From the Casein kinase I The casein kinase I
(CKI)1 gene family
encompasses an increasing number of genes expressed in eukaryotes
including yeast Caenorhabditis elegans and mammals. Two
subgroups of the CKI family have been separated by functional analysis
and complementation of mutations in yeast. One group encoding nuclear
kinases appears in yeast to be involved in the response to DNA damage.
Mutations in these genes, including HRR25 and YCK3
in Saccharomyces cerevisiae and hhp1 and
hhp2 in Schizosaccharomyces pombe, lead to
sensitivity to DNA damaging agents such as x-rays and methyl
methanesulfonate (1-4). The mammalian genes encoding CKI The structure of the CKI family suggests several potential mechanisms
for the regulation of activity. All family members contain a short
amino-terminal domain of 9-76 amino acids, a highly conserved kinase
domain of 284 amino acids, and a highly variable carboxyl-terminal domain that ranges from 24 to more than 200 amino acids in length. The
carboxyl terminus of a CKI isoform may serve several functions, including regulation of substrate recognition, modulation of catalytic activity, and/or determination of kinase subcellular localization. Prenylation of the tail of the YCK1/YCK2 family has been
shown to be of functional importance in yeast (3, 8, 9).
Phosphorylation of CKI may also be an important regulatory mechanism.
Most of the CKI proteins are phosphoproteins, and several of the yeast kinases can autophosphorylate on serine, threonine, and tyrosine residues (10). Studies using synthetic substrates and the artificial substrate casein have indicated that these phosphoryl residues may
inhibit kinase activity (11, 12), although the location of these
inhibitory groups remains unclear. Kuret and co-workers (11) found that
an unphosphorylated truncation of Cki1 containing only the kinase
domain was twice as active as the phosphorylated form, whereas Graves
et al. (12) mapped a phosphorylation-dependent inhibitory domain in CKI One in vitro functional assay of CKI activity is its ability
to phosphorylate simian virus 40 (SV40) large T antigen. We have previously shown that CKI We have now overexpressed and purified active CKI We also report that CKI Ni2+-nitrilotriacetate-agarose was obtained from
Qiagen. Trypsin (T8642), calcineurin, and calmodulin were from Sigma.
PP1c and inhibitor 2 were from New England Biolabs. Okadaic
acid was from Life Technologies, Inc. Restriction enzymes were from
Life Technologies, Inc. and New England Biolabs. Plasmids expressing I Cloning and Escherichia coli Expression of CKI Purification and Partial Proteolysis of Recombinant
CKI Division of Molecular Biology and Genetics,
Program in Human Molecular Biology and
Genetics,
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
(CKI
) is a
member of the CKI gene family, members of which are involved in the
control of SV40 DNA replication, DNA repair, and cell metabolism. The
mechanisms that regulate CKI
activity and substrate specificity are
not well understood. We report that CKI
, which contains a highly
phosphorylated 123-amino acid carboxyl-terminal extension not present
in CKI
, is substantially less active than CKI
in phosphorylating
a number of substrates including SV40 large T antigen and is unable to
inhibit the initiation of SV40 DNA replication. Two mechanisms for the
activation of CKI
have been identified. First, limited tryptic
digestion of CKI
produces a protease-resistant amino-terminal 39-kDa
core kinase with several-fold enhanced activity. Second, phosphatase treatment of CKI
activates CKI
5-20-fold toward T antigen.
Similar treatment of a truncated form of CKI
produced only a 2-fold
activation. Notably, this activation was transient;
reautophosphorylation led to a rapid down-regulation of the kinase
within 5 min. Phosphatase treatment also activated CKI
toward the
novel substrates I
B
and Ets-1. These mechanisms may serve to
regulate CKI
and related forms of CKI in the cell, perhaps in
response to DNA damage.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
and
CKI
complement HRR25-deleted yeast, suggesting they
too may be involved in DNA repair pathways (5). A second group in
S. cerevisiae encodes prenylated membrane-bound isoforms
involved in bud growth and includes YCK1 and YCK2
(6); deletions in these genes are complemented by the mammalian
genes encoding CKI
(7).
to a 26-amino acid domain in the
carboxyl-terminal tail. Polyanions such as heparin can regulate kinase
activity; activation by heparin appears dependent on the presence or
absence of the carboxyl-terminal domain (12, 13). Additionally,
membrane bound forms of the kinase may be regulated by
phosphatidylinositol-4,5-bisphosphate (14).
purified from HeLa cell extracts
phosphorylates T antigen on physiologic sites and inhibits the
initiation of viral DNA replication (15-17). As SV40 DNA replication
is regulated in the cell cycle and by DNA damage (18-21), it was of
interest to determine whether forms of CKI implicated in DNA repair
pathways could also regulate in vitro DNA replication.
. The data show
that this form of CKI, although active on peptide substrates, is
markedly hindered in its ability to phosphorylate and inhibit the
origin unwinding function of T antigen. This decreased activity of
CKI
is apparently due to an inhibitory effect of the
carboxyl-terminal domain not present in CKI
, since limited tryptic
digestion of CKI
released a catalytically active amino-terminal
39-kDa fragment able to both phosphorylate T antigen and inhibit its
replication initiation function.
is activated by dephosphorylation in a
tail-dependent manner. Treatment of recombinant CKI
with the catalytic subunits of PP1, PP2A, or PP2B (calcineurin) leads to as
much as a 20-fold increase in activity toward T antigen, casein, and
two novel substrates, the Ets-1 transcription factor and recombinant
I
B
. Activation of the kinase by phosphatases was transient
and self-limited; reautophosphorylation of the kinase led to
inactivation within 5 min. Activation was dependent on the presence of
the carboxyl-terminal domain of the kinase; a truncation mutant of
CKI
was activated by phosphatase 4-fold less than was full-length
kinase. These findings support the hypothesis that the
carboxyl-terminal domain of CKI
inhibits its activity on key protein
substrates and suggests that in the cell, CKI
may be regulated in a
self-limited manner by phosphatases and in a more sustained manner by
intracellular proteolysis.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
B
(22) were the generous gift of John Hiscott.
--
The
cDNA encoding wild type human CKI
was isolated as a
NcoI/SalI 1333-base pair fragment and ligated
into NcoI/XhoI-digested pET16b (a T7-based
expression vector from Novagen) as described previously (5). This
construct (pKF115) removes the hexahistidine sequence present in the
pET16b vector downstream from the NcoI site. The 1317-base
pair NcoI/HindIII fragment from pKF115 was ligated into the same sites in pRSET-B (a T7-based expression vector
from Invitrogen). This construct (pV71) encodes CKI
with a 41-amino
acid amino-terminal fusion that contains a hexahistidine tag and an
enterokinase cleavage site. The d305 truncation mutant was created by
the introduction of a stop codon after amino acid 305 in a derivative
of pV71 (pKF162) by site-directed mutagenesis. All recombinant proteins
were expressed in BL21(DE3) cells (23). Bacteria were grown in Luria
broth containing 50 µg/ml carbenicillin at 37 °C to an
A600 of 0.3 and induced overnight at 28 °C
with 1 mM
isopropyl-1-thio-
-D-galactopyranoside.
--
Hexahistidine-tagged CKI
was expressed at low levels in
BL21(DE3) cells. Lysates in 20 mM Hepes, pH 7.5, 25 mM NaCl, 1 mM DTT, 1 mM EDTA,
0.02% Nonidet P-40, 10% sucrose (buffer B) with 0.1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml
pepstatin were applied to DEAE-cellulose and batch-eluted with the same
buffer containing 200 mM NaCl. Kinase-containing fractions
were dialyzed into buffer B with 10 mM NaCl and then applied to a S-Sepharose column equilibrated with the same buffer. Kinase activity was batch-eluted with buffer B with 250 mM
NaCl (without EDTA or DTT) and loaded directly onto
Ni2+-nitrilotriacetate-agarose from which it was eluted
with buffer B (without EDTA or DTT) containing 80 mM
imidazole. Untagged CKI
was purified by a similar procedure except
that the Ni2+-nitrilotriacetate-agarose step was omitted.
The typical yield was 100 µg of CKI
from a 2-liter culture.
Hexahistidine-tagged CKI
was used in all assays except where
specifically indicated. Histidine-tagged and untagged kinase were found
to behave identically in all assays tested.
was
performed in the standard kinase reaction buffer (30 mM Hepes, pH 7.5, 7 mM MgCl2, 0.5 mM
DTT for 30 °C for 15 min either without or with previous
autophosphorylation of substrate. Trypsin was then inhibited by soybean
trypsin inhibitor added to a final concentration of 10 µg/ml.
Proteolysis with trypsin at 1 µg/ml gave almost quantitative scission
of CKI
into its major digestion products; this concentration of
trypsin was routinely used for the production of the active tryptic
fragment.
Immunoblot Analysis--
For immunoblot analysis, proteins and
trypsin-generated peptides were separated by SDS-PAGE on a 17% gel and
then transferred to nitrocellulose membrane in 12.5 mM
Tris, 86 mM glycine, pH 8.3, 0.1% SDS, 20% methanol.
After a 15-min fixation in 0.5% glutaraldehyde in phosphate-buffered
saline, the membrane was blocked with 3% bovine serum albumin and then
incubated with a 1:500 dilution of UT31 antiserum raised against the
amino-terminal CKI peptide MELRVGNKYRLGC (5). Immunoreactive
peptides were detected using an alkaline phosphatase-conjugated
goat-anti-rabbit IgG (Bio-Rad) followed by incubation with
bromo-chloro-indolylphosphate and nitroblue tetrazolium (24).
Kinase and Phosphatase Assays--
Kinase reactions were
performed in buffer containing 30 mM Hepes, pH 7.5, 7 mM MgCl2, 0.5 mM DTT, 100 µg/ml
bovine serum albumin, 150 or 250 µM ATP, and 1-5 µCi
of [-32P]ATP at 37 °C or as indicated. Reactions
were stopped by the addition of SDS-PAGE sample buffer and analyzed by
SDS-PAGE and autoradiography as described previously (15, 16).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Substrate Specificity--
In previous studies we purified CKI
based on its ability to phosphorylate SV40 large T antigen and thus
inhibit the initiation of SV40 DNA replication (15, 16). In the process
of characterizing human CKI genes, we cloned a novel member of the CKI
family, CKI
, that encodes a 48-kDa kinase (5). CKI
was of
interest because, unlike CKI
, it was able to functionally complement
yeast deleted for the DNA repair-related kinase HRR25. In preliminary
studies on recombinant CKI
, we found virtually identical specific
activity to CKI
when tested on peptide substrates (data not shown).
However, although CKI
was found to readily phosphorylate T antigen,
CKI
was approximately 9-fold less active on this protein substrate (Fig. 1). Similar results were obtained
when a partially purified non-tagged form of CKI
was tested (data
not shown), indicating the histidine tag has no significant effect on
the ability of CKI
to phosphorylate T antigen. Interestingly, CKI
autophosphorylation was also consistently stimulated severalfold by the
addition of T antigen (Fig. 1, lane 4 compared with
lanes 5 and 6), suggesting a specific
protein-protein interaction between the carboxyl-terminal domain and
the substrate.
|
Effect of Carboxyl-terminal Tail on CKI Activity--
CKI
and CKI
are closely related in the kinase domain (86% similar and
74% identical) but differ in the length of the carboxyl-terminal tail.
CKI
has only a 24-amino acid extension beyond the kinase domain, whereas CKI
has a 123-amino acid tail. Recent studies of
carboxyl-terminal domains in the related kinases CKI
and Cki1 have
indicated that they perform a regulatory function (11, 27). The
differing activities of the CKI
and CKI
isoforms suggested that
the carboxyl-terminal domain of CKI
was responsible for the
decreased activity of the kinase on T antigen and secondly, that this
region might form a discrete structure separable from the kinase domain
by limited proteolysis. To test this, intact autophosphorylated CKI
was subjected to limited digestion with various concentrations (0-5
µg/ml) of trypsin. As demonstrated in Fig.
2, digestion of
32P-autophosphorylated CKI
with increasing amounts of
trypsin results in the production of a series of higher mobility
species that are further digested to a relatively stable 39-kDa
phosphoprotein that finally disappears during incubation at the highest
trypsin concentration (5 µg/ml). This 39-kDa product appears to be
activated CKI
since (a) its appearance coincided with a
3-fold increase in kinase activity toward T antigen, (b) it
reacts with polyclonal antibody UT31 that recognizes the amino terminus
of CKI
, and (c) the purified 39-kDa polypeptide retains
kinase activity (Fig. 3 and data not
shown). The 3-fold activation achieved by trypsin treatment may
under-represent the actual activation since (a) there may
have been partial loss of the kinase domain as well, and (b)
proteolytic tail fragments may retain some inhibitory activity (data
not shown). These results suggest that CKI
contains a discrete
inhibitory domain that blocks specific substrate phosphorylation.
|
|
Functional Activity of the 39-kDa Amino-terminal Fragment of
CKI Protein--
T antigen catalyzes the initial steps in SV40 DNA
replication, the unwinding of the duplex origin of replication to
single-stranded DNA. CKI
inhibits T antigen function by
phosphorylating it on at least two inhibitory sites, serines 120 and
123 (15, 16). The finding that proteolytic cleavage of CKI
stimulated its activity on T antigen led to the further functional
characterization of the 39-kDa tryptic fragment. Specifically, we
wished to determine whether its increased protein kinase activity
translated into an increased ability to inhibit T antigen-catalyzed
SV40 origin unwinding. CKI
, CKI
, and the 39-kDa CKI
fragment
(CKI
-39) were pre-incubated with T antigen and ATP, and then T
antigen-dependent unwinding of the SV40 minimal origin of
replication was assayed (Fig. 3). As previously demonstrated,
phosphorylation of T antigen by CKI
inhibited its origin unwinding
activity with 50% inhibition occurring at about 0.4 pmol of
kinase/reaction (40 nM in the preincubation mixture). In
contrast, CKI
very inefficiently inhibited the origin unwinding
activity of T antigen with 50% inhibition occurring at about 7 pmol of
kinase/reaction (700 nM in preincubation mixture). The
39-kDa fragment of CKI
was substantially more active than full-length CKI
, producing 50% inhibition of T antigen origin unwinding activity at about 0.9 pmol of kinase/reaction (90 nM in preincubation mixture, Fig. 3B). These
results indicate that the catalytic core of CKI
is in fact similar
in activity to CKI
and that proteolytic removal of the carboxyl
terminus of CKI
partially activates it to phosphorylate the same or
similar inhibitory sites on T antigen.
Diverse Serine/Threonine Phosphatases Activate CKI toward T
Antigen--
Recent studies on the related CKI isoform CKI
have
demonstrated that the full-length kinase can be activated 2-5-fold
toward casein or a synthetic peptide (D4) by treatment with the
catalytic subunit of protein phosphatase 1 (PP1c) (27). We
tested whether removal of phosphoryl groups from CKI
stimulated its
kinase activity toward SV40 large T antigen
(Fig.4, A and B).
Purified CKI
was incubated with increasing amounts of the catalytic
subunit of either PP1 or PP2A. At the end of the dephosphorylation
reaction, okadaic acid was added to a final concentration of 250 nM, and the activity of the kinase on T antigen was
assessed. As demonstrated in Fig. 4, A and B,
pretreatment of CKI
with either phosphatase stimulates its activity
on T antigen up to 20-fold, with half-maximal activation occurring with
less than 2 ng (~4 nM) of PP2Ac. The stimulation requires phosphatase catalytic activity, since inclusion of
okadaic acid in the preincubation fully blocks the effect of PP2Ac (Fig. 4A, lane 7). The
activation by 16 ng (~30 nM) of PP1c is only
partially blocked by 250 nM okadaic acid, a not unexpected result since PP1c is 10-100-fold less sensitive than
PP2Ac to inhibition by okadaic acid. Of note, alterations
in the phosphorylation state of CKI
had a significant effect on
kinase mobility (Fig. 4A). This appears to be due to tail
phosphorylation, as truncated CKI
can autophosphorylate without a
significant change in electrophoretic mobility (Fig. 7A and
data not shown).
|
Autophosphorylation Rapidly Inactivates CKI--
CKI
is
capable of autophosphorylation of up to approximately 12 mol/mol (Fig.
4C and see below). Since phosphatase treatment of the
bacterially expressed kinase leads to marked activation toward protein
substrates, we asked how rapidly reautophosphorylation of the kinase
occurs and whether this reautophosphorylation correlates with a
decrease in protein kinase activity. CKI
was dephosphorylated with
PP2Ac and then allowed to reautophosphorylate in the
presence of okadaic acid. T antigen was either added at the same time
as [
-32P]ATP (time 0) or at the indicated times after
ATP addition. As Fig. 5 demonstrates,
CKI
reautophosphorylation was complete within 5 min, coincident with
the re-suppression of kinase activity. Thus, CKI
appears to
re-autophosphorylate and autoinhibit itself rapidly in the absence of
phosphatase activity. Dilution studies and experiments with CKI
kinase dead mutants indicate that CKI
autophosphorylation is
entirely intramolecular (data not shown). Of note, neither activation
nor autoinhibition of kinase activity toward a peptide substrate was
seen in similar time course reactions (data not shown), suggesting
autoinhibition is effective toward protein but not peptide substrates.
|
Bacterially-produced CKI Is Heavily
Autophosphorylated--
Dephosphorylation of bacterially expressed
CKI
had a significant activating effect on the kinase. To determine
the number of phosphates on CKI
that contribute to kinase
inhibition, CKI
autophosphorylation was quantitated after
pretreatment with increasing amounts of PP2Ac or
PP1c (Fig. 4C). Untreated kinase was able to add
about 2.5 mol of phosphate/mol of kinase, whereas
PP2Ac-treated kinase added about 12 mol/mol. These data
suggest that the kinase was close to maximally autophosphorylated at
approximately 10 mol/mol when it was purified from E. coli.
Half-maximal activation of the kinase toward T antigen was seen when
the kinase added approximately 8 mol/mol, suggesting that 4 mol/mol of
phosphate had not been removed by the pretreatment. Similar results
were seen with PP1c.
PP2A Activates CKI Toward an Array of Protein
Substrates--
To determine whether dephosphorylation of CKI
activated it toward multiple protein substrates, the kinase was tested
using casein, replication protein A (the gift of Marc Wold, U. Iowa), and recombinant Ets-1 (the gift of Barbara Graves, U. Utah) as substrates. As demonstrated in Fig.
6A, CKI
is activated toward T antigen, casein, and Ets-1 but not replication protein A (RP-A) by
PP2Ac treatment. Similarly, treatment of CKI
with PP1,
PP2A, or PP2B catalytic subunit activated the kinase toward
GST-I
B
(Fig. 6B). The activation of CKI
by
phosphatases is therefore a general phenomenon. Whereas CKI substrate
specificity on peptides can be determined by phosphoryl groups, we note
that the increase in activity occurs toward unphosphorylated
(bacterially produced Ets-1 and GST-I
B
) as well as phosphorylated
(T antigen) proteins.
|
CKI Phosphorylates the Carboxyl Terminus of
I
B
--
Although genetic studies have suggested a role for
CKI
-related proteins in DNA damage response pathways, few
physiologic substrates of CKI
have been identified. As I
B
is
phosphorylated and degraded in response to many signals including DNA
damage and it contains sequences in the amino and carboxyl termini
similar to CKI phosphorylation consensus sites found in peptide
substrates, we tested whether CKI
could be activated to
phosphorylate I
B
. As shown in Fig. 6B, CKI
phosphorylates GST-I
B
in a phosphatase-activable manner. The
serine/threonine phosphatases PP2Ac, PP1c, and
PP2B/calcineurin were all able to activate CKI
to phosphorylate
recombinant I
B
.
Full Activation by Phosphatases Requires the Carboxyl Terminus of
CKI--
One model to explain both the inhibitory effect of the
carboxyl-terminal tail and the stimulatory effect of dephosphorylation is that phosphate groups on the carboxyl terminus of CKI
interact with the kinase domain, leading to inhibition of protein substrate binding. This model suggests that a tail-less CKI
should not be
activated by phosphatases. To test this, a stop codon was introduced after amino acid 305 in CKI
, and the truncated histidine-tagged protein (denoted CKI
-d305) was expressed in E. coli and
purified on Ni2+-nitrilotriacetate-agarose. Roughly equal
amounts of full-length and truncated CKI
were used to phosphorylate
T antigen, either without or with prior phosphatase treatment. As shown
in Fig. 7, CKI
was activated 9-fold by
PP2Ac, whereas CKI
-d305 was activated only 2-fold.
Similarly, CKI
(76% identical to CKI
over the kinase domain and
lacking a carboxyl-terminal tail) was not activated by
PP2Ac (data not shown). The data suggest that much of the
observed autoinhibition of CKI
requires the carboxyl-terminal tail,
but that an inhibitory phosphoryl group on the CKI
kinase domain also contributes to autoinhibition. These results are consistent with
those seen by Kuret and co-workers (11) on the S. pombe homolog Cki1-d298 but differ slightly from those of Graves and Roach
(27) who found that truncated CKI
was not activated by phosphatase
treatment.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The casein kinase I family is characterized by a conserved core
kinase domain and a series of variable carboxyl-terminal extensions. This study indicates that one major function of the carboxyl terminus is to regulate the activity of CKI on protein substrates. The ability
of the carboxyl terminus to inhibit protein but not peptide phosphorylation suggests that this tail region interacts with the
substrate binding face of the kinase, as illustrated in Fig. 8, rather than directly in the catalytic
cleft. The data suggest CKI autoinhibition may be relieved by at least
three mechanisms; (a) proteolytic cleavage of the tail (this
study), (b) dephosphorylation of the kinase (this study and
Refs. 11 and 27), and (c) binding of heparin (and presumably
other polyanions) to the tail (27). Which, if any, of these mechanisms
function in vivo is the subject of ongoing investigation.
One reason for the diversity in CKI carboxyl-terminal domains may be to
allow distinct activation mechanisms for the different family members.
Alternatively, the function of the tail may be to constitutively
restrict access to the catalytic cleft to all but a limited number of
substrates, and no further regulation of the kinase may occur in
vivo. Differentiation between these mechanisms will require the
demonstration and characterization of CKI activation in
vivo. Given the functional similarity between CKI and HRR25,
one possibility is that agents that trigger the DNA repair response
will lead to CKI
activation.
|
Activation of forms of CKI lacking a carboxyl-terminal extension by
dephosphorylation suggests that at least one inhibitory phosphoryl
group is present on the core kinase domain and may interact directly
with the kinase tail. Additionally, the finding that multiple
phosphatases can activate CKI raises the possibility that the kinase
may be activated by diverse signal transduction pathways. Since the
inhibitory phosphoryl groups are placed in an intramolecular
autophosphorylation reaction, kinase activation by dephosphorylation
allows a very tight temporal control over the duration of kinase
activity. As we have demonstrated, once the activating phosphatase is
removed, autophosphorylation leads to rapid kinase inactivation. Such
self-limited bursts of kinase activity allow rapid down-regulation of
kinase activity, a mechanism that may be required to turn off a pathway
once its inciting agent is removed. Alternatively, if CKI
itself
activates a phosphatase, for example by CKI phosphorylation of the
protein phosphatase 1 inhibitor 2 (data not shown and Ref. 30), then a
positive feedback loop may be established that can further increase the activity of CKI
.
The observation that trypsin can activate CKI supports the model
(shown in Fig. 8) that the inhibitory tail is much more accessible to
proteolysis than is the kinase domain itself. Cleavage of CKI
by
regulated intracellular proteolysis would lead to a long lived
activation of CKI
in response to the appropriate stimulus. Proteolytic activation has been demonstrated for an increasing number
of intracellular proteins, including protein kinase C, mitogen-activated protein kinase kinase kinase 1 (MEKK1), and NF-
B
(31-33). DNA damage resulting in protease activation might lead to
CKI
cleavage. Notably, caspases with DEVD sequence specificity (34)
may be activated during apoptosis and cleave in the acidic region of
CKI
at the beginning of the carboxyl terminus. Such cleavage might
either activate CKI
or release it from its normal anchoring
site.
Previous studies have suggested that CKI recognizes peptide and protein
substrates differently. CKI phosphorylates acidic peptides, with the
best peptide substrates containing either phosphorylated or acidic
residues amino-terminal to the target site (35, 36). In this case,
peptide recognition requires interaction of the phosphorylated region
with the catalytic fold (37, 38). However, this localized charge
interaction does not appear operative in the case of T antigen
phosphorylation. We have previously shown that (a) CKI
can phosphorylate bacterially expressed (and hence unphosphorylated) T
antigen and (b) CKI
does not recognize its target site in
the amino terminus of T antigen unless the carboxyl-terminal residues
of T antigen are present (15). Thus, CKI
could not phosphorylate a T
antigen-derived peptide, an amino-terminal tryptic fragment of T
antigen, nor T259, an active recombinant amino-terminal fragment of T
antigen that contains the target sites and the DNA binding domain.
Similarly, in the current study we found that removal of the carboxyl
terminus of I
B
blocks phosphorylation of upstream residues. These
results are consistent with a more complex picture of kinase-substrate
interactions, where the tertiary structure of the substrate interacts
with multiple surface features of the kinase. Therefore, additional
structural elements of the kinase may interfere with docking of
substrate proteins at sites distant from the target residues. One
function of the CKI
tail may be to restrict the access of protein
substrates to the active site until the kinase is activated by
transient dephosphorylation or limited proteolysis. However, these
results do not exclude the possibility that in vivo, the
inhibitory effect of the tail observed in vitro could also
be overcome by constitutive active dephosphorylation or additional
mechanisms such as noncovalent binding of regulatory molecules.
Identification of potential cellular substrates of CKI has been
problematic. CKI isoforms can phosphorylate SV40 large T antigen (15,
16), p53 (39), inhibitor-2 (30), and glycogen synthase, among others.
The present study extends the list by identifying the carboxyl terminus
of IB
as an in vitro substrate. Whether CKI is as
important as CKII in the phosphorylation of I
B
in vivo
is unclear. In very few cases has a clear functional role for the
mammalian CKI family been defined. One approach to this question has
been to overexpress kinases in cells or organisms and examine them for
changes in phosphorylation patterns. An implication of this current
study is that overexpression of CKI
and related CKI family members
may produce inactive, autoinhibited kinase with no phenotype or effect
on in vivo substrates unless point mutations, truncations,
or activating conditions are first introduced. One possible route to
the identification of additional in vivo-specific substrates
of CKI
is therefore the development of autophosphorylation site
point mutants that retain an intact regulatory domain but are
constitutively active.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank John Hiscott for IB
constructs
and Erica Vielhaber, Paola Dal Santo, Mylynda Schlessinger, and Brent
McCright for help along the way.
![]() |
FOOTNOTES |
---|
* Oligonucleotide synthesis was supported by NCI, National Institutes of Health Grant 3P30 CA42014, and the research was supported by National Institutes of Health Grant CA71074.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.
§ Contributed equally to this work.
** To whom correspondence should be addressed: University of Utah, 15 North 2030 East, Room 2100, Salt Lake City, UT 84112-5330. E-mail: David.Virshup{at}genetics.utah.edu.
1 The abbreviations used are: CKI, casein kinase I; SV40, simian virus 40; PP2A, PP1, and PP2B, protein phosphatase 2A, 1, and 2B, respectively; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase.
2 A. Cegielska, E. Vielhaber and D. M. Virshup, unpublished results.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|