(Received for publication, February 27, 1995; and in revised form, August 8, 1995)
From the
Neuronal Cdc2-like kinase is a heterodimer of Cdk5 and a 25-kDa
subunit that is derived from a 35-kDa brain- and neuron-specific
protein called the neuronal Cdk5 activator
(p35/p25) (Lew, J., Huang, Q.-Q., Qi, Z.,
Winkfein, R. J., Aebersold, R., Hunt, T., and Wang, J. H.(1994) Nature 371, 423-426; Tsai, L. H., Delalle, I., Caviness,
V. S., Jr., Chae, T., and Harlow, E.(1994) Nature 371,
419-423). Upon screening of a human hippocampus library with a
bovine Nck5a cDNA, we uncovered a distinct clone encoding a 39-kDa
isoform of Nck5a. The isoform, designated the neuronal Cdk5 activator
isoform (p39
), showed a high degree of
sequence similarity to p35
with 57% amino acid
identity. Northern blot analysis detected its mRNA transcript in bovine
and rat cerebrum and cerebellum, but not in any other rat tissues
examined. In situ hybridization showed that Nck5ai was
enriched in CA1 to CA3 of the hippocampus, but absent in the fimbria of
hippocampal formation. Among seven cell lines in proliferating
cultures, only PC12 and N2A, two cell lines capable of differentiating
into neuron-like cells, were found to contain Nck5ai mRNA. A 30-kDa
truncated form of Nck5ai expressed as a glutathione S-transferase fusion protein in Escherichia
coli] was found to associate with Cdk5 to form an active Cdk5
kinase. Thus, the isoform shares many common characteristics with
p35
, including Ckd5 activating activity and
brain- and neuron-specific expression. Both proteins show limited
sequence homology to cyclins, suggesting that they define a new family
of cyclin-dependent kinase-activating proteins.
The cell division cycle gene, cdc2, in fission yeast
performs rate-limiting functions in both G/S and
G
/M transitions. The protein product of the gene,
p34
, is a protein kinase catalytic subunit that
associates with specific cyclins to form functional protein kinases,
which are activated at discrete phases of the cell
cycle(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) .
In mammalian cells, seven Cdc2 homologous proteins have been
discovered; most have been shown to depend on cyclin for activity.
These Cdc2 homologous proteins are called cyclin-dependent kinases
(Cdks) (
)and are individually identified by a numerical
system as Cdk1 (Cdc2), Cdk2 up to Cdk7 (12, 13, 14) . In addition to Cdks, an
extended family of cyclins has been demonstrated to exist in mammalian
cells. Combinatorial association between members of these two protein
groups gives rise to a large number of heterodimeric protein kinases
that are called Cdc2-like
kinases(15, 16, 17) .
While most of the
Cdc2-like protein kinases are cell cycle regulators, the concept has
emerged recently that some of the Cdc2-like kinases may have major
functions unrelated to cell division(18) . A novel Cdc2-like
kinase has been discovered, purified from mammalian brains, and
extensively characterized
recently(19, 20, 21, 22) . The
purified kinase has been shown to be a heterodimer of Cdk5 and a 25-kDa
regulatory subunit that is essential for kinase activity(19) .
Molecular cloning studies have revealed that the 25-kDa protein is
derived proteolytically from a 35-kDa precursor protein that is
expressed specifically in brain neurons(23, 24) .
Since this brain Cdc2-like kinase is considered neuron-specific on
account of its regulatory subunit, it has been designated neuronal
Cdc2-like kinase, and the regulatory subunits, neuronal Cdk5 activator
(Nck5a or p25/35).
In addition to its
tissue specificity, p25/35 possesses certain
unique properties that distinguish it from members of the cyclin
family. The amino acid sequence of the protein displays minimal
homology to cyclins(23) . While the activation of Cdk1, Cdk2,
and Cdk4 by various cyclins has been shown to depend on the
phosphorylation of the Cdks by a specific Cdk-activating
kinase(25, 26, 27, 28, 29, 30) ,
the activation of Cdk5 by p25
is independent
of Cdk5 phosphorylation (31) . Northern blot analysis has
revealed two populations of mammalian brain mRNA (4.0- and 2.4-kb
mRNAs) that hybridize with a Nck5a probe, suggesting the existence of
isoforms of Nck5a(23) . In this study, we document the
discovery, cloning, and preliminary characterization of an isoform of
Nck5a. The isoform, a 39-kDa protein, shows 57% amino acid sequence
identity to Nck5a. The protein, tentatively designated the neuronal
Cdk5 activator isoform (Nck5ai or p39
), is
similar to Nck5a in displaying Cdk5 activation activity and brain- and
neuron-specific expression, but distinct from Nck5a in cell culture
distribution.
During the screening of a human hippocampus library with a
randomly labeled DNA probe from a bovine Nck5a cDNA encoding the active
domain of the protein(23) , several clones (9I4, 3I4, 10I4,
15I23, and 7II2) were obtained. Restriction map analysis of the clones
suggested that these clones represented two different mRNA transcripts.
Partial nucleotide sequences and the deduced amino acid sequences of
clones 15I23, 3I4, and 10I4 indicated that these clones encoded a
protein that was identical to human Nck5a(24) . On the basis of
their nucleotide sequences and deduced amino acid sequences, clones 9I4
and 7II2 were found to encode a distinct Nck5a homologous protein.
Clone 7II2 appeared to contain the complete protein reading frame. Fig. 1shows the nucleotide sequence of clone 7II2 and the
deduced amino acid sequence of the protein. The protein contains 367
amino acid residues with a calculated M of
39,000. The assignment of the initiation codon is based on the
observation that the sequence flanking the ATG codon contains an A at
position -3 and a G at position +4, characteristic of a
functional initiation codon(39) . A GeneBank
search with the nucleotide sequence and the protein sequence has
shown that this is a novel protein.
Figure 1:
Nucleotide sequence
and deduced amino acid sequence of
p39.
The sequence alignment of
p35 and the isoform, p39
, is presented
in Fig. 2A. The two proteins show 57% amino acid
identity in the overall sequence, with a higher level of homology (65%
amino acid identity) in the region of the protein corresponding to the
25-kDa subunit of the purified neuronal Cdc2-like kinase. This region
of Nck5a contains the Cdk5-activating domain(23) . The
amino-terminal sequence of 9 amino acid residues and the
carboxyl-terminal sequence of 3 residues of the two proteins are
identical. The difference in molecular weights of the two proteins can
be attributed mainly to the presence of a number of gaps in the Nck5a
sequence when the proteins are aligned for optimal homology. Most of
these gaps are located close to the protein termini. Like
p35
, p39
shows no overall amino acid
sequence homology to cyclins. A small region of 17 residues in Nck5a
and Nck5ai sequence is the only region with detectable sequence
similarity to the equivalent region of the cyclin box consensus
sequence (Fig. 2B). Amino acid identity of Nck5a and
Nck5ai in this region is 88.2%.
Figure 2:
Amino acid sequence comparison of
p35, p39
, and
cyclin-like proteins. A, alignment of Nck5a and Nck5ai
sequences. The top and bottom sequences are for human
p35
(24) and
p39
, respectively. Vertical lines and dots indicate the conserved (identical) and similar
amino acid residues between these two proteins. Shaded areas indicate regions representing bacterially expressed proteins in
this study: p21
and
p30
. B, comparison of Nck5a and
Nck5ai sequences with cyclin and cylin-like sequences. The cyclin
consensus sequence is derived from cyclins A, B, D, and E, with the
highly conserved Leu and Lys residues highlighted with asterisks. Shaded residues indicate matches to Nck5a
and Nck5ai.
In a previous study, Northern blot analysis of bovine brain mRNA using a Nck5a cDNA probe detected a 4-kb mRNA band, whereas human brain was found to contain two populations of mRNA of 4 and 2.4 kb(23) . In the present study, mRNAs from bovine cerebrum and cerebellum and rat cerebrum and cerebellum were probed by the randomly labeled Nck5a cDNA. Fig. 3shows that the two populations of mRNA could be detected in both bovine and rat cerebrum, with the Northern blot intensity of the 2.4-kb transcript much weaker than that of the 4-kb transcript. Bovine and rat cerebellum also contained the transcripts, but at much lower levels, and only the 4-kb transcript was clearly seen on the Northern blot. When the blots of rat and bovine brain mRNAs were probed by a randomly labeled Nck5ai cDNA probe, the same two populations of mRNA of 4.0 and 2.4 kb were detected in bovine and rat cerebrum. However, contrary to what was revealed by the Nck5a cDNA probe, the smaller transcript displayed far higher intensity than the larger transcript on the Northern blot probed by the Nck5ai cDNA (Fig. 3). Weak Northern blot signals of the 2.4-kb transcript could also be detected in bovine and rat cerebellum. whereas no signal was detected at the 4.0-kb mRNA positions (Fig. 3). These observations indicate that the 4.0- and 2.4-kb mRNAs represent Nck5a and Nck5ai transcripts, respectively.
Figure 3: Northern analysis of the tissue distribution of Nck5a and Nck5ai. Messenger RNA samples from different bovine and rat tissues were isolated, and 5 µg of each was probed with randomly labeled Nck5a (A) or randomly labeled Nck5ai (B) DNA probes.
Tissue distribution study in humans has revealed that the mRNA transcript of Nck5a is expressed specifically in the brain(23) . Fig. 3shows that Nck5a mRNA displays a similar brain-specific expression in rats. Essentially identical tissue expression patterns were observed for the 4- and 2.4-kb transcripts. Both transcripts were found to be expressed exclusively in brains, with levels in the cerebrum markedly higher than those in the cerebellum. No expression of either of the two transcripts was detected in non-neuronal tissues even upon increasing the exposure time by 10 times that used for the experiment of Fig. 3. The tissue distributions of Nck5a and Nck5ai mRNAs are markedly different from that of Cdk5. Previous studies showed that Cdk5 was widely distributed in humans as all human tissues examined were found to contain Cdk5 mRNA. Northern blot analysis of Cdk5 tissue distribution in rats showed that Cdk5 mRNA expression could also be readily detected in all the rat tissues examined (data not shown).
A number of proliferating cell lines were examined
previously by Northern blot analysis and Western immunoblot analysis
for Cdk5 distribution; all were found to contain Cdk5 mRNA and Cdk5
protein (40) . To determine whether the cultured cells
contained any of the neuronal Cdk5 activators, several cell lines were
examined for the expression of p35 and p39
mRNAs by Northern blot analysis. Fig. 4A shows that
none of the cell lines expressed p35
mRNA, with the
exception of Jurkat cells, a human T cell line, which showed a very
low, nonetheless detectable Northern blot signal. On the other hand,
N2A cells, a neuroblastoma cell line, and PC12 cells, a
pheochromocytoma cell line that can be differentiated by nerve growth
factor treatment into sympathetic neuron-like cells, expressed
significant levels of p39
mRNA (Fig. 4B). The level of the mRNA transcript was
especially high in PC12 cells. None of the other cell lines, including
a glioma cell line (C6), contained detectable p39
transcript. The observation that only cell lines of neural origin
or with neuron characteristics express p39
suggests
that the protein is neuron-specific.
Figure 4: Northern analysis of the cell line distribution of Nck5a and Nck5ai. 5 µg of mRNAs from bovine and rat cerebrum and cerebellum (serving as positive controls) and from different cell lines was hybridized with the randomly labeled Nck5a (A) and Nck5ai (B) DNA probes.
In situ hybridization
procedures were used to examine the regional distribution of
p39 mRNA in the brain of a 3-month-old rat. The result
indicated that the messenger transcript was highly expressed in CA1 to
CA3 of hippocampal formation, an area enriched in neurons (Fig. 5). The presence of the Nck5ai mRNA in neurons can be
clearly seen with a higher magnification of the CA3 region (Fig. 5C). Some expression of the p39
mRNA could also be seen in the dentate gyrus of the hippocampus.
In contrast, no expression of the mRNA was detected in the fimbria
hippocampi, an area containing axons of neurons and glial cells. These
results on the regional distribution of p39
mRNA are
compatible with the suggestion that p39
is a
neuron-specific protein.
Figure 5:
Expression of p39 in adult rat hippocampus and dentate gyrus. A, in situ hybridization using the digoxigenin-labeled antisense
p39
probe in adult rat hippocampus; B, high magnification of the CA2 region in the hippocampus; C, in situ hybridization using a digoxigenin-labeled
sense p39
probe in the
hippocampus.
The active neuronal Cdc2-like kinase
purified from bovine brain contains a 25-kDa subunit
(p25)(19) , a truncated form of
p35
(23) . Bacterially expressed p25
or a further truncated form of 21 kDa (p21
) can
activate bacterially expressed monomeric Cdk5 to an activity similar to
that of the purified neuronal Cdc2-like kinase(31) . This
observation suggests that p21
contains the active
domain essential for Cdk5 activation. A cDNA clone encoding a 30-kDa
truncated form of p39
containing the region
corresponding to p25
was expressed as a GST fusion
protein in E. coli. The expressed fusion protein,
GST-p30
, was purified by glutathione affinity
chromatography and tested for Cdk5 activating activity. Fig. 6shows that the protein could activate bacterially
expressed Cdk5 to a maximal activity about the same as that achieved by
a GST-p21
fusion protein. Three different preparations
of GST-p30
were tested for the ability to activate
Cdk5; essentially identical levels of maximal Cdk5 activation were
obtained. However, the dose dependence of the Cdk5 activation by the
bacterially expressed fusion protein varied from preparation to
preparation, probably because different preparations contained
different proportions of the active protein.
Figure 6:
Activation of Cdk5 by
GST-p30. 900 ng of GST-Cdk5 was reconstituted
with various amounts of GST-p21
(p21)
or GST-p30
(p30i) in 30 µl of
phosphate-buffered saline containing 1 mM DTT, 1 mM EDTA, and 5 µg of bovine serum albumin at room temperature for
2 h, and 3-µl aliquots were withdrawn at the intervals indicated
for histone H1 kinase activity assay in 30 mM Mops, pH 7.4, 10
mM MgCl
, 100 µM histone H1 peptide,
and 100 µM ATP (1000 cpm/ml) at 30 °C for 30
min.
Bovine brain extract
contains high levels of monomeric Cdk5. Monomeric brain Cdk5 has been
partially purified and shown to associate readily with p25 or p21
to form active Cdk5 kinases. When the
GST-p30
fusion protein was incubated with an aliquot
of bovine brain extract and then affinity-precipitated by glutathione
beads, Cdk5 in the brain extract was found to coprecipitate with the
fusion protein as revealed by a specific Cdk5 antibody probe (Fig. 7A). The precipitate displayed good histone H1
peptide kinase activity, suggesting that brain Cdk5 could be activated
by the fusion protein (Fig. 7B). In a separate
experiment, a monomeric Cdk5 preparation partially purified from bovine
brain was incubated with the GST-p30
fusion protein
for 30 min, and the kinase activity of the sample was then determined.
While neither monomeric Cdk5 nor the bacterially expressed fusion
protein alone had any kinase activity, incubation of the two proteins
together resulted in high histone H1 kinase activity (data not shown).
This observation provides direct proof that Nck5ai is capable of
activating the native form of Cdk5.
Figure 7:
Coprecipitation of Cdk5 from bovine brain
extract with GST-p21 and
GST-p30
. 1 ml of bovine brain extract (10
mg/ml protein) was incubated with 100 µg of GST or GST fusion
protein at 4 °C for 2 h and then precipitated with glutathione
beads. The beads was washed four times at 4 °C with MTPBS
containing 2 mM DTT, 1 µg/ml leupeptin, and 2 µg/ml
antipain and resuspended in 100 µl of the same washing buffer. A, histone H1 kinase activities of glutathione bead affinity
precipitates of the various GST and GST fusion proteins indicated; B, immunoblot analysis of GST and GST fusion protein
precipitates with a Cdk5-specific polyclonal
antibody.
Until now, Nck5a has been the only protein known to be
capable of reconstituting highly active Cdk5 kinases. In this study, we
demonstrate the existence of an isoform of Nck5a, a 39-kDa protein that
displays a high degree of amino acid sequence homology to p35 with 57% amino acid identity. Like p35
, this
protein, called p39
, is capable of associating with
Cdk5 to form functional kinases of high activity and shows
brain-specific mRNA expression as revealed by the Northern blot
analysis of its tissue distribution in both bovine and rat tissues. On
the basis of detailed immunohistochemical studies and in situ hybridization analysis, p35
has been suggested to
be expressed specifically in neurons. (
)At least two lines
of evidence support the suggestion that p39
is also
present specifically in animal brain neurons. Among a number of cell
lines examined in culture, only N2A cells (a cell line of neural
origin) and PC12 cells (a pheochromocytoma cell line capable of
differentiating with nerve growth factor into neuron-like cells) were
found to contain p39
mRNA. Significantly, a cell line
of glial origin, the C6 glioma cell line, did not show detectable
p39
mRNA signal on its Northern blot. The in situ hybridization patterns of Nck5ai indicate that p39
is greatly enriched in the CA1 to CA3 zone of hippocampal
formation of adult rat brain (a region rich in neurons) and absent in
the fimbria hippocampi, where glial cells predominate.
The existence
of two brain- and neuron-specific Cdk5 activators has raised the
question as to the physiological significance of the isoforms. As the
brain is a highly complex organ containing many different cell types,
one possible reason for the existence of isoforms of neuronal Cdk5
activators may be that the isoforms have distinct cell-type or brain
regional distributions. Alternatively, the isoforms may coexist in the
same neurons, but have differential subcellular localizations. We have
recently observed that Cdk5 and p35 are localized in
different compartments in neurons of adult rat brains. While Cdk5 is
enriched in the axons of central nervous neurons, p35
has been seen mostly in the cell body.
It is tempting
to speculate that p39
may have an axonal localization
so that the cell body and axonal function of Cdk5 in neurons are
regulated by Nck5a and Nck5ai, respectively. The situation is somewhat
similar to the regulation of Cdc2 by phase-specific cyclins in yeast
during cell division cycle progression(10, 11) ,
except that the phase-specific cyclins are temporal specific regulators
of Cdc2, whereas Nck5a and Nck5ai are spatial specific regulators of
Cdk5. Work is under way to determine whether p39
indeed has an axonal localization.
The suggestion that
p35 and p39
have different functional
roles in brain neurons appears to be supported by the observation that
the two proteins are differentially expressed in cultured proliferating
cells. Of all the cells examined, only the two cell lines with neuronal
characteristics and capable of induced differentiation into neuron-like
cells were found to contain p39
. While it is tempting
to suggest that the protein contributes to the neuronal differentiation
of the cells, supporting evidence for such a suggestion has not been
forthcoming. In contrast to Nck5ai mRNA, Nck5a mRNA was not detected in
any of the cell lines examined, except in the human T cell line (Jurkat
cells), albeit at a very low level. The significance of the presence of
p35
in a human T cell line is not clear. It should be
noted that neither p35
nor p39
mRNA
has been detected in bovine or rat thymus, an organ with concentrated T
cells. The question as to why p35
mRNA is absent in
proliferating N2A and PC12 cells is not addressed in this study. The
possibility that the protein and its mRNA are expressed in
differentiated N2A and PC12 cells will be of considerable significance
and interest.
Although p35 and p39
are activators of a cyclin-dependent kinase, they display only
marginal sequence homology to members of the cyclin family. The two
proteins also appear to distinguish themselves from members of the
cyclin family in the mechanism of kinase activation. It has been well
documented that the activation of Cdk2 by cyclin A, Cdc2 by cyclin B,
and Cdk4 by cyclin D is dependent on the phosphorylation of the Cdks on
a threonine residue (Thr-160 of Cdc2 or its equivalent in the other
Cdks) by a specific Cdk-activating kinase for full kinase
activity(25, 26, 27, 28, 29, 30) .
The activation of Cdk5 by the Cdk5 activators, on the other hand, can
bring out high Cdk5 kinase activity in the absence of Cdk5
phosphorylation (31) . The unique amino acid sequences and
distinctive activation characteristics together suggest that the two
proteins may define a new family of kinase activator proteins. As none
of the cyclins and no other proteins have been shown to activate Cdk5,
this new family of kinase activators may be specific and unique for
Cdk5 or Cdk5-like kinases. While Cdk5 has a wide tissue and cell
distribution, only brain- and neuron-specific Cdk5 activators have been
identified to date. It seems reasonable to suggest that there are
additional members of this protein family that function as Cdk5
activators in non-neuronal cells. If so, degenerated polymerase chain
reaction primers derived from some of the most conserved regions of
these two neuronal Cdk5 activators may be useful in the search for
additional Cdk5 kinase activators.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U34051[GenBank].