(Received for publication, May 4, 1995; and in revised form, November 6, 1995)
From the
Neuronal cdc2-like kinase (Nclk) purified from bovine brain is a
heterodimer of Cdk5 and an essential 25-kDa regulatory subunit (Lew,
J., and Wang, J. H.(1995) Trends Biochem. Sci. 20,
33-37). The regulatory subunit is an N-terminal truncated
derivative of a 35-kDa protein expressed specifically in brain, hence
the name neuronal Cdk5 activator, p25/p35. In
this study, we probe the relationship between the two different forms
of Nck5a and their interaction with and activation of Cdk5 in bovine
brain extract. Using protein fractionation procedures in combination
with Western blot analysis and protein kinase assay, three forms of
Cdk5 have been detected in bovine brain: a monomeric Cdk5 that can be
activated by bacterially expressed GST-p21
, a
heterodimer of Cdk5 and p25
that displays high
kinase activity, and a Cdk5
p35
complex
that is inactive and refractory to GST-p21
activation. Analysis of the Cdk5
p35
complex by gel filtration chromatography indicated that the
complex was part of a macromolecular structure with a molecular mass of
670 kDa. When the macromolecular complex was subjected to gel
filtration chromatography in the presence of 10% ethylene glycol, the
fractions containing both p35
and Cdk5,
although eluting at the same position as control, displayed high kinase
activity. The result is compatible with the suggestion that the
macromolecular complex contained a kinase inhibitory factor that
dissociated from the complex in 10% ethylene glycol.
The animal cell division cycle is controlled by the coordinated
actions of a family of protein Ser/Thr kinases, called cdc2-like
kinases, which are activated and deactivated at discrete phases of the
cell cycle. Members of the cdc2-like kinase family are heterodimers of
a cdc2-related protein called cyclin-dependent kinase (Cdk) ()and a cyclin. The activity of a cdc2-like kinase in
dividing cells is usually dictated by the level of the cyclin subunit
as well as the phosphorylation state of the Cdk subunit. In general,
the catalytic activities of cell cycle cdc2-like kinases depend on the
phosphorylation of a threonine residue (Thr-161 or its equivalent) by a
specific CDK-activating kinase, whereas phosphorylation of a threonine
or a tyrosine residue at the nucleotide binding loop of Cdk subunit by
specific inhibitory kinases results in the inactivation of the kinases
(for review, see (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) ).
In addition, cdc2-like kinases undergo functional and regulatory
significant associations with a large number of cellular proteins,
which include a family of low molecular weight Cdk inhibitory
proteins(11, 12, 13, 14, 15, 16, 17) ,
transcription factors, and Rb and Rb-related
proteins(18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) .
In a number of reports, Cdks and cdc2-like kinases have been shown to
exist within macromolecular
complexes(26, 27, 28, 29, 30, 31, 32) .
While it is logical to suggest that these macromolecular complexes
represent the functional and regulatory significant association of Cdks
and/or cdc2-like kinases with defined cellular proteins, systematic
studies of such complexes have not been carried out.
Recently, a
cdc2-like kinase has been discovered and purified from bovine brain as
a proline-directed kinase (33, 34) or a tau protein
kinase II(35) . This enzyme has been implicated in the
regulation of neurocytoskeletal proteins, and a loss in the regulation
of the enzyme has been suggested to be involved in Alzheimer
pathology(34, 36) . The purified protein kinase is a
heterodimer of a 33-kDa catalytic subunit and a 25-kDa regulatory
subunit(33) . Molecular cloning has identified the 33-kDa
subunit as Cdk5 and the 25-kDa regulatory subunit as a proteolytic
derivative of a novel neuron-specific 35-kDa
protein(31, 34, 37) . The regulatory subunit
is essential for the kinase activity and only expresses in the central
nervous system. Therefore, it has been designated as the neuronal Cdk5
activator, Nck5a
(p25/p35
), and the
heterodimeric holoenzyme as the neuronal cdc2-like kinase,
Nclk(38, 39, 40) . Since neurons in adult
brain do not divide, Nclk is unique in being a cdc2-like kinase not
involved in cell division.
As a cdc2-like kinase, Nclk is expected to share many common regulatory and functional properties with other family members. Thus, the enzyme is composed of a catalytic subunit and a positive regulatory subunit. All the regulatory phosphorylation sites found in other Cdks are conserved or conservatively substituted (Ser to Thr) in Cdk5(34, 41) . On the other hand, for its neuronal functions, Nclk may possess unique regulatory properties that distinguish itself from the cell cycle regulatory cdc2-like kinases. For example, while Nck5a displays a cyclin-like activity in that it activates the catalytic subunit, it shows little, or at best, marginal amino acid sequence homology to cyclins(37, 41) . In addition, the activation of Cdk5 by Nck5a is independent of the Cdk-activating kinase, whereas cyclin activation of Cdks in many cases have been found to depend on the Cdk-activating kinase(38) . A comparison of the regulatory properties of Nclk with those of the cell cycle regulatory cdc2-like kinases may shed new light on both cell cycle regulation and the control of neuronal functions.
Although the
purified bovine brain Nclk is composed of Cdk5 and
p25, the intact p35
is the predominant form of the protein in crude brain
extract(37) . The relationship between the two forms of Nck5a
and their interaction with Cdk5 in the brain extract are not known. In
this study, we have examined the states of Cdk5 in crude bovine brain
extract using protein fractionation procedures in combination with
kinase activity and immunological analysis. The results suggest that
Cdk5 exists in at least three forms: a free Cdk5 monomer that is
readily activated by a bacterially expressed
p21
, a heterodimer of Cdk5/p25
that is highly active, and a Cdk5/p35
form in association with a macromolecular complex that is
neither enzymatically active nor activable by the bacterially expressed
GST-p21
.
For the ECL system, the blot was probed with 1/2000 dilution of a rabbit polyclonal Cdk5 (C-8) antibody from Santa Cruz Biotechnology, Inc., followed by incubation with goat anti-rabbit IgG conjugated with horseradish peroxidase and developed as specified by the manufacturer (Amersham Corp.).
To analyze the relationship between p25 and
p35
and their interactions with Cdk5 in brain extracts,
we attempted the fractionation of the two forms of Nck5a in a 120,000
g fraction of the brain extract on various columns.
Initial characterization using specific antibodies showed that both
Cdk5 and Nck5a were quantitatively absorbed on a hydroxylapatite column
or a cation exchanger such as an FPLC Mono S column but negligibly
bound to anion exchangers such as DEAE-Sepharose and FPLC Mono Q
columns. When a small amount of crude sample (40 mg) of fresh bovine
brain 120,000
g extract was applied to an FPLC Mono S
(HR 5/5) column, and the column eluents from a linear gradient of
0-0.5 M NaCl were analyzed by Western blot analyses
using Cdk5- and Nck5a-specific antibodies, Cdk5 was found to be eluted
from the column over the entire salt gradient, whereas p25
and p35
were eluted separately, each within a
narrow range of the salt gradient. The result was in agreement with a
postulate that there were at least three forms of Cdk5 in the crude
bovine brain sample: a p35
-complexed form, a
p25
-complexed form, and a form not associated with
Nck5a. To analyze the activity states of the different forms of Cdk5,
the column eluents were assayed for histone H1 kinase activity in the
presence and absence of GST-p21
, a bacterially
expressed truncated form of p25
previously shown to
activate the bacterially expressed Cdk5(38) . When the kinase
assay was carried out in the absence of GST-p21
, a
small amount of kinase activity was detected, exclusively in fractions
containing p25
. In fractions containing Cdk5 but
without p25
or p35
, high levels of the
kinase activity could be demonstrated in the presence of
GST-p21
. On the other hand, fractions containing Cdk5
and p35
appeared to have little or no activity in the
presence and absence of GST-p21
. Thus, it may be
further suggested that the three forms of Cdk5 have different histone
H1 kinase activity characteristics. The Cdk5/p25
is the
only active species of Cdk5, the uncomplexed Cdk5 is inactive but can
be activated by Nck5a, and Cdk5/p35
is inactive as well
as refractory to Nck5a activation.
To further test this suggestion,
the 120,000 g bovine brain extract was analyzed by
different column chromatographic procedures, a procedure combining both
DEAE-Sepharose and hydroxylapatite column. In comparison with a Mono S
chromatography, this procedure has the advantage that a large amount of
the sample can be processed, and the separated fractions may be further
analyzed. A sample (1,300 ml) of the 120,000
g fraction was loaded onto a 400-ml DEAE-Sepharose column directly
coupled to a 100-ml hydroxylapatite column. After sample application,
the columns were disconnected, and the hydroxylapatite column was
eluted with a linear gradient of K
HPO
(0-400 mM). The eluted proteins were analyzed for
histone H1 peptide-phosphorylating activity in the presence and absence
of GST-p21
, a bacterially expressed truncated form of
p25
that is capable of activating a bacterially
expressed Cdk5(38) , and immunoblotted with either a Nck5a- or
Cdk5-specific antibodies. As shown in Fig. 1A, in the
absence of GST-p21
, a small peak of endogenous kinase
activity eluted at 280 mM K
HPO
. In the
presence of GST-p21
, the kinase activity was markedly
increased. The peak of the GST-p21
-activated kinase
activity eluted slightly before that of the endogenous kinase activity
(see Fig. 1A, inset). This indicates that the
majority of Cdk5 exists in an inactive form but can be activated by
GST-p21
.
Figure 1:
DEAE-hydroxylapatite
column chromatography. Protein fractionation of the 120,000 g supernatant by DEAE-hydroxylapatite columns was carried out
as described under ``Experimental Procedures.'' A,
-, A
value;
-
, endogenous kinase activity;
-
,
kinase activity in the presence of GST-p21
;
&cjs1359;-&cjs1359;, kinase activity in the presence of
GST-Cdk5; -, a linear gradient of 0-0.4 M
K
HPO
,. Inset represents the kinase
activity at different scales indicating the separation of two different
activity peaks. B, immunoblot analysis of the eluted proteins
with a Nck5a-specific antibody. C, immunoblot analysis of the
eluted proteins with a Cdk5-specific antibody. D, immunoblot
analysis of the eluted proteins with a Cdk5-specific antibody, C-8, and
developed by the ECL system as described under ``Experimental
Procedures.''
Immunoblot analysis of the eluted proteins
with a Nck5a-specific antibody showed two major immunoreactive bands
with molecular weights corresponding to p25 and
p35
(Fig. 1B). These two proteins were
partially separated by hydroxylapatite column chromatography: the
25-kDa protein eluted between 250 and 330 mM
K
HPO
, whereas the 35-kDa protein eluted between
280 and 400 mM K
HPO
. On the other
hand, Cdk5 eluted between 140 and 400 mM
K
HPO
(Fig. 1, C and D). At K
HPO
> 250 mM, Cdk5
eluted with one or both forms of Nck5a; however, at
[K
HPO
] < 250 mM, Cdk5
eluted without Nck5a. Interestingly, only the fractions that contained
p25
appeared to have significant endogenous kinase
activity (Fig. 1, A and B). The peak of the
GST-p21
-activated kinase activity, however, was
observed in fractions containing Cdk5 but neither p25
nor p35
. At
[K
HPO
] > 330 mM, Cdk5
eluted only with p35
and showed no kinase activity in
the presence and absence of GST-p21
(Fig. 1, A-D). Together, these results support the existence of
three distinct forms of Cdk5 in bovine brain extract: (i) a free form
that is inactive but activable by Nck5a, (ii) an active p25
complexed form, and (iii) a p35
complexed form
that is neither active nor activable by Nck5a.
To further test the
existence of three distinct forms of Cdk5 in bovine brain extract, the
hydroxylapatite fractions were pooled into three different groups;
fraction I (nos. 28-34), fraction II (nos. 36-42), and
fraction III (nos. 44-54), which were enriched with free Cdk5,
p25/Cdk5, and p35
Cdk5 complex,
respectively. Each of the fractions was concentrated and further
analyzed by FPLC Superose-12 gel filtration chromatography. As shown in Fig. 2, A and C, the majority of Cdk5 in
fraction I eluted at a volume corresponding to the molecular mass of
30 kDa and became highly active when the kinase assay was carried
out in the presence of GST-p21
. This indicates that
Cdk5 exists mainly as a monomeric form. When fraction II was analyzed
by Superose-12 gel filtration chromatography, a single peak of
endogenous kinase activity containing both p25
and Cdk5
eluted at a volume corresponding to the molecular mass of
60 kDa (Fig. 3). Addition of GST-p21
to the
p25
/Cdk5-containing fractions did not increase the
kinase activity, suggesting that Cdk5 and p25
exists as
a complex. There was a peak of GST-p21
-activated kinase
activity at an elution volume corresponding to the molecular weight of
30 kDa. However, this is expected because the separation of the
free and p25
complexed Cdk5 forms on the
hydroxylapatite column was incomplete, and the fraction II sample
contained free Cdk5 (see Fig. 1). Analysis of fraction III by
Superose-12 gel filtration chromatography showed that most of
p35
and Cdk5 of the sample co-eluted at the void volume
of the column (Fig. 4), thus raising the possibility that these
proteins were in a complex of over 400 kDa. There was little or no
histone H1 kinase activity associated with these fractions in the
presence and absence of GST-p21
. Occasionally, a small
amount of free Cdk5, which possessed GST-p21
-activated
kinase activity, was observed.
Figure 2:
Superose-12 gel filtration column
chromatography of fraction I. A, protein fractionation of
fraction I by a Superose-12 column was carried out as described under
``Experimental Procedures.'' A, -, A value;
-
, endogenous
kinase activity;
-
, kinase activity in the presence of
GST-p21
. B, immunoblot analysis of
the eluted proteins with a Nck5a-specific antibody. C,
immunoblot analysis of the eluted proteins with a Cdk5-specific
antibody.
Figure 3:
Superose-12 gel filtration column
chromatography of fraction II. A, protein fractionation of
fraction II by a Superose-12 column was carried out as described under
``Experimental Procedures.'' A, -, A value;
-
, endogenous
kinase activity;
-
, kinase activity in the presence of
GST-p21
. B, immunoblot analysis of
the eluted proteins with a Nck5a-specific antibody. C,
immunoblot analysis of the eluted proteins with a Cdk5-specific
antibody.
Figure 4:
Superose-12 gel filtration column
chromatography of fraction III. A, protein fractionation of
fraction III by a Superose-12 column was carried out as described under
``Experimental Procedures.'' A, -, A value;
-
, endogenous
kinase activity;
-
, kinase activity in the presence of
GST-p21
. B, immunoblot analysis of
the eluted proteins with a Nck5a-specific antibody. C,
immunoblot analysis of the eluted proteins with a Cdk5-specific
antibody.
While the existence of
GST-p21-activable Cdk5 and the highly active
p25
/Cdk5 form was expected, the presence of the
inactive p35
/Cdk5 form was a somewhat surprising
finding. One possible explanation for the lack of kinase activity in
these fractions is that the two proteins, although co-eluted by the gel
filtration column chromatography, did not form a complex. A number of
observations, however, argued against this explanation. For example,
the fractions containing both proteins could not be activated by
GST-p21
, suggesting that Cdk5 did not exist as a free
form. Similarly, the addition to these column fractions of a
bacterially expressed GST-Cdk5, which could be readily activated by
GST-p21
(see Fig. 1and Fig. 5), did not
result in active kinase, thus arguing against the presence of free
p35
. Furthermore, the fraction III sample was analyzed
by FPLC Superose-6 gel filtration chromatography, and p35
and Cdk5 again co-eluted at a volume corresponding to a molecular
mass of
670 kDa (Fig. 5). Although co-immunoprecipitation
analysis with either Nck5a-specific or Cdk5-specific antibodies could
provide a direct evidence for the existence of the
p35
Cdk5 complex, we have been unable to produce a
precipitating Cdk5 or Nck5a antibody. A Cdk5-specific antibody (C-8)
from Santa Cruz Biotechnology was tried and found to precipitate the
p25
Cdk5 complex, but the antibody did not
immunoprecipitate Cdk5 from the fractions containing Cdk5 and
p35
.
Figure 5:
Superose-6 gel filtration column
chromatography of fraction III. A, protein fractionation of
fraction III by a Superose-6 column was carried out as described under
``Experimental Procedures.'' A, -, A value;
-
, endogenous
kinase activity;
-
, kinase activity in the presence of
GST-p21
; &cjs1359;-&cjs1359;, kinase
activity in the presence of GST-Cdk5. B, immunoblot analysis
of the eluted proteins with a Nck5a-specific antibody. C,
immunoblot analysis of the eluted proteins with a Cdk5-specific
antibody.
Another possibility that might account for the
lack of kinase activity in fractions containing both p35 and Cdk5 is that the complex contains a protein(s) that inhibits
the kinase. Recently, a number of inhibitory proteins for the family of
cdc2-like kinases have been discovered: p21
(also known
as CAP20, SDI1, or WAF1) as a general inhibitor of
Cdks(11, 12, 13, 14, 15) ,
p16
as an inhibitor of cyclin D1-D3/Cdk4 (16) ,
and p27
as an inhibitor of cyclin E/Cdk2(17) .
Some of these exist complexed with the holoenzyme form of the kinases
to form inactive kinase
complexes(14, 29, 41) . Therefore, attempts
were made to remove the putative inhibitor protein(s) from the complex
using either high concentrations of NaCl or ethylene glycol. When
fraction III was incubated with 1 M NaCl and then
chromatographed on Superose-6 FPLC column in the presence of 1 M NaCl, the protein elution (data not shown) and enzyme activity
profiles (see Fig. 6) were unaltered from the control sample (Fig. 5). However, when the sample was treated with 10% ethylene
glycol and then subjected to gel filtration chromatography in the
presence of 10% ethylene glycol, the fractions containing both
p35
and Cdk5, although eluting at the same position as
the untreated sample (Fig. 5), displayed high kinase activity (Fig. 6). The observation was compatible with a suggestion that
a low molecular weight inhibitor had been removed from the complex. An
alternative explanation that ethylene glycol incubation caused a
conformational change of the kinase from an inactive to an active state
is unlikely since incubation of the sample in 10% ethylene glycol
without subjecting the gel filtration chromatography did not result in
activation of the kinase (data not shown). Taken together, our results
suggest a potential interaction between a low molecular weight
inhibitor and the p35
Cdk5 complex to form the
inactive high molecular weight complex.
Figure 6:
Superose-6 gel filtration column
chromatography of fraction III in the presence of 10% ethylene glycol. A, protein fractionation of fraction III by a Superose-6
column was carried out as described under ``Experimental
Procedures.'' A, -, A value;
-
, endogenous kinase activity.
Kinase activity values (&cjs1359;-&cjs1359;) after Superose-6
gel filtration column chromatography of fraction III in the presence of
1 M NaCl are presented. B, immunoblot analysis of the
eluted proteins with a Nck5a-specific antibody. C, immunoblot
analysis of the eluted proteins with a Cdk5-specific
antibody.
In summary, we observed
three distinct Cdk5 molecular species in bovine brain extract: (i) a
monomeric Cdk5 that can be activated by Nck5a, (ii) a
p25Cdk5 form that is intrinsically active, and
(iii) a p35
Cdk5 form that is inactive and
refractory to Nck5a activation. Further analysis of the fractions
containing both p35
and Cdk5 indicated the presence of
an inactive p35
Cdk5 high molecular weight
complex. A number of recent papers support the presence of the
p35
Cdk5 high molecular weight complex: (i) the
existence of inactive macromolecular complex-containing multiple p21
subunits in insect Sf9 cell lysate where p21 has been suggested to act
as a Cdk assembly factor to promote the association of cyclin and Cdk
subunits(30) , (ii) the identification of Cdk-activating kinase
complex as a component of TFIIH (26, 27, 28) , (iii) the identification of
cyclin-dependent kinase binding protein (p15
) as a
part of a high molecular weight complex in starfish oocyte
lysate(32) , and (iv) the existence of additional
Cdk5-regulatory subunits (p60, p62, and p180) aside from p25
and p35
in rat neuronal
cells(31, 40) . (
)Although the molecular
basis for the lack of kinase activity of the macromolecular complex is
not known, initial experiments are compatible with the suggestion that
the complex contains kinase inhibitory factor(s). Tests are underway to
identify a possible low molecular weight inhibitor.