1 ICRF Clare Hall Laboratories, South Mimms, Herts EN6 3LD, UK
2 Department of Biochemistry, Hong Kong University of Science and Technology,
Clear Water Bay, Hong Kong
3 Division of Biomedical and Clinical Laboratory Sciences, Section of Biomedical
Sciences, The University of Edinburgh, Hugh Robson Building, George Square,
Edinburgh EH8 9XD, UK
* Present address: Target Identification and Validation, ProQinase GmbH,
Breisacher Str. 117, D-79106 Freiburg, Germany
Present address: Institut Curie Section Recherche, CNRS UMR 144, 26
rue d'Ulm, 75248 Paris Cedex 05, France
Author for correspondence (e-mail:
r.graeser{at}proqinase.com)
Accepted 12 June 2002
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Summary |
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Key words: PCTAIRE-1, CDK-related, Protein kinase A, 14-3-3, Neurite outgrowth
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Introduction |
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Messenger RNAs encoding the 45-58 kDa PCTAIRE family of protein kinase
subunits have been identified in frogs, mice and humans, and consist of two or
three isoforms. The mRNAs for PCTAIRE-2 and 3 are found almost exclusively in
the brain, whereas PCTAIRE-1 is located in both brain and testis of adult mice
and rats (Hirose et al., 1997;
Rhee and Wolgemuth, 1995
).
Unlike CDK1, which has only 10 residues before the Walker A motif, the PCTAIRE
family have 127-198 residues in N-terminal extensions
(Meyerson et al., 1992
;
Okuda et al., 1992
). No cyclin
partner has been identified for any of the PCTAIREs, and these extensions may
play the role of an attached cyclin for these kinases, although they do not
display obvious homology to known cyclin sequences. However, interacting
proteins have been identified in 2-hybrid screens; Sladeczek and co-authors
isolated 14-3-3 proteins and the annexin II-interacting protein p11 in a
screen using the full-length mouse PCTAIRE-1 cDNA as a bait for a mouse brain
cDNA library (Sladeczek et al.,
1997
). Although the interaction of PCTAIRE-1 with the 14-3-3
proteins was strong enough to allow the affinity purification of PCTAIRE-1
(Le Bouffant et al., 1998
), no
functional significance could be ascribed in the absence of any clues about
the function of PCTAIRE-1 itself. In another 2-hybrid screen, which used the
isolated N-terminal region of rat PCTAIRE-2, a protein was pulled out with
tudor repeats that showed some sequence homology to AKAP proteins
(Hirose et al., 2000
). This
protein, called Trap, interacted in vitro with PCTAIRE-1 and -2, but not with
PCTAIRE-3. Trap co-localized with PCTAIRE-2 in the brain
(Hirose et al., 2000
), but the
kinase activity of PCTAIRE-2 was not influenced by Trap
(Hirose et al., 2000
). Thus,
the PCTAIREs do form complexes with a number of other proteins, but none of
them seem to act as a PCTAIRE-specific cyclin.
Although expression of the PCTAIREs is restricted to the brain and testis,
our attention was aroused by the finding that high levels of expression
occurred in a wide range of transformed and immortalised cell lines of
epithelial origin (Charrasse et al.,
1999; Hirose et al.,
1997
; Rhee and Wolgemuth,
1995
). There is great uncertainty about the function of the
PCTAIREs, some authors suggesting a role in the cell cycle
(Charrasse et al., 1999
),
others in the differentiation of neurons or testicular cells
(Besset et al., 1999
;
Rhee and Wolgemuth, 1995
).
Equally, the natural substrate for this family of protein kinases is not
known, but some investigators found that PCTAIRE-1, and PCTAIRE-2 could
phosphorylate histone H1 (Hirose et al.,
1997
; Le Bouffant et al.,
1998
), whereas others only detected MBP phosphorylation
(Besset et al., 1999
;
Rhee and Wolgemuth, 1995
). The
levels of kinase activity, compared to CDK1 or protein kinase A (PKA), are
extremely low. This makes it difficult to be sure that the observed kinase
activity is due to PCTAIRE, rather than traces of contaminating protein
kinases. At least one of the antibodies used in studies of PCTAIRE recognises
a completely unrelated protein (Le
Bouffant et al., 1998
).
