(Received for publication, April 27, 1995; and in revised form, July 8, 1995)
From the
Casein kinase I is a member of the casein kinase I (CKI)
family, a group of second messenger independent protein kinases. We
present evidence that the COOH-terminal domain of CKI
has
regulatory properties. CKI
expressed in Escherichia coli was activated by heparin, as found previously, and by treatment
with the catalytic subunit of type-1 protein phosphatase (CS1).
Concomitant with activation by CS1, there was a reduction in the
apparent molecular weight of CKI
from 55,000 to 49,000 as judged
by polyacrylamide gel electrophoresis in the presence of sodium dodecyl
sulfate. Truncation of CKI
by removal of the COOH-terminal 110
amino acids eliminated the ability of CS1 to activate or to increase
electrophoretic mobility. Casein kinase I
, a 37-kDa isoform that
lacks an extended COOH-terminal domain, was not activated by CS1 or the
presence of heparin. However, a chimeric enzyme consisting of CKI
fused to the COOH-terminal domain of CKI
was activated by both
heparin and CS1. Analysis of the effects of CS1 on a series of CKI
COOH-terminal truncation mutants identified an inhibitory region
between His
and Pro
, which contained six
potential phosphorylation sites. From analysis of the specific
activites of these truncation mutants, removal of the same region
resulted in enzyme with a specific activity nearly 10-fold greater than
wild-type. Thus, CKI
activity can be regulated by phosphorylation
of its COOH terminus, which may serve to create an autoinhibitory
domain. This mechanism of regulation could have important consequences in vivo.
Casein kinase I (CKI) ()is one of two classes of
protein kinase discovered over 20 years ago for their ability to
phosphorylate of casein in vitro (for reviews, see (1) and (2) ). It is now known that CKI and casein
kinase II, the other major casein kinase form, are distinct gene
products and, by amino acid sequence, are no more related than other
protein kinases. CKI has been detected in numerous animal and plant
cells including those of mammals, yeast, broccoli(3) , Dictyostelium(4) , and Paramecium(5) . The enzyme is found in cytosolic,
membrane, mitochondrial, and nuclear fractions(1) . Cytosolic
CKI is a monomeric polypeptide of
34-37 kDa, but nuclear CKI
has been reported with molecular masses from 23 to 55 kDa(1) .
The heterogeneity in size earlier reported for CKI can be explained, at
least in part, by the presence of multiple isoforms. In mammals, at
least six different genes are known, encoding isoforms termed CKI
and -
of 37 and 38 kDa(6, 7, 8) ,
CKI
1, -
2, and -
3 of 45, 47, and 51 kDa(9) , and
CKI
of 49 kDa(10) . Saccharomyces cerevisiae has
four different genes, HRR25(11) , YCK1 and YCK2(12, 13) , and YKS1, (
)encoding proteins ranging in size from 57
to 62 kDa. In Schizosaccharomyces pombe, four genes have also
been identified, CKi1 and CKi2(14) and Hhp1 and Hhp2(15, 16) , encoding
proteins from 42 to 50 kDa. HRR25, Hhp1, and Hhp2 have been implicated in DNA strand break repair, and yeast
defective in these genes show hypersensitivity to DNA damaging
agents(11, 15) . Interestingly, CKI
shows 73%
identity to Hhp1, the highest identity between any mammalian and yeast
CKI isoform, raising the possibility that mammalian CKI
might have
a role in DNA repair. All CKI isoforms have a similar structural
architecture consisting of a conserved catalytic domain of
300
amino acids and differently sized NH
- and COOH-terminal
extensions. The COOH-terminal noncatalytic domains range in size from
13 to 188 amino acids and in general show little amino acid identity
between isoforms.
Many proteins are CKI substrates in vitro. Included are cytosolic proteins such as glycogen synthase, acetyl-CoA carboxylase, and the inhibitor-2 protein of type-1 protein phosphatase(1) ; cytoskeletal proteins such as myosin, troponin, ankyrin(1) ; membrane-associated proteins such as spectrin, neural cell adhesion molecule(17) , and the insulin receptor; nuclear proteins such as p53 (18) , cAMP response element modulator (CREM)(19) , SV40 large T antigen (20) , and RNA polymerases I and II(21) ; and proteins involved in protein synthesis such as initiation factors 4E(22) , 3, 4B, and 5, aminoacyl-tRNA synthetases and ribosomal protein S6(1). Those proteins for which there is evidence for phosphorylation at the CKI sites in vivo include glycogen synthase(23) , SV40 large T antigen (24) , cAMP response element modulator(19) , and p53(18) . CKI phosphorylation is known to inhibit glycogen synthase(25) , inhibit DNA replication by SV40 large T antigen(24) , and enhance DNA binding of cAMP response element modulator(19) .
Unlike most protein Ser/Thr kinases, CKI recognizes acidic amino
acids in its substrates and has therefore been termed an
``acidotropic'' protein kinase(26) . Synthetic
peptide substrates that contain several acidic amino acids
NH-terminal to the target residue, such as the D4 peptide
(DDDDVASLPGLRRR), are relatively specific CKI substrates(27) .
These substrates are, however, usually much less efficient than
phosphorylated substrates in which the phosphate group is found in the
sequence motif (Ser(P)/Thr(P))-Xaa-Xaa-(Ser/Thr)- (26, 28) . CKI's ability to utilize a phosphate
group as a recognition determinant could link its activity to that of
other protein kinases, which in turn could be regulated by classical
second messengers(29) . Some effective CKI substrates do not
require prior phosphorylation. The clearest example here is inhibitor-2
of protein phosphatase 1 (30) which contains a cluster of
acidic residues NH
-terminal to the target site.
Relatively little is known about the control of CKI enzymes. In this
report, we define an autoinhibitory region in the COOH terminus of
CKI through which activity can be modulated by phosphorylation.
Figure 7:
Localization of an autoinhibitory region
in the COOH-terminal domain of CKI. A, the indicated
CKI
COOH-terminal truncation mutants or wild-type CKI
(WT) were treated with CS1 for 60 min, and their activities
toward the D4 peptide were measured. The activation is normalized to
the activity measured without dephosphorylation. B, the
specific activity of the untreated samples using the D4 peptide as
substrate.
Figure 1:
Effect of autophosphorylation on the
electrophoretic mobility of CKI. CKI
, after the indicated
treatment, was subjected to SDS-PAGE, transferred to nitrocellulose,
and probed with anti-CKI
antibody as described under
``Experimental Procedures.'' An autoradiogram is shown. Lane 1, untreated recombinant CKI
; lane 2,
CKI
after treatment with CS1; lane 3, sample from lane 2 incubated with ATP, Mg
, and 1
µM microcystin.
Figure 2:
Activation of CKI by catalytic
subunit of type 1 phosphatase. Recombinant CKI
was incubated with
CS1 (filledcircles) for the time indicated and then
assayed for protein kinase activity as described under
``Experimental Procedures.'' The control incubation (opencircles) lacked phosphatase.
However, even though the 55-kDa
form of CKI was already phosphorylated, it could still undergo
additional autophosphorylation in vitro, to a stoichiometery
of 2-3 mol of phosphate/mol of protein, again exclusively at Ser
and Thr residues (data not shown). This latter autophosphorylation did
not change the electrophoretic mobility of the enzyme or significantly
affect its activity (data not shown). Enzyme allowed to
autophosphorylate in the presence of
[
-
P]ATP was digested with endolysine C, and
the resulting peptides were resolved on SDS-PAGE. After transfer to a
polyvinylidine difluoride membrane, the predominant
P-labeled species was subjected to protein sequencing. A
unique CKI
sequence, FGA, was obtained, which corresponds to amino
acids Phe
-Ala
. The next lysine
residue occurs at Lys
, thus localizing these in vitro autophosphorylation sites to the region from residue 295 to 368.
In other experiments, the initial rate of CKI
autophosphorylation
was shown to be first order with respect to protein concentration over
a 20-fold range of enzyme (2.5-50 µg/ml) (data not shown).
Thus, the specific activity is independent of concentration, suggesting
that in vitro autophosphorylation of CKI
occurs by an
intramolecular mechanism.
Figure 3:
Effect of heparin or phosphatase
treatment on CKI, CKI
, CKI
317, and CKI
-
.
The effect of 100 µg/ml heparin (cross-hatchedbars) or incubation with CS1 for 60 min (solidbars) on D4 peptide phosphorylation was analyzed.
Activities are normalized to controls in the absence of heparin or
phosphatase treatment. Conditions were as described under
``Experimental Procedures.''
Since there was a correlation between heparin and CS1 activation of
CKI (Fig. 3), we determined whether the phosphorylation
state of CKI
affected its activation by heparin. CKI
was
treated with CS1, and then protein kinase activity was measured with
increasing concentrations of heparin. Once CKI
was activated by
CS1, it could no longer be activated by heparin (Fig. 4). This
finding suggests that heparin and CS1 activate CKI
, at least in
part, via a common mechanism.
Figure 4:
Effect of heparin on dephosphorylated
CKI. CKI
was treated with CS1 for 60 min (filledcircles) and D4 peptide phosphorylation analyzed in the
presence of the indicated concentrations of heparin. In a control (opencircles), similar measurements were made of
CKI
, which had not been exposed to
phosphatase.
Figure 5:
Amino acid sequence of an autoinhibitory
domain in CKI. Amino acids His
to Met
of CKI
are shown. Potential autophosphorylation sites are underlined; arrows denote the locations of the
different truncations described in this
study.
Figure 6:
Effect of phosphatase treatment on the
electrophoretic mobility of CKI COOH-terminal truncation. The
indicated CKI
COOH-terminal truncation mutants were treated with
CS1 for 60 min and separated on SDS-PAGE as described under
``Experimental Procedures.'' Untreated samples were similarly
analyzed. The migration of molecular mass standards, indicated in kDa,
is shown.
COOH-terminal
deletion itself affected the activity of CKI as judged by the
specific activities of the purified truncated proteins (Fig. 7B). Thus, CKI
317, in which essentially
all of the COOH-terminal tail was removed, had a specific activity
nearly 10-fold greater than that of wild-type enzyme. CKI
327
had a specific activity 5-fold greater than wild-type. However,
tuncations up to residue 342 did not alter the specific activity
compared with wild-type enzyme. Thus, CKI
contains an
autoinhibitory region between His
and ProP
(Fig. 5), the same region identified as inhibitory from
CS1 treatment (Fig. 7A).
Casein kinase I has, for many years, been considered to be a
constitutively active enzyme since it is spontaneously active after
isolation from native tissues or after expression of recombinant enzyme
in a prokaryotic system. Moreover, this spontaneous activity is not
generally found to be affected by second messengers or by association
with any known proteins. There is a report of the inhibition of a
37-kDa CKI species by phosphatidylinositol bisphosphate(41) ,
although this result has been questioned by others(42) . In the
present study, we provide evidence that the CKI isoform is a
regulatable enzyme whose COOH terminus acts as an autoinhibitory domain
in which phosphorylation at Ser and Thr residues causes inactivation of
the enzyme.
The CKI family consists of isoforms with a conserved
catalytic domain and variably sized NH- and COOH-terminal
extensions. The NH
-terminal extensions are less than 15
amino acids, except in the case of the recently identified
isoforms(9) , with lengths of
43 amino acids, and the
yeast Yck1p and Yck2p enzymes (12, 13) , with
73-amino acid extensions. The COOH-terminal domains of most CKI
isoforms are considerably larger. For example, two of the mammalian (9, 10) and six of the yeast(11, 12, 13, 14, 15, 16) CKI
isoforms have COOH-terminal tails of at least 100 amino acids. CKI
and -
, which are most likely the commonly studied 37-kDa forms of
CKI, have COOH-terminal domains of only 13-25 amino
acids(6) .
The first indication that the COOH-terminal
region of CKI might be regulatory came from earlier studies with
heparin. Effects of heparin on CKI isoforms are complicated and
substrate-specific (see (10) ). However, we observed that
full-length CKI
was activated by heparin when the D4 peptide was
used as a substrate, whereas mutant CKI
lacking the COOH terminus
was insensitive(10) . Furthermore, the CKI
isoform, with
its minimal COOH-terminal extension, was not activated by heparin and
in fact was partially inhibited (Fig. 3; (7) ).
Therefore, we asked if the COOH-terminal domain of CKI
conferred
the heparin activation and tested the hypothesis by creating a chimeric
kinase consisting of CKI
fused to the portion of the
isoform
removed by truncation. Heparin now activated the
-
chimeric
enzyme. Whatever heparin does mechanistically, the COOH-terminal domain
of CKI
is clearly critical, being both necessary and sufficient
for heparin activation.
That CKI, like most protein kinases, can
autophosphorylate has been known for many years but had never been
linked with any change in enzyme activity(43, 44) . We
previously reported that recombinant CKI purified from E. coli could autophosphorylate without change in electrophoretic
mobility(10) . It is now apparent that CKI
does contain
autophosphorylation sites that affect both electrophoretic mobility
and, more importantly, activity toward exogenous substrates. The key
observation was that treatment of purified recombinant CKI
with
type 1 protein phosphatase activated the enzyme and reduced its
apparent M
from 55,000 to its predicted value of
49,000. Thus, the sites that influence both activity and mobility are
already modified, the phosphorylation presumably occurring inside the E. coli cells. This phosphorylation could be due to
autophosphorylation or the action of E. coli protein kinases.
Arguing against the latter possibility is the observation that a
kinase-dead mutant of CKI
(K
N) had apparent M
49,000, indicative of the protein not being
phosphorylated. Interestingly, a nuclear form of CKI(N1) was reported
to have an apparent M
of 55,000 on SDS-PAGE (45) and not to autophosphorylate, which could be possible if
the enzyme were already fully modified.
Using a series of
COOH-terminal truncation mutants, we were able to localize the
phosphorylation sites responsible for anomalous electrophoretic
migration as being COOH-terminal to His, and several
different sites within this region contribute to this behavior. Similar
results were observed with a completely different isoform, S. pombe CKi1, in which anomalous migration on SDS-PAGE was attributed to
the COOH-terminal domain(46) . By analyzing the ability of
protein phosphatase to activate the COOH-terminally truncated forms of
CKI
, the inhibitory phosphorylation was localized to a region
between His
and Pro
, which contains six
potential phosphorylation sites. Although we did not determine which of
these six sites has the greatest inhibitory affect on the enzyme,
multiple autophosphorylation sites are involved. Exactly the same
region, His
to Pro
, was inferred to be
autoinhibitory from consideration of the specific activities of the
truncation mutants. Thus, activation of the enzyme by truncation may
result from removal of inhibitory autophosphorylation sites. Heparin
activates CKI
toward the D4 peptide by a mechanism that requires
the COOH-terminal region. Possibly, heparin interacts with the COOH
terminus, which carries a high positive charge, to cause an overall
conformational change that mimics dephosphorylation. Heparin is
unlikely to be a physiological regulator of the enzyme, but we cannot
exclude the possibility that some other compound interacts with the
regulatory COOH terminus. Likewise, it is not known in vivo whether the inhibitory phosphorylations discussed above would
result from autophosphorylation or the action of a separate protein
kinase. There is a parallel in the extracellular
signal-regulated/mitogen-activated protein kinase enzymes, which can
activate by autophosphorylation (47) even though enzymes of the
MAPK or ERK family are thought to be responsible
physiologically(48, 49) .
Other CKI enzymes may
also be regulated via their COOH-terminal domains, even though these
have no sequence similarity whatsoever. For example, CKI3 can be
activated 3-4-fold by type-1 protein phosphatase concomitant with
a shift in its apparent molecular weight from 60 to 55 kDa on SDS-PAGE. (
)Similarly, the S. pombe CKi1 isoform undergoes an
inhibitory autophosphorylation that was localized to its COOH
terminus(46) . Truncation of the enzyme and removal of the
COOH-terminal domain resulted in a 3-fold activation in the catalytic
rate of the enzyme. Autophosphorylation, the majority of which was
localized to the COOH-terminal domain, resulted in a 4-fold decrease in
the affinity for protein substrate(46) . Inhibitory
COOH-terminal phosphorylation could therefore be a common regulatory
mechanism for CKI isoforms.
Given the unique substrate recognition
characteristics of CKI, as discussed in the Introduction, it is of
interest to survey the sequences surrounding the potential
autophosphorylation sites between His and Pro
(Fig. 5). None is preceded by a cluster of acidic
residues, precluding this modality for recognition. The other motif
recognized by CKI requires prior phosphorylation and so cannot account
for the initial autophosphorylation. In any case, only one such site is
available in this region: phosphorylation at Ser
would
create a potential site at Ser
. We conclude, therefore,
that different substrate recognition constraints must be operative for
the autophosphorylation reaction, which is likely to be an
intramolecular process, than when the enzyme acts on exogenous
substrates. Interestingly, introduction of phosphate at five of the
sites between His
and Pro
would generate a
sequence (Ser(P)/Thr(P))-Xaa-Xaa-Yaa, (where Yaa is not Ser or Thr)
that could then act as a pseudosubstrate. Better understanding of the
interactions between the catalytic and regulatory domains of CKI will
have to await the solution of the three-dimensional structure of
full-length CKI.