From the
Neuronal Cdc2-like kinase is a heterodimer of Cdk5 and a 25-kDa
subunit which is derived from a brain-specific 35-kDa novel protein,
p35 (Lew, J., Huang, Q.-Q., Qi, Z., Winkfein, R. J., Aebersold, R.,
Hunt, T., and Wang, J. H. (1994) Nature 371, 423-426).
Three truncated forms of p35 including the one corresponding to the
25-kDa subunit of the kinase have been expressed in Escherichia
coli and shown to activate a bacteria-expressed Cdk5 with equal
efficacy. The shortest truncated form of p35, p21, spanning amino acid
residues 88 to 291, has been used to reconstitute active Cdk5 kinase
and to characterize the activation reaction. The purified kinase
displays similar specific enzyme activity and similar phosphorylation
site specificity as the neuronal Cdc2-like kinase purified from bovine
brain. Bovine brain extract contains Cdk5 uncomplexed with p35 or p25
which has also been found to be activated by p21 or p25. The results
substantiate the previous suggestion that p35 is a specific Cdk5
activator. Several observations suggest that, unlike other well
characterized Cdc2-like kinases whose activities depend on the
phosphorylation of the catalytic subunits at a specific site by a
distinct kinase, the reconstituted Cdk5/p21 does not depend on the
phosphorylation of Cdk5 for activity. The reconstitution of the highly
active Cdk5 kinase was achieved without requiring any other kinase in
the reconstitution reaction. The possibility of autophosphorylation of
Cdk5 on the putative activation site has been ruled out as no
phosphorylation occurred on Cdk5 during the enzyme reaction. The rate
and extent of the kinase reconstitution were not significantly affected
by Mg
The regulation of cell division cycle in eukaryotes is governed
by a universal mechanism in which a family of closely related kinases
play rate-limiting functions at discrete phases of the cell cycle (for
reviews, see Refs. 1-7). In yeast cells, p34
In
addition to depending on cyclin for activity, Cdc2 and Cdk2 are
regulated by complex phosphorylation mechanisms involving a network of
protein kinases and phosphatases
(21, 25, 26, 27, 28, 29) .
The phosphorylation of a specific threonine residue,
Thr
Not all
Cdc2-like kinases are cell cycle regulators. Many Cdc2-related proteins
are present in tissues where proliferative activity is very low
(19, 43, 44) . Neurons of mammalian central
nervous system contain a Cdc2-like kinase, called neuronal Cdc2-like
kinase, which has been implicated in the regulation of
neurocytoskeleton dynamics
(45, 46, 47) .
Purified active neuronal Cdc2-like kinase is a heterodimer of Cdk5 and
a 25-kDa regulatory protein
(48, 49, 50) .
Molecular cloning studies have revealed that the 25-kDa protein is
derived from a novel 35-kDa protein, p35
(51) . The mRNA
transcript of p35 has been shown to be specifically expressed in the
central nervous system. The primary structure of p35 shows only limited
homology to members of the cyclin family; yet, the protein appears to
perform a cyclin-like role in neuronal Cdc2-like kinase. A
bacteria-expressed, truncated form of p35 has been found to activate a
bacteria-expressed Cdk5.
In the present study, we demonstrate the
activation of Cdk5 by a number of bacterially expressed, truncated
forms of p35, including one corresponding to the 25-kDa subunit of
neuronal Cdc2-like kinase purified from bovine brain, and carry out
detailed characterization of the kinase activation reaction. The study
has revealed a major difference between the mechanism of Cdk5
activation and that of Cdc2 or Cdk2 activation. Although the amino acid
sequence of Cdk5 contains a phosphorylatable residue, serine, at the
position corresponding to the positive regulatory threonine of Cdc2 or
Cdk2, the activation of Cdk5 does not appear to be dependent on the
presence of a Cdk activating kinase. The active form of the kinase does
not catalyze autophosphorylation of Cdk5, thus ruling out the
possibility that the specific serine residue is phosphorylated by an
autocatalytic reaction.
For further
purification of GST-p21 to remove most of the proteolyzed products, the
concentrated GST-p21 was clarified by centrifugation and loaded on an
FPLC Superose 12 gel filtration column (Prep grade, Pharmacia Biotech
Inc.) equilibrated in PBS with 1 mM EDTA and 1 mM DTT
and eluted at a flow rate of 0.5 ml/min. Fractions containing the
45-kDa polypeptide of GST-p21, as determined by SDS-PAGE, were pooled
and then concentrated by dialysis against Aquacide II to a small
volume.
The expressed proteins were
purified from the soluble fractions of bacteria lysates by glutathione
affinity column chromatography. As shown previously
(51) , the
expressed Cdk5 fusion protein did not exhibit any kinase activity by
itself, nor did any of the p35-related fusion proteins. Protein kinase
activity, however, could be readily detected upon mixing Cdk5 with the
fusion proteins of the truncated forms of p35, and the fusion proteins
of p21, p23, and p25 showed similar activity in Cdk5 activation
(Fig. 2 A). To achieve maximal activation, the mass ratio
of activating proteins to Cdk5 of 8:1 was used in these experiments,
and the protein mixtures were preincubated for 30 min before kinase
assay. Dose-dependent activation of Cdk5 by p21 indicated that close to
maximal activation could be approached at a mass ratio of Cdk5/p21 of
1:1.5 (Fig. 2 B). The optimal activation of Cdk5 by the
truncated forms of p35 also requires preincubation of the proteins. For
example, the time course of the kinase reaction of a mixture of Cdk5
and p21 displays low kinase activity and pronounced upward curvature
indicative of a slow activation of the kinase during the reaction. If
the two proteins have been incubated for 1 h before the kinase reaction
is initiated, a linear initial time course with greatly increased
kinase activity will result (Fig. 2 C). Since our
attempts in bacteria expression of the full-length p35 had not been
successful, a similar reconstitution experiment with the full-length
protein was not possible. p25 corresponds to the 25-kDa subunit of the
purified bovine brain neuronal Cdc2-like kinase which exhibits specific
kinase activities similar to or higher than those of the fully
activated Cdc2 and Cdk2 kinases. Thus, the results of
Fig. 2
suggest that the three truncated forms of p35 contain the
complete Cdk5-activating domain. The activation of Cdk5 by p35
derivatives appeared to be specific, the bacteria-expressed
His
We have previously used a set of peptide
analogues to elucidate the substrate structural determinants for bovine
brain neuronal Cdc2-like kinase
(53) . These peptides were
prepared by systematic substitution of a parent peptide derived from
the Cdc2 kinase phosphorylation site of histone H1. Kinetic
characterization of neuronal Cdc2-like kinase-catalyzed peptide
phosphorylation reactions was carried out, and the results were used to
assess the contribution of individual residues of the parent peptide to
substrate activity. To further characterize the enzymological property
of the reconstituted kinase, the phosphorylation reactions of these
peptides by the reconstituted kinase were examined. The kinetic
parameters were determined for these reactions and compared to those of
the bovine brain enzyme-catalyzed reactions. shows that
the reconstituted kinase displayed very similar, if not identical,
K
While the reconstitution of highly active kinase from
bacteria-expressed protein components may rule out the involvement of
an activating kinase in the enzyme reconstitution, the possibility that
Ser
When neuronal Cdc2-like kinase was first purified and shown
to be a heterodimer of a Cdc2-related 33-kDa and a 25-kDa subunit
(48, 49, 50) , it was speculated that the 25-kDa
protein played the role of an essential activating subunit. This
suggestion is supported by the recent observation that histone H1
kinase activity could be obtained from mixing a bacteria-expressed
truncated form of the 25-kDa protein (p21) with a bacteria-expressed
Cdk5
(51) . The reconstituted kinase, however, displayed very
low specific kinase activity, about 2 orders lower than that of the
neuronal Cdc2-like kinase purified from bovine brain. In this study, we
have extended the previous observation and carried out detailed
characterization of the kinase reconstitution reaction. The results
have shown that the main reason for the lower-than-expected kinase
activity may be attributed to the fact that most of the
bacteria-expressed proteins are present in the degraded and/or
incorrectly folded forms. While this finding is hardly surprising, we
have succeeded, by removing the inactive protein derivatives, in the
purification of a highly active reconstituted kinase which has similar
specific activity, as well as similar phosphorylation site specificity
as the enzyme purified from brain. This has provided a convenient
source for neuronal Cdc2-like kinase. The amount of reconstituted
kinase obtained from a typical preparation with 1 liter each of the
bacteria culture of the protein components is equivalent to 2
preparations of the brain enzyme, each with 500 g of tissue.
Purification of the enzyme from bovine brain routinely requires about 8
days to complete.
Three truncated forms of p35, p21, p23, and p25,
have been expressed as GST fusion proteins in E. coli and
tested as Cdk5 activator. p25 has the amino acid sequence corresponding
to the 25-kDa subunit of the purified bovine brain neuronal Cdc2-like
kinase. The facts that all these p35 derivatives showed similar Cdk5
activation activity and that the purified neuronal Cdc2-like kinase
containing the 25-kDa subunit is highly active suggest strongly that
all the derivatives contain complete Cdk5-activating domain. Although
the purified reconstituted Cdk5 kinase used for most of the detailed
characterizations was the Cdk5-p21 heterodimer, other reconstituted
kinases, Cdk5-p23 and Cdk5-p25, are expected to have similar general
characteristics. Similarly, although the recombinant and bacterially
expressed Cdk5 was used for the reconstitution of the highly active
Cdk5 kinase, Cdk5 monomer isolated from bovine brain could also be
activated by the truncated forms of p35.
The best characterized
members of the Cdc2-like kinase family, Cdc2/cyclin B and Cdk2/cyclin A
have been shown by many investigators to depend on the phosphorylation
of Thr
The
recently elucidated crystal structure of Cdk2 shows that Thr
It is tempting to suggest that the unique,
phosphorylation-independent kinase activation is due to unique
structural properties of the regulatory component of the protein
complex, as p35 is only remotely related to cyclins. This suggestion,
unfortunately, is difficult to test, since Cdk5 has not been shown to
be activated by any cyclins. The fact that p35 expression is restricted
to neurons of central nervous systems, whereas Cdk5 is widely
distributed among human tissues
(51) , suggests that there might
be other protein activators of Cdk5. It is significant to determine
whether these protein activators activate Cdk5 in a
phosphorylation-dependent or independent manner. Similarly, if further
studies uncover other Cdc2-like kinases which are activated by p35, it
will be important to determine whether those kinase activations are
phosphorylation-dependent or independent. In any case, detailed
structural information about the Cdk5-p21 protein complex may shed
important new light on the molecular basis of the
phosphorylation-dependent activation of Cdc2-like kinases. In addition,
Cdk5-associated activity, measured as histone H1 kinase, has been found
only in brain extract, thus raising the possibility that the kinase
specificity of Cdk5 is dictated by its regulatory partner.
The kinetic parameters
were determined as detailed under ``Materials and Methods''
with the purified enzymes. Those parameters for the kinase purified
from bovine brain were determined by Beaudette et al. (53).
We thank Drs. Li-Huei Tsai and Ed Harlow
(Massachusetts General Hospital) for providing the GST-Cdk5 plasmid,
Dr. Tim Hunt (ICRF Clare Hall Laboratories, United Kingdom) for the
GST-cyclin D1 and hexahistidine-tagged cyclin A plasmids, and Dr. Anna
A. DePaoli-Roach (Indiana University School of Medicine) for
phosphatase 2A.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
ATP.
associates with phase-specific cyclins, G
or M phase
cyclins, to regulate cell cycle progression at specific phases
(4, 8, 9, 10) . In animal cells, while
the regulation of the entry and exit of mitotic phase by
p34
is conserved, a distinct Cdc2 homolog, Cdk2 is
primarily involved in the control of G
phase
(11, 12, 13, 14, 15, 16) .
In addition, recent studies have revealed the existence of extended
families of both Cdc2 homologous proteins and cyclins
(17, 18, 19, 20) . Several Cdc2 family
members with molecular masses in a range of 33 kDa to 35 kDa have been
suggested to depend on cyclin for activity and designated as
cyclin-dependent kinases, Cdks; individual members of this group are
distinguished by numbers, such as Cdk1 (Cdc2), Cdk2 etc.
(21, 22, 23, 24) . Members of the cyclin
family, on the other hand, are categorized into subfamilies by letters
as, cyclin A, cyclin B etc. A large number of active protein kinases
may be derived from heterologous interaction between members of the two
protein families. Cell cycle progression of animal cells appears to be
coordinated by the concerted action of many of these kinases.
/Thr
, by a distinct kinase, Cdk
activating kinase, is essential for, or markedly enhances, the activity
of the Cdc2/Cdk2 kinase
(30, 31) . Cdk activating kinase
has recently been identified to contain a Cdc2 homologous protein,
MO15, as catalytic subunit and a cyclin partner
(32, 33, 34, 35, 36) . On the
other hand, phosphorylation of a threonine, Thr
or a
tyrosine, Tyr
, in the amino-terminal region results in
inactivation of the Cdc2/Cdk2 kinase
(25, 26, 29) . A protein kinase catalyzing the
inhibitory tyrosine phosphorylation has been identified and shown to be
related to the yeast wee1 gene product
(37, 38, 39, 40) , whereas the enzyme
responsible for the phosphorylation of Cdc2/Cdk2 kinase on Thr
has not been identified. Since the regulatory phosphorylation
sites are mostly conserved, or conservatively substituted among Cdc2
family members, it seems possible that some other Cdc2-like kinases are
also regulated by similar phosphorylation mechanisms. For example, a
recent study has shown that the activation of Cdk4 by cyclin D is also
dependent on an activating kinase
(41, 42) .
Fusion Plasmid Construction and Escherichia coli
Expression
Four sets of polymerase chain reaction primers were
designed according to the full sequence of neuronal regulatory subunit
described previously
(51) for plasmid constructions to express
the full-length or truncated forms of the neuronal regulatory subunit
(Fig. 1). Primer 1, 5`-CCCGGGGATCCATGGGCACGGTGCTG-3`, and primer
3, 5`-CCCGGGAATTCTCACCGGTCCAGCCC-3`, are for the full-length protein of
307 amino acids, p35. Primer 2, 5`-CCGGGGATCCGCCCAGCCCCCGCCGG-3`, and
primer 3 are for the 25-kDa protein (p25) found in the purified
neuronal Cdc2-like kinase. Primer 4, 5`-CCCGGGGATCCAAGTCGCTGTCGTGC-3`,
and primer 6, 5`-CCCGGGAATTCTAGTTCTTCAGGTCGGAGA-3`, are for the 23-kDa
truncated form (p23) of p35, which encodes amino acids 88-291.
Primer 5, 5`-CCCGGATCCTGTCCCCCTGCCAGC-3`, and primer 6 are for the
21-kDa fragment (p21) of p35, which encodes amino acids 108-291.
These expressed fragments as well as the 35-kDa full-length sequence
are schematically shown in Fig. 1. The polymerase chain reaction
products were cloned in pGEX-2T vector at BamHI and
EcoRI sites.
Figure 1:
Schematic
representation of p35 and the various truncated forms of
p35.
The recombinant pGEX plasmid constructs,
encoding glutathione S-transferase (GST,(
)
26 kDa) fusion proteins, GST-p21, GST-p23, GST-p25,
GST-Cdk5, and GST-cyclin D1, were individually expressed in E. coli strain BL21 (DE3). The host cells were cultured in 1 liter of LB
medium containing 100 µg/ml ampicillin to A
of 0.5-0.8 at 37 °C. The synthesis of GST recombinant
protein was induced with 0.4 mM
isopropyl-1-thio-
-D-galactopyranoside (Life Technologies,
Inc.) for a subsequent incubation of 2-3 h. The cells were
harvested by centrifugation at 2000
g. The cell
pellets were washed with PBS and pelleted again for subsequent
purification of the recombinant proteins.
Purification of the Recombinant
Proteins
Purification of the GST fusion proteins was carried out
essentially as described by Smith and Johnson
(52) . The cell
pellets were suspended in 20 ml of 50 mM Tris-HCl (pH 7.4)
buffer containing 2 mM EDTA, 1% Tween 20, 1 mM DTT,
0.25 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each of
leupeptin, aprotinin, and pepstatin. All procedures for the
purification were carried out at 0-5 °C. The cells were lysed
using a French press at 1000 p.s.i. and then centrifuged at 18,000
g for 15 min. The supernatant was applied onto a 2-ml
column of glutathione-agarose (Sigma) which had been equilibrated with
20 mM Tris-HCl (pH 7.4) supplemented with 0.5 M NaCl,
0.5% Nonidet P-40, 1 mM EDTA and DTT, and the mixture of
protease inhibitors described above. The column was sealed and allowed
to sit on an end-over-end shaker for 1 h. After loading, the column was
washed with 20 bed volumes of the same buffer, followed by 5 bed
volumes of PBS supplemented with 1 mM DTT. The GST fusion
protein was eluted by 5 mM reduced glutathione (Sigma) in 50
mM Tris-HCl, pH 8.0, and 1 mM DTT. To remove
glutathione, the pooled samples were dialyzed against PBS containing 1
mM EDTA and DTT and then concentrated to a small volume by
dialysis against Aquacide II (Calbiochem). His
-tagged
cyclin A was expressed and purified as described by Poon et
al.
(33) except that cells were lysed by passing through a
French Press in the lysis buffer without lysozyme.
Reconstitution and Purification of Neuronal Cdc2-like
Kinase
2 mg of GST-Cdk5 affinity-purified by a
glutathione-agarose column was mixed with 4 mg of GST-p21 purified from
a glutathione-affinity column and gel filtration in a volume of 5 ml in
PBS buffer containing 1 mM EDTA and 1 mM DTT. The
mixture was allowed to sit at 30 °C for 1 h for reconstitution and
subsequently treated with human thrombin (Sigma) in a concentration of
10 units of thrombin per mg of recombinant protein at room temperature
for 1 h to cut out the GST fusion part. The GST-free complex of Cdk5
and p21 was dialyzed against 50 mM Tris-HCl, pH 7.4, 1
mM EDTA, 1 mM DTT, 0.25 mM
phenylmethylsulfonyl fluoride, and 100 µg/ml benzamidine at 4
°C for 2 h. The dialyzed sample was clarified by centrifugation and
then loaded on an FPLC Mono S column (HR5/5, Pharmacia) equilibrated
with the same buffer at a flow rate of 0.5 ml/min. The column was
washed with 10 bed volumes of the same buffer, and the bound kinase
activity was eluted by a linear gradient of NaCl (0-0.3
M/10 ml). Protein kinase activity was monitored by
phosphorylation assay of the histone peptide.
Isolation of the Native Cdk5 Monomer
The bovine
brain extract in Buffer A (25 mM Hepes (pH 7.2), 1 mM
EDTA, 1 mM DTT, 0.6 mM phenylmethylsulfonyl fluoride,
0.3 mg/ml benzamidine, 1 µg/ml leupeptin, 2 µg/ml antipain)
supplemented with 0.1 mg/ml soybean trypsin inhibitor was clarified by
centrifugation at 100,000 g. The supernatant
containing 40 mg of protein was injected at the flow rate of 0.5 ml/min
into an FPLC Mono S column (HR 5/5, Pharmacia) equilibrated with Buffer
A. The column was washed with 20 ml of Buffer A and eluted with a salt
gradient (0-0.5 M NaCl) in 30 ml of Buffer A. The
fractions containing Cdk5 monomer were identified by immunoblots with
the Cdk5 and p25 antibodies. The pooled sample was concentrated by
Amicon Centricon-10 concentrator and then applied to an FPLC Superose
12 column (HR 10/30, Pharmacia) equilibrated in Buffer A supplemented
with 0.15 M NaCl at 0.5 ml/min. This column was calibrated in
the same condition using a Bio-Rad calibration kit (bovine
thyroglobulin, 670 kDa; bovine
-globulin, 158 kDa; chicken
ovalbumin, 44 kDa; horse myoglobin, 17 kDa; vitamin B12, 1.35 kDa).
Immunoblotting and reconstitution assay with GST-p21 or GST-p25 were
performed with the column fractions.
Protein Kinase Activity Measurement
The protein
kinase activity and substrate specificity were determined according to
the procedure described previously
(48, 53) . The
histone H1-derived peptide,
HS
(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) ,
KTPKKAKKPKTPKKAKKL, was used as substrate for the protein kinase
activity assay
(48) . This sequence contains a repeat of the
proline-directed phosphorylation site. A set of peptide analogues,
derived from the parent peptide
HS
(9, 10, 11, 12, 13, 14, 15, 16, 17, 18) ,
PKTPKKAKKL, was used for determination of the substrate specificity
(53) . The kinase reaction was performed at 30 °C, and the
reaction mixture contained 20 mM MOPS, pH 7.4, 5 mM
MgCl, 100 µM
[
-
P]ATP (
300 dpm/pmol), peptide
substrate as indicated, and the protein kinase sample. When assay of
purified kinase was performed, 0.5 mg/ml bovine serum albumin was
included in the reaction mixture for enzyme stability. Phosphate
incorporation into substrate peptide was quantitated by liquid
scintillation using a scintillation counter (Beckman LKB 1215).
Protein Concentration Determination
Protein
concentration was measured either by absorbance at A or by the method of Bradford
(54) . The Bradford assay was
performed in an alternate way. The samples were mixed with Bradford
reagent in a microtiter plate and read at A
using a Dynatec MR 600 microtiter plate reader. The amount of
pure enzyme in the purified sample was estimated by scanning
densitometry of Coomassie Blue-stained gels using a Pharmacia LKB 2202
Ultrascan laser densitometer. The stained protein bands were compared
with carbonic anhydrase as the protein standard.
SDS-PAGE and Immunoblot
SDS-PAGE was performed by
the method of Laemmli
(55) in 10% vertical slab gels, and gels
were stained with silver
(56) or 0.2% Coomassie Brilliant Blue.
For immunoblot, proteins were transferred to polyvinylidene difluoride
membranes (Millipore) and immunostained with the indicated antibodies.
Antibodies against Cdk5 and the p35 derivatives were rabbit polyclonal
antibody preparations raised against the bacteria-expressed Cdk5 and
21-kDa fragment of p35, respectively.
Protein Kinase Activity of Cdk5 and p35-related
Proteins
The expression of GST-fused Cdk5 and p35, as well as
p25, p23, and p21 truncated forms of p35 (Fig. 1) were attempted
in E. coli strain BL21 (DE3). The bacteria lysates were
centrifuged to separate into a soluble and a particulate fraction, and
expression of the recombinant proteins was analyzed by SDS-PAGE and
immunoblot. The expression of the full-length p35 fusion protein was
not successful; essentially no fusion protein was detectable in the
bacteria lysate (data not shown). Most of the expressed truncated forms
of p35 were found in the insoluble fraction. In all cases, the soluble
recombinant proteins appeared to have undergone considerable
proteolytic degradation. Lowering the induction temperature from 37
°C to room temperature or decreasing the protein induction time to
1 h did not significantly affect the extent of the protein degradation.
Attempts to express the full-length p35 using a different vector, PQE30
(QIAGEN), was also unsuccessful.
-tagged cyclin A and GST-cyclin D1 could not substitute
for the p35 derivatives in Cdk5 activation under the same condition
(Fig. 2 A).
Figure 2:
Activation of bacteria-expressed Cdk5.
A, 0.5 µg of GST-Cdk5 and 5 µg each of GST-p21,
GST-p23, GST-p25, His-cyclin A, and GST-cyclin D1 were
preincubated alone or together at 30 °C for 30 min in a volume of
15 µl of PBS buffer containing 1 mM EDTA and 1 mM
DTT. 10
concentrated protein kinase assay mixture was added to
each sample to make the ATP and substrate peptide HS(1-18)
concentration 100 µM and the Mg
concentration 5 mM in the kinase reaction. B,
dose-dependent activation of Cdk5 by p21. 2 µg of GST-Cdk5 was
reconstituted with various amounts of GST-p21 at 30 °C for 1 h,
other conditions were the same as in A. The kinase reaction
was performed at 30 °C for 20 min as described under
``Materials and Methods.'' C, effect of
preincubation on kinase activity. GST-Cdk5 (10 µg) and GST-p21 (20
µg) in PBS buffer containing 1 mM EDTA and 1 mM
DTT were mixed in a final volume of 150 µl. Kinase reaction was
initiated by the addition of [
-
P]ATP,
Mg
, and peptide substrate immediately (
-
- -
) or after incubation of the mixture at 30 °C
for 1 h (
--
). At the indicated time after
initiation of the kinase reaction, aliquots (15 µl) were withdrawn
for the analysis of phosphate incorporation.
Bovine brain extract contains a high
concentration of Cdk5 that does not complex with p25 or p35. The
p25/p35-free Cdk5 in the 100,000 g fraction can be
separated from the complexed forms by FPLC on a Mono S column (see
``Materials and Methods''). When the Mono S column-purified
Cdk5 fraction was analyzed on an FPLC Superose 12 column, all Cdk5
appeared in a single peak (as revealed by Western immunoblot analysis)
at a position indicative of Cdk5 monomer (Fig. 3 A).
While the fractions containing Cdk5 displayed no histone H1 peptide
kinase activity, high kinase activity could be demonstrated after
incubation of the fractions with the bacterially expressed p21 for 40
min. The activation of brain Cdk5 shows characteristics similar to that
of the recombinant Cdk5 expressed in bacteria. p21 and p25 have been
found to activate the brain Cdk5 with essentially identical efficiency
(Fig. 3 B).
Figure 3:
Analysis of the p25/p35-free Cdk5 from
bovine brain extract. A, Superose 12 gel filtration
chromatography. The crude brain extract was applied on Mono S column
and the fractions containing Cdk5 which was uncomplexed with p25 or p35
were pooled and concentrated. 0.5 ml of the sample was injected into
the FPLC Superose 12 column (bed volume = 25 ml) equilibrated in
Buffer A with 0.15 M NaCl. The column was precalibrated in the
same condition as described under ``Materials and Methods.''
--
, A
. Fractions were
collected in 0.5 ml. The column fractions were incubated with
(
--
) or without (
--
)
GST-p21 at 30 °C for 40 min before the histone peptide kinase
activity was assayed. The inset is the immunoblots with
antibodies against Cdk5 ( top panel) and p25/p35 ( bottom
panel), respectively. B, activation of the monomeric Cdk5
isolated from brain extract by GST-p21 and GST-p25. Aliquots (5.4
µg) of the sample pooled from the Superose 12 collections
containing the monomeric Cdk5, was incubated alone or reconstituted
with 1 µg of GST-p21 or GST-p25 at 30 °C for 40 min. Histone
peptide kinase activity was assayed at 30 °C for 20 min as
described under ``Materials and
Methods.''
Specific kinase activity of the
close-to-homogeneous neuronal Cdc2-like kinase from bovine brain is
about 4 µmol of PO/min/mg of protein using histone H1
peptide
HS
(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) as substrate
(48) . This value is similar to the
specific kinase activity of purified Cdk2/cyclin A protein kinase
(57) . The maximal kinase activity obtained by the mixture of
recombinant Cdk5 and p21, however, was only several tens of nanomoles
per min per mg of protein. A number of factors may contribute to the
low specific activity displayed by the reconstituted enzyme. More than
50% of the expressed GST-Cdk5 and GST-p21 proteins purified by
glutathione beads were degraded forms which, upon being separated from
the intact protein by gel filtration, were found to have little
p21-activated histone kinase activity and Cdk5-activating activity,
respectively. Moreover, gel filtration analysis showed that only the
trailing edge of the protein peak enriched with the intact Cdk5 or p21
fusion proteins displayed activities (data not shown), suggesting that
most of the proteins in the affinity-purified GST-Cdk5 and GST-p21 were
present in incorrectly folded states.
Purification and Characterization of the Active Form of
Reconstituted Cdk5/21-kDa Kinase
Since the low specific activity
of the reconstituted enzyme appeared to be due, at least in part, to
the existence of a large amount of degraded and incorrectly folded
recombinant proteins, attempts were made to purify the active form of
the enzyme from the reconstitution reaction. It was found that a single
step FPLC Mono S column chromatography of an optimally reconstituted
and thrombin-treated sample could achieve a 65-fold purification of the
active kinase (for detailed procedures, see ``Materials and
Methods''). Most of the protein in the reconstitution mixture did
not bind to the Mono S column, whereas about 85% of the total kinase
activity bound. The bound activity could be quantitatively eluted as a
sharp peak, along with a small amount of protein from the column by a
salt gradient (Fig. 4 A).
Figure 4:
FPLC Mono S chromatography of the
reconstituted kinase. The reconstitution and purification of the
Cdk5-p21 complex were carried out as described in detail under
``Materials and Methods.'' After loading and column washing,
the collection was started by fractions of 0.5 ml. A:
- - -
, histone peptide phosphorylation
activity;
--
, A
. Protein
was determined by the Bradford method performed in a microtiter plate.
B, SDS-PAGE of the indicated fractions from A. os is the original sample loaded on the column. ft is the
flow-through of the loaded sample, and w is the collected
column wash after sample loading. The gel was stained with
silver.
Analysis by SDS-PAGE
revealed that the 33-kDa and 21-kDa proteins were abundant in the
histone kinase active fractions (Fig. 4 B), which were
identified as Cdk5 and p21, respectively, by immunoblots (data not
shown). Densitometric scanning of the Coomassie Blue-stained gels
indicated that Cdk5 and p21 were about 50% of the total protein, and
the molar ratio of the 21-kDa protein to the 33-kDa protein was about
1.3. Purification data for a typical preparation are summarized in
. Protein concentration of the activity peak fraction
(Fraction 14) was determined by both scanning densitometry of the
Coomassie Blue-stained gels and the Bradford assays performed in a
microtiter plate. The purified, reconstituted Cdk5/p21 kinase had a
specific kinase activity of 3.8 µmol of PO/min/mg of
protein, 65-fold higher than that of the crude reconstituted enzyme.
Since about 50% of the proteins of the purified sample were represented
by Cdk5 and p21, a homogeneous active enzyme is expected to have a
specific kinase activity of about 7 µmol of PO
/min/mg
of protein. Thus, the active form of the kinase reconstituted from
bacteria-expressed Cdk5 and a truncated form of p35 has a specific
activity as high as that of neuronal Cdc2-like kinase purified from
bovine brain, about a few micromoles of PO
transferred per
min per mg of enzyme.
values for these peptides as the enzyme
purified from brain.
The Activation of Cdk5 by Reconstitution with p21 Is
Independent of Cdk5 Phosphorylation
The activation of Cdc2 by
cyclin B shows an absolute dependence on the phosphorylation of
Thr of Cdc2 by Cdk activating kinase
(30, 31, 57) . Although Cdk2 exhibits some
activity upon activation by cyclin A, the activity can be greatly
increased by the Cdk activating kinase-catalyzed phosphorylation of the
protein at Thr
(57) . Sequence alignment of Cdk5
with Cdc2 and Cdk2 has shown that the residue corresponding to
Thr
/Thr
of Cdc2/Cdk2 is substituted in Cdk5
by a serine, Ser
. Since sequence surrounding Ser
is highly conserved in Cdk5 in comparison to those of Cdc2 and
Cdk2, it has been suggested that Cdk5 may also be regulated by Cdk
activating kinase
(49) . The isolation of the highly active,
reconstituted Cdk5-p21 complex, however, appears to argue against such
a suggestion.
phosphorylation is required in the enzyme activation
cannot be ruled out completely. Thus, the enzyme may undergo
autophosphorylation at this serine residue during reconstitution to
result in increased kinase activity. Since the time dependence of Cdk5
activation by p21 can be readily monitored (see
Fig. 2C), one approach to test the autoactivation
mechanism is to examine the effect of Mg
ATP in the
preincubation on the kinase activation reaction. Fig. 5 A shows that the time course of the activation of recombinant Cdk5
by p21 was not significantly affected by Mg
ATP. The
activation of brain Cdk5 monomer by the recombinant p21 appears to be
depressed somewhat by the addition of Mg
ATP in the
activation incubation (Fig. 5 B). The reason for this is
not clear. In any case, the results of Fig. 5argue against the
notion that serine 159 of Cdk5 is phosphorylated by an
autophosphorylation reaction during the reconstitution reaction.
Figure 5:
MgATP effect on Cdk5/p21
reconstitution and kinase activation. A, time dependence of
Cdk5/p21 kinase reconstitution. GST-Cdk5 (20 µg) and GST-p21 (40
µg) were mixed in a volume of 300 µl in PBS buffer containing 1
mM EDTA, 1 mM DTT, and 0.5 mg/ml bovine serum
albumin. The preincubation was performed at 30 °C with
(
--
) or without (
--
)
Mg
ATP at a concentration of 100 µM.
Aliquots (30 µl) were removed at indicated intervals for the
measurement of protein kinase activity. Protein kinase reaction time
was 5 min. B, dose-dependent activation of the isolated native
Cdk5 by GST-p21. 7.5 µg of the Superose 12 column pooled sample
containing monomeric Cdk5 from brain extract (see ``Materials and
Methods'') was reconstituted with various amounts of GST-p21 at 30
°C for 1 h with (
--
) or without
(
--
) 100 µM Mg
ATP.
There was GST protein instead of GST-p21 in the control tubes
(
--
). Histone H1 peptide phosphorylation assays
were performed at 30 °C for 15 min as described under
``Materials and Methods.''
To
probe further whether or not the reconstituted kinase could undergo
autophosphorylation on Cdk5, a sample of the purified, reconstituted
kinase was incubated with [-
P]ATP and
Mg
at 30 °C, aliquots of the sample were
withdrawn at various intervals and analyzed by SDS-PAGE and
autoradiography. Fig. 6 A shows that while p21 was
rapidly phosphorylated, no radioactivity was seen to associate with
Cdk5 even after prolonged incubation. Similar results were also
obtained with a partially purified kinase reconstituted from GST-p21
and bovine brain Cdk5 (Fig. 6 B). The possibility that
autophosphorylation on Cdk5 could occur during the kinase reaction has
also been examined and ruled out. Fig. 6, A and
B, shows that kinase samples taken from kinase reactions were
autophosphorylated on the 21-kDa subunit but not on Cdk5. The ability
of the reconstituted kinase to autocatalyze the phosphorylation of p21
is compatible with results of a previous study showing that the
regulatory subunit of bovine brain neuronal Cdc2-like kinase is
phosphorylated under autophosphorylation conditions
(48) . The
phosphorylation of p21 does not appear to affect the activity of the
enzyme. As shown in Fig. 6 C, the phosphorylation of p21
in the kinase reaction was significantly suppressed in the presence of
the substrate peptide. While the extent of p21 phosphorylation
decreased with the increase in the peptide substrate concentration,
there was no indication of substrate-dependent activation or
inactivation for this enzyme.
Figure 6:
Autophosphorylation of the reconstituted
Cdk5/p21 kinase. Time course of the autophosphorylation reaction is
shown in A and B. A, aliquots (1 µg) of
the reconstituted enzyme purified by Mono S column from the bacterially
expressed proteins were incubated at 30 °C with 100 µM
[-
P]ATP (
5000 dpm/pmol) in 20
mM MOPS (pH 7.4), 5 mM MgCl
for different
intervals as indicated. B, 0.4 mg of GST-p21 and 10 mg of the
brain Cdk5 sample partially purified by Mono S and Superose 12 columns
were mixed and incubated at 30 °C for 1 h. The mixture was then
mixed with the GSH-agarose beads at 4 °C for 30 min. After the
beads were washed intensely, Cdk5/GST-p21 was released by 5 mM
glutathione in 50 mM Tris (pH 8.0) and 1 mM DTT.
Aliquots containing 2.6 µg of the affinity-purified Cdk5/GST-p21
were incubated at 30 °C with 20 µM
[
-
P]ATP (
5000 dpm/pmol) in 20
mM MOPS (pH 7.4) and 5 mM MgCl
for
different intervals as indicated. The immunoblots were done with the
anti-Cdk5 ( I) and anti-p21 ( II) antibodies
individually. C, substrate effect on autophosphorylation.
Aliquots (0.4 µg) of the reconstituted enzyme which was purified
with the bacterially expressed Cdk5 and p21 were incubated at 30 °C
for 5 min in the same condition as above with substrate peptide
HS(1-18) of 0 µM, 50 µM, 100
µM, 500 µM, and 1000 µM.
Reactions were stopped with SDS-PAGE sample buffer, boiled at 95 °C
for 5 min, and then analyzed by SDS-PAGE. Phosphorylated proteins were
visualized by an autoradiogram which was carried out at -70
°C for 3 h with intensifying screens.
To further substantiate the suggestion
that the activation of Cdk5 by p21 does not depend on the
phosphorylation of Cdk5, the possibility, albeit remote, that the
bacterially expressed Cdk5 used for reconstitution had already been
phosphorylated was tested by examining the effect of protein
phosphatase on the reconstituted kinase. The protein phosphatase 2A
catalytic subunit, which had been shown to inactivate and
dephosphorylate the isolated p34-cyclin complex
previously
(59) , was applied in such experiments. The
bacterially expressed GST-Cdk5 and GST-p25 were mixed and reconstituted
at 30 °C for 1 h in the presence of 0.5 mg/ml bovine serum albumin.
20-µl aliquots containing 1 µg of GST-Cdk5 and 1.5 µg of
GST-p21 were then treated with 20 ng of protein phosphatase 2A
catalytic subunit at 30 °C for 30 min. Okadaic acid (100
nM in final concentration) was then added to the mixture to
stop the phosphatase activity before the kinase activity was measured.
The kinase reaction was carried out for a short interval, 6 min, to
minimize the possibility of autophosphorylation during the reaction. It
was found that the kinase activity of the phosphatase-treated sample
was identical with that of the control samples which had been treated
in the same way except that okadaic acid was added before the addition
of the phosphatase.
/Thr
of Cdc2/Cdk2 by a distinct
kinase, Cdk activating kinase, for full kinase activity
(32, 33, 34, 35, 36, 57) .
More recently, the kinase, Cdk4/cyclin D has also been documented to
depend on an activating kinase for full activity
(41, 42) . Although the amino acid residue in Cdk5
corresponding to Thr
/Thr
of Cdc2/Cdk2,
Ser
, is also a potential kinase phosphorylation residue,
several observations of the present study suggest that the activity of
neuronal Cdc2-like kinase does not depend on phosphorylation of Cdk5. A
highly active kinase could be isolated from the reconstitution reaction
of Cdk5 and p21 without requiring any additional kinase. The enzyme
reconstitution and Cdk5 activation were not affected by
Mg
ATP. Moreover, no phosphorylation of Cdk5 could be
detected when the highly active, reconstituted enzyme was incubated
under phosphorylation conditions, ruling out the possibility that Cdk5
is phosphorylated at Ser
by an autocatalytic reaction.
Similar results were obtained with either the bacterially expressed
recombinant Cdk5 or the partially purified brain monomeric Cdk5
indicating that the difference in the mechanism of Cdk5 and Cdc2/Cdk2
activation cannot be attributed to an artifact arising from the use of
bacterially expressed Cdk5. Since we were unable to express the
full-length p35 to test how it might activate Cdk5, the possibility
that the activation of Cdk5 by the full-length protein is dependent on
the phosphorylation of Cdk5 at Ser
cannot be completely
ruled out. In addition, our results do not exclude the possibility that
the highly active, reconstituted neuronal Cdc2-like kinase can be
further activated by a Cdk activating kinase-like kinase.
is in a large loop, T-loop, expanding residues 152-170
(58) . Comparing the structure of Cdk2, an inactive form of
kinase, with the crystal structure of the catalytic unit of
cAMP-dependent protein kinase, a fully active kinase has revealed that
T-loop is a region of major conformational difference between the two
structures. Like that in Cdc2 or Cdk2, this region of cAMP-dependent
kinase contains a regulatory phosphorylation residue,
Thr
; autophosphorylation of the enzyme at this site
brings about ionic interactions between the phosphothreonine residue
with a number of cationic residues: His
,
Arg
, and Lys
to stabilize the active
conformation. It is suggested that cyclin activation of Cdk2 involves a
displacement of the loop to result in a conformation similar to
cAMP-dependent kinase. Phosphorylation of Thr
may bring
about ionic interactions to stabilize the active conformation. Two of
the three cationic amino acids in cAMP-dependent kinase that undergo
ionic interaction with P-Thr
are conserved in Cdk2. The
observation that Cdk5-21-kDa protein displays high kinase
activity in the absence of phosphorylation of Cdk5 is somewhat
unexpected. Amino acid sequence of Cdk5 in the T-loop region is highly
homologous to those of Cdc2 and Cdk2, and the two arginines postulated
to be involved in interacting with P-Thr
of Cdk2 are also
conserved in Cdk5.
Table:
Purification of the reconstituted kinase
by Mono S chromatography
Table:
Kinetic parameters of NCLK purified from
bovine brain and the reconstituted kinase
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.