIPYXEDKNLTGT,
and
YASINAXLGIEQS
(where
X
denotes
a
cycle
at
which
no
assignment
could
be
made);
the
peptide
positions
depicted
in
bold
lettering
differ
from
the
testis
sequence.
30
Genetics:
Robinson
et
al.
Proc.
Natl.
Acad.
Sci.
USA
89
(1992)
31
segregants
showed
growth
differences
from
Ura-
segregants
on
any
carbon
source
tested
at
300C
or
37TC.
Regulation
of
invertase
expression
was
identical
in
four
yckl
segregants
and
four
wild-type
siblings.
Tetrad
analysis
was
also
carried
out
for
progeny
of
two
diploid
transformants
heterozygous
for
the
yck2:
:HIS3
allele.
All
spore
clones
were
viable,
and
His'
spore
clones
showed
no
obvious
growth
differential
from
wild-type
(His-)
siblings
under
any
conditions
tested,
including
high
salt,
fermentable
and
nonfermentable
carbon
sources,
and
high
(37TC)
and
low
(16TC)
temperature.
To
determine
the
phenotype
of
strains
lacking
both
YCKI
and
YCK2
function,
two
diploid
strains
heterozygous
for
both
yckl
and
yck2
were
sporulated
and
tetrads
were
analyzed.
The
pattern
of
spore
viability
observed
(2:2,
3:1,
and
4:0)
was
consistent
with
the
presence
of
two
unlinked
mutations
that
together
are
lethal.
Single
yckl
and
yck2
disruption
mutants
were
recovered
with
the
expected
frequency
but
no
His'
Ura'
double
disruption
mutants
were
recovered.
To
confirm
that
yckl
yck2
is
a
lethal
combination,
we
examined
the
meiotic
progeny
of
strains
LRB271,
MCY2394,
and
MCY2395
(yckl/yckl
yck2/+)
and
LRB272
(yckl/+
yck2/
yck2).
In
each
case,
we
observed
the
2:2
segregation
for
viability
that
is
expected
if
the
double
mutant
is
inviable
(45/45
total
tetrads;
Fig.
3).
None
of
the
viable
spore
clones
tested
carried
both
mutations.
The
spore
clones
corresponding
to
the
predicted
double
mutants
showed
an
unusual
growth
pattern
(Fig.
3).
All
such
spores
germinated
and
divided
several
times
on
rich
medium
containing
glucose
at
30°C,
but
normal
division
ceased
after
3-10
rounds.
Most
cells
then
formed
one
or
multiple
projec-
tions
that
continued
to
elongate
over
24-48
hr,
after
which
growth
ceased.
The
lethality
of
the
double
mutation
was
not
rescued
by
alteration
of
carbon
source
(ethanol
or
raffinose),
temperature
of
incubation
(16°C,
25°C,
or
37°C)
or
ionic
(0.1
or
1
M
KCI;
0.9
M
NaCI)
or
osmotic
(1
M
sorbitol)
conditions.
To
confirm
that
mitotic
growth
requires
YCK
function,
we
analyzed
the
mitotic
progeny
of
double
mutants
carrying
pLS2.33
for
retention
of
the
plasmid
under
nonselective
conditions.
Four
transformants
of
the
strain
LRB271
(yckl/+
yck2/yck2)
carrying
pLS2.33
were
sporulated
and
all
yckl
yck2
(Ura+
His+)
segregants
carried
the
plasmid
(Leu+).
Four
such
spore
clones
were
grown
in
nonselective
medium
for
20
generations,
and
cultures
of
each
were
plated
on
nonselective
medium.
All
of
the
resulting
colonies
(>400
colonies
per
strain)
carried
the
plasmid
marker.
In
contrast,
identical
experiments
with
single
yckl
(LRB263)
or
yck2
(LRB266)
mutant
strains
and
the
wild-type
strain
JC482
carrying
pLS2.33
resulted
in
only
30-50%
plasmid
retention.
These
results
indicate
that
plasmid
loss results
in
inviability
and
thus
that
YCK
function
is
required
for
mitotic
growth.
YCK2
Affects
Casein
Kinase
I
Activity.
The
extensive
se-
quence
similarity
between
the
YCK
gene
products
and
rabbit
casein
kinase
I
suggests
that
YCKI
and
YCK2
encode
casein
kinase
I
species.
We
therefore
tested
the
effects
of
increased
dosage
of
one
of
the
two
genes,
YCK2,
on
casein
kinase
I
activity.
A
synthetic
peptide
substrate
that
is
phosphorylated
specifically
by
mammalian
casein
kinase
I
(8)
was
used
to
assay
casein
kinase
I
activity
in
yeast
cell
extracts.
Extracts
from
cells
overexpressing
YCK2
and
from
control
strains
carrying
the
vector
were
assayed.
Increased
gene
dosage
of
YCK2
was
correlated
with
a
2-
to
3-fold
increase
in
casein
kinase
I
activity
(Fig.
4).
DISCUSSION
In
this
report,
we
describe
two
structurally
and
functionally
homologous
genes
that
were
isolated
in
independent
genetic
screens.
Together,
these
genes
are
essential
for
yeast
cell
growth.
The
predicted
products
of
the
two
genes
show
extensive
sequence
similarity
to
partial
sequence
obtained
FIG.
3.
Loss
of
YCK
function
is
lethal.
(Upper)
Ten
tetrads
that
are
progeny
of
diploid
strain
LRB271
(yckl/yckl
yck2/+).
(Lower)
Clusters
of
cells
corresponding
to
inviable
double
mutants,
illustrat-
ing
both
the
elongated
terminal
morphology
and
the
variability
in
number
of
normal
divisions
before
the
terminal
morphology
was
manifested.
The
plate
was
incubated
at
300C,
and
photographs
were
taken
after
72
hr
(plate)
or
96
hr
(spore
clones)
of
incubation.
(x480.)
for
rabbit
casein
kinase
I,
and
increased
gene
dosage
of
YCK2
increases
casein
kinase
I
activity
in
cell
extracts.
Therefore,
we
propose
that
the
two
yeast
genes
encode
casein
kinase
I
homologues,
and
designate
them
YCKI
and
YCK2.
The
YCKI
and
YCK2
predicted
products
are
-62
kDa,
significantly
larger
than
mammalian
casein
kinase
I,
which
exists
as
a
monomer
of
30-38
kDa.
However,
nuclear
casein
kinase
I
species
from
plants
show
a
range
of
sizes
up
to
60
kDa
(1).
Also,
a
size
difference
is
not
unusual
between
yeast
and
mammalian
homologues;
yeast
products
often
show
N-
or
C-terminal
extensions
relative
to
their
mammalian
coun-
terparts
(37-39).
The
high
degree
of
sequence
similarity
between
the
YCK
predicted
products
and
rabbit
casein
kinase
I
indicates
that
the
three
proteins
are
members
of
the
same
protein
kinase
subfamily.
Biochemical
studies
of
casein
kinase
I
activity
in
yeast
have
detected
multiple
isoforms of
30-50
kDa
(10-12).
The
YCKI
and
YCK2
products
are
predicted
to
be
larger,
and
their
relationship
to
these
activities
is
not
clear.
We
observed
additional
cross-hybridizing
sequences
on
genomic
Southern
blots,
hybridized
with
YCKJ
or
YCK2
sequence
(L.C.R.,
unpublished
work).
These
related
sequences
may
encode
related
proteins
with
different
biological
function.
We
do
not
understand
why
the
YCK
genes
were
isolated
using
the
genetic
screens
described
here.
The
ability
of
casein
kinase
I
homologues
to
suppress
the
raffinose
growth
defect
caused
by
decreased
SNF1
protein
kinase
activity
could
be
due
simply
to
promiscuous
phosphorylation
by
the
overex-
pressed
YCK
products
that
compensates
for
loss
of
phos-
phorylation
by
SNF1.
However,
the
observation
that
in-
creased
dosage
of
YCKI
or
YCK2
allows
growth
of
a
snf4
mutant
on
raffinose
without
significantly
restoring
invertase
activity
suggests
another
mode
of
suppression.
The
snf4
Genetics:
Robinson
et
al.
Ir
i
Proc.
Natl.
Acad.
Sci.
USA
89
(1992)
C
Ii1500
CE1000
O
0
0
--
0
5
1
0
1
5
20
Time
(min)
FIG.
4.
Casein
kinase
I
activity
is
elevated
in
strains
overex-
pressing
YCK2.
Incorporation
of
32P
into
casein
kinase
I
substrate
peptide
by
yeast
extracts
was
measured
as
a
function
of
time.
Extracts
were
prepared
from
the
wild-type
strains
JC482
(circles)
and
LRB188
(triangles)
transformed
with
the
multicopy
YCK2
plasmid
pLS2.3
(filled
symbols)
or
the
vector
YEp352
(open
symbols).
Data
shown
are
from
one
of
three
experiments
that
gave
similar
results.
growth
defect
reflects
the
inability
of
the
mutant
both
to
derepress
invertase
and
to
utilize
a
low
concentration
of
hexose
effectively.
Thus,
it
is
possible
that
overexpression
of
the
YCK
genes
results
in
more
efficient
hexose
metabolism
without
affecting
the
glucose
repression
mechanism
directly.
The
mechanism
responsible
for
the
effects
of
increased
YCKJ
and
YCK2
dosage
on
salt
tolerance
is
also
not
obvious.
Hyperaccumulation
of
glycerol
is
the
best
defined
response
that
is
required
for
adaptation
of
S.
cerevisiae
to
high-salinity
medium
(31).
Much
of
the
glycerol
is
synthesized
from
glycolytic
intermediates,
since
cells
adapting
to
high
salt
show
increased
activities
both
of
glycolytic
enzymes
and
of
enzymes
responsible
for
glycerol
synthesis
(30,
31).
It
is
possible
that
increased
YCK
activity
causes
changes
in
hexose
metabolism
that
account
for
both
suppression
of
snf4
and
increased
tolerance
to
high
salt.
Together
YCKI
and
YCK2
are
essential
for
the
viability
of
yeast
cells.
Neither
of
the
phenotypes
with
which
these
genes
are
associated
suggests
an
essential
function,
since
neither
the
response
to
glucose
deprivation
nor
tolerance
of
high
salt
is
required
for
viability
under
appropriate
conditions.
That
casein
kinase
I
activity
is
essential
for
growth
in
all
conditions
tested
could
reflect
a
requirement
for
phosphorylation
of
a
wide
variety
of
substrates
or
of
a
single
essential
substrate.
The
isolation
of
the
yeast
casein
kinase
I
homologues
will
allow
genetic
analysis
of
the
functions
of
these
protein
kinases
in
eukaryotic
cells.
Note
Added
in
Proof.
The
S.
cerevisiae
HRR25
protein
(40)
is
40%o
identical
to
YCK1
and
YCK2
and
80%o
identical
to
the
rabbit
sequence.
We
thank
S.
L.
Agellon
for
help
with
analysis
of
YCKI
and
P.
Leeds
for
critical
reading
of
the
manuscript.
Research
was
supported
by
National
Institutes
of
Health
Grants
GM26217
(M.R.C.),
GM34095
(M.C.),
DK27221
(P.J.R.),
GM40219
(C.H.H.),
and
GM45460
(M.G.),
Postdoctoral
Fellowship
GM14117
(L.C.R.),
and
Predoctoral
Training
Grant
CA09503
(E.J.A.H.);
a
Lucille
P.
Markey
Trust
Award
(M.R.C.
and
C.K.);
an
American
Cancer
Society
Faculty
Research
Award
(M.C.);
National
Institutes
of
Health
Research
Career
Development
Award
DK01690
(A.A.D.-R.);
and
a
Veterans
Administration
Associate
Investigator
Career
Develop-
ment
Award
(D.W.H.).
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