We originally came across PCTAIRE in an early screen for CDK homologues in X. laevis that revealed two frog homologues of PCTAIRE (R. Y. C. Poon, Cyclin-dependent kinase family and its regulation, PhD. thesis, University of Cambridge, 1993). The protein was only detectable after the start of neurogenesis, and had no detectable kinase activity when expressed in bacteria. We discovered the overexpression of PCTAIRE-1 in cell lines, and decided to revisit the subject, in case there might be a connection with cellular transformation. This paper presents our finding about the mode of activation and function of PCTAIRE-1 in cells. We present evidence that PCTAIRE-1 is regulated by PKA and may play a role in the control of neurite outgrowth.
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Materials and Methods |
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Expression of PCTAIRE-1 in E. coli and phosphorylation by
brain extracts
The PCTAIRE-1 constructs in pET 21d were transformed into BL21(DE3), and
the proteins were expressed and purified using nickel agarose according to
standard methods (Crowe et al.,
1994; Hochuli et al.,
1987
).
Lysates of brain or testis from mice were produced by homogenizing fresh
tissue in an equal volume of lysis buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1
mM EDTA, 0.1% NP-40, 1 mM DTT, 1 mM PMSF, 1 µg/ml pepstatin and leupeptin).
Aliquots of these extracts containing 100 µg of total protein were
incubated with bacterial extracts containing 1 µg of PCTAIRE-1 for 30
minutes at 30°C. The reaction was diluted with buffer A (20 mM Tris pH
7.4, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, 1 mM DTT), and the recombinant
PCTAIRE-1 recovered by affinity chromatography using monoclonal antibody 9E10
covalently coupled to Affi-Prep beads (BioRad)
(Schneider et al., 1982). The
beads were washed twice with buffer A, twice with buffer A plus 500 mM NaCl,
twice with buffer C (50 mM Tris pH 7.4, 15 mM MgCl2) and assayed
for kinase activity as described below.
To investigate the interaction of PCTAIRE-1 with GST-tagged 14-3-3 proteins
[produced as described (Dubois et al.,
1997)], the PCTAIRE-1 proteins were bound to 9E10 beads as
described above. The beads were then incubated with 1 unit of purified PKA
(Sigma) or buffer, washed twice with buffer A, incubated with purified
GST-tagged 14-3-3
or
, washed again with buffer A, and the bound
material was analysed by SDS-PAGE and immunoblotting using an antiserum
against GST.
Cell lines, transfections and immunoprecipitations
For most experiments involving expression in mammalian cells, the cell line
Neuro-2A (ATCC CCL-131) was used (Klebe and Ruddle, 1969). Transfections were
carried out using CaPO4 as described
(Ausubel et al., 1999).
Briefly, the precipitates were left on the cells for 16 hours, the medium was
replaced, and the cells were left for 48 hours to express the transfected
construct. Transfection efficiencies were generally around 30-50%. All other
cell lines were transfected with Superfect (Gibco) according to the
manufacturer's instructions.
For immunoprecipitations, the cells were extracted in lysis buffer, the
insoluble material was removed by centrifugation, and the YFP-tagged
PCTAIRE-1-constructs were immunoprecipitated using a rabbit serum against GFP
covalently coupled to Affi-Prep protein A beads with dimethylpimelimidate
(Schneider et al., 1982). The
material bound to the beads was washed twice with buffer A, twice with buffer
A plus 500 mM NaCl, and twice with buffer C.
Kinase assays
Proteins bound to beads were then assayed for kinase activity by incubating
them in 10 µl of buffer C containing 1 mg/ml myelin basic protein (MBP), 20
µM ATP, 1 mM DTT and 1 µCi [-32P]ATP for 30 minutes at
30°C.
Mass spectrometry analysis of phosphorylation sites
Recombinant PCTAIRE phosphorylated by PKA was loaded on a 12.5% SDS-PAGE to
separate the phosphorylated protein from ATP. PCTAIRE was visualised on the
gel by GelCODE (Pierce) staining and digested with trypsin as described
(Aitken and Learmonth,
1997).
High performance liquid chromatography-electrospray mass spectrometry
(LC-ESMS) was performed as follows. A modified Perkin Elmer/ABI 130A HPLC was
coupled to a Micromass Platform electrospray mass spectrometer. Chromatography
was performed with a Vydac C18 0.25x250 mm PEEK column (slurry packed
in-house) at a flow rate of 10 ml/minute over a 0-60% B linear gradient in 60
minutes [buffer A is 0.12% formic acid, 98% water and 2% acetonitrile (ACN);
buffer B is 10% formic acid, 15% water and 85% ACN] with in-line UV monitoring
at 215 nm. The low flow rate was achieved by incorporating a valco tee stream
splitter coupled to a microbore dummy column, just prior to the Rheodyne
injection. For the identification of the phosphorylation sites, reverse phase
chromatography was performed on a Beckman Gold HPLC using a 4.6x250 mm
Merck superspher 100 RP18 end-capped column at 0.8 ml/min and a 1%/minute
gradient [buffer A is 0.10% trifluoroacetic acid (TFA); buffer B is 0.10% TFA,
15% water and 85% ACN]. Fractions were dried then resuspended in 50% ACN,
0.10% formic acid and analysed by ESMS. The elution positions of the
32P-labeled peptides were determined by Cerenkov counting and the
phosphopeptide fractions were analysed by electrospray mass spectrometry as
described (Dubois et al.,
2001).
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Results |
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PCTAIRE-1 is phosphorylated by protein kinase A (PKA)
The recombinant PCTAIRE-1 phosphorylated by the tightly-bound kinase was
analysed by mass spectrometry, which identified two serine residues, S12
(KRQLSM) and S153 (RRVSL) as the major sites of phosphorylation
(Fig. 2A). Both sites conformed
to the recognition sequence for phosphorylation by PKA, R-R/K-X1-2-
hydrophobic-S/T, -R-X1-2-S/T-, or -R-R/K-X-S/T- hydrophobic, where
hydrophobic stands mainly for I, L or V residues. Inspection of the N-terminus
revealed two additional PKA sites, S110 (RKIST) and S119 (KRLSLP). We noted
that the latter site might form part of a 14-3-3 binding motif (RLpSLP),
consistent with the report that PCTAIRE-1 interacted with 14-3-3 proteins in a
2-hybrid screen (Sladeczek et al.,
1997).
|
In order to test whether PCTAIRE-1 was a substrate for PKA, we incubated bacterially expressed proteins with purified PKA. Mutants were prepared that had S119A/S153A, S12A/S110A/S153A or all four potential PKA phosphorylation sites replaced by alanines. Fig. 2B shows that wild-type PCTAIRE-1 was an excellent substrate for PKA, and addition of the PKA inhibitor, PKI, reduced the phosphorylation of PCTAIRE-1 to the background autophosphorylation level (lane 1). The quadruple mutant could no longer be phosphorylated (lane 5), but S12A/S110A/S153A was still weakly labelled, showing that PKA was able to phosphorylate PCTAIRE-1 on its putative 14-3-3 binding site, S119 (lane 4). However, it should be noted that S12 and S153 are much stronger phosphorylation sites under the conditions of this experiment, as found for the kinase derived from the extract. The N-terminus of PCTAIRE-1 has other potential phosphorylation sites, and we found evidence for the existence of additional contaminating protein kinase(s) when the protein was expressed in neuroblastoma cell lines. As this activity was unable to phosphorylate MBP, its origin was not explored further. However, we should note that the activity of these substoichiometric, tight-binding protein kinases was often comparable with that of PCTAIRE-1 itself, depending on the substrate used, and could give misleading results if misinterpreted.
PKA phosphorylation of PCTAIRE-1 on S153 inhibits its kinase
activity
Incubation of the bacterially expressed PCTAIRE-1 with PKA and ATP did not
stimulate the MBP kinase activity of the PCTAIRE-1 (data not shown). To check
whether PKA phosphorylation had an effect on the kinase activity of PCTAIRE-1,
wild-type, kinase-dead, and PKA phosphorylation-site mutated forms of
PCTAIRE-1 were introduced into Neuro-2A cells by transfection. Two days later,
the cells were harvested, lysed, and the YFP-fused PCTAIRE-1 was
immunoprecipitated with antibodies against GFP covalently coupled to Affi-prep
protein A beads. The beads were then assayed for kinase activity towards MBP.
Fig. 3A shows that wild-type
PCTAIRE-1 was active in this assay, whereas the K194R and YFP-alone controls
were not (compare lane 1 with 2 and 12). Three out of the four PKA
phosphorylation site mutants had somewhat reduced activity (S12A, S110A, and
S119A; lanes 3-5), but the S153A mutation increased the protein kinase
activity of the immunoprecipitated PCTAIRE-1 by at least three-fold (lane 6).
The increased activity of the S153A mutant can be accounted to PCTAIRE-1
kinase activity, as the S153A/K194R double mutant had no detectable protein
kinase activity (lane 10).
|
These results suggested that phosphorylation of PCTAIRE-1 on S153 might regulate its kinase activity. To check this, wild-type YFP-PCTAIRE-1 was transfected into Neuro-2A cells. After 48 hours, the cells were treated with 10 µM forskolin, which should increase PKA activity in the cells. Wild-type, S12A, S110A, S119A and S480A mutants showed about half the activity from forskolin-treated cells compared with the untreated control (Fig. 3B, compare lanes 1,3,4,5 with 8,10,11,12), whereas the S153A mutant showed the same increased activity with or without the forskolin treatment (lanes 6,13).
As a further test of these effects, YFP-PCTAIRE-1 was recovered from transfected Neuro-2A cells and incubated with purified PKA. Fig. 3C shows that wild-type PCTAIRE-1 kinase activity was reduced by 50%, whereas the S153A mutant, which was about twice as active as the wild-type enzyme, was not significantly affected by incubation with PKA.
Characterisation of the PCTAIRE-1 protein kinase
Previous investigators have reported that either MBP
(Le Bouffant et al., 1998) or
histone H1 (Rhee and Wolgemuth,
1995
) were substrates for PCTAIRE-1. To resolve this discrepancy,
we compared MBP, histone H1, casein, the C-terminal domain of RNA polymerase
II, c-jun and two neuronal forms of stathmin as substrates for the YFP-tagged
S153A mutant form of PCTAIRE-1 isolated from Neuro-2A cell lysates. Only MBP
and histone H1 were good substrates for this form of PCTAIRE-1
(Fig. 4A, and data not shown).
In our hands, MBP was a better substrate for PCTAIRE-1 than histone H1
(Fig. 4A, lanes 4,5) because,
as mentioned previously, contaminating protein kinases seemed to prefer
histone H1 over MBP.
|
Given a reliable source of PCTAIRE-1 and a reliable assay, we investigated the role of other parts of the molecule for its activity: in particular, the N-terminal extension and the role of S319 in the so-called activation or T-loop domain (the equivalent of T160 in CDK2). We produced two truncated versions of PCTAIRE-1 as YFP fusion proteins: the isolated N-terminus, residues 1-161-YFP, and the catalytic domain comprising residues 162-491-YFP. Neuro-2A cells were transfected with plasmids encoding these mutants as well as with wild-type, S153A and K194R full-length PCTAIRE-1-YFP. Fig. 4B shows that the S153A mutant displayed at least fourfold elevated kinase activity compared with the wild-type enzyme (lane 4), while both K194R and S319A mutants were completely inactive (lanes 2,3). The N-terminus of PCTAIRE-1 displayed significant autophosphorylation despite lacking a functional kinase domain, which must be due to a contaminating protein kinase that bound to the immunoprecipitate (lane 5). By contrast, the C-terminal portion of PCTAIRE-1, which includes the catalytic domain, was almost completely devoid of MBP kinase activity, and displayed no autophosphorylation (lane 6).
We next produced a series of N-terminal truncations of PCTAIRE-1. The
constructs were transfected into Neuro-2A cells, and assayed for MBP kinase
activity. Similar activity was shown by full-length and truncated versions up
to residue 71 (Fig. 4C, lanes
1-3), but a version starting at residue 111 showed enhanced activity towards
MBP, although this version was expressed at higher levels than the longer
constructs (lane 4). The version of PCTAIRE-1 lacking all 162 residues of the
N-terminus was catalytically inactive (lane 5). Thus, a minimum of 52 residues
of the N-terminus are required for kinase activity. This region is
well-conserved amongst all three PCTAIRE genes and contains both the potential
14-3-3-binding motif, and the phosphorylation site at S153. Lane 12 shows that
the N106/S153A mutant was about four times more active against MBP than
the N
106 form, confirming the importance of S153 as a potential
regulator of kinase activity.
Does PCTAIRE-1 kinase require a partner subunit?
Preparations of the PCTAIRE-1 protein kinase never contained significant
amounts of other polypeptides, suggesting that the N-terminus might be acting
as an intrinsic cyclin. By contrast, the kinase activity of PCTAIRE-1 was so
low that it was possible that only very low levels of a heterologous
activating subunit were present in the cells we were using as a source of the
activity. To measure the size of the PCTAIRE-1 kinase activity we investigated
the elution profile of endogenous PCTAIRE-1 in brain extracts by gel
filtration. The PCTAIRE-1 protein was immunoprecipitated from the individual
fractions using a monoclonal antibody specific for the N-terminus of
PCTAIRE-1, G6, and the bound material was assayed for kinase activity. The
reaction mix was then applied to an SDS-PAGE, and immunoblotted for PCTAIRE-1
using a polyclonal antibody against the C-terminus.
Fig. 5A shows the elution
pattern of PCTAIRE-1 on an AcA34 column, which suggested that some of the
PCTAIRE-1 protein was present in high molecular weight complexes, but the
active form of the enzyme eluted in the position expected for the monomeric
protein.
|
An alternative way to test whether the PCTAIRE-1 protein kinase required a
specific partner was to examine its activity in a variety of non-neuronall
cell lines. Indeed, Besset et al. found that PCTAIRE-1 expressed in COS-7
cells lacked detectable activity, which they ascribed, by analogy with CDK5,
to the absence of an activating partner subunit
(Besset et al., 1999).
Wild-type, K194R or the S153A mutants were introduced by transfection into a
variety of cell lines, and tested for MBP kinase activity.
Fig. 5B shows that in many cell
lines, the wild-type protein was essentially inactive as a protein. However,
the S153A mutant displayed significant kinase activity in all the cell lines
we tested, suggesting that the lack of PCTAIRE-1 kinase activity was due to
inactivation by PKA rather than a lack of a partner subunit
(Fig. 5B, lanes
3,6,9,12,15).
For a comparison with a related neuronal protein kinase that is known to require an activating subunit, YFP-tagged CDK5 was expressed in either Neuro-2A cells or HEK 293 cells. Fig. 5C shows that CDK5 was inactive unless its partner, in the form of bacterially expressed GST-p21nck, was added to the kinase assays. PCTAIRE-1 preferentially phosphorylated MBP, whereas CDK5 preferred histone H1, and the kinase activity of the S153A mutant was comparable with that of the activated CDK5.
The RLpS119LP motif is a 14-3-3-binding site in vitro and
in vivo
There is one report about a potential regulatory subunit for PCTAIRE-1:
Sladeczek and co-workers reported that the PCTAIRE-1 protein interacted with
14-3-3 proteins (Sladeczek et al.,
1997). The motif surrounding S119 (RLS*LP) might
present a phosphorylation-dependent 14-3-3 protein-binding site. The ability
of PKA to phosphorylate PCTAIRE-1 on S119 enabled us to check whether this
created a 14-3-3 protein-binding site. We bound wild-type recombinant
PCTAIRE-1 or the S119A mutant to 9E10 beads, incubated an aliquot of each with
PKA and ATP, and finally added recombinant GST-14-3-3
and
proteins. The beads were washed, and the bound material was analysed by
SDS-PAGE followed by immunoblotting using antiserum against GST. Both 14-3-3
isoforms bound to the PCTAIRE-1 protein in a phosphorylation-dependent manner
(Fig. 6A). To check whether
PCTAIRE-1 bound endogenous 14-3-3 proteins in vivo, neuroblastoma cells were
transfected with constructs encoding various YFP-tagged forms of wild-type or
mutant PCTAIRE-1. Fig. 6B shows
that anti-GFP immunoprecipitates from lysates of cells expressing wild-type or
K194R PCTAIRE-1 contained 14-3-3 proteins, which were absent from the
immunoprecipitates of cells expressing the S119A mutant (compare lanes 1 and 2
with lane 3). Replacing S119 by aspartic acid to mimic a phosphorylated
residue did not restore the binding (lane 4). Surprisingly, mutation of S153
also inhibited binding of 14-3-3 proteins (lane 5). By contrast, mutation of a
serine residue in a second putative 14-3-3 binding site, S480, did not
significantly affect 14-3-3 binding (lane 6).
|
However, it is unlikely that binding of 14-3-3 proteins regulated the kinase activity of PCTAIRE-1 directly, given the results shown previously in Fig. 3A. Both S119A and S153A mutants lost the ability to bind 14-3-3 proteins, yet the former mutation slightly inhibited and the latter strongly stimulated the kinase activity of PCTAIRE-1 (Fig. 3A, compare lanes 5 and 6).
Effect of transfected PCTAIRE-1 constructs on neurite outgrowth of
Neuro-2A cells
Despite the lack of an effect on kinase activity, binding of 14-3-3
proteins might still play a role in the regulation of PCTAIRE-1 function in
cells. Since PCTAIRE-1 is found mainly in brain and testis, we used the
neuroblastoma cell line Neuro-2A for these examinations. Neuro-2A cells were
transfected with a variety of PCTAIRE-1-YFP constructs and the effects on the
morphology or the cell cycle distribution of the cells were examined 2 days
later. No change was caused in the cell cycle distribution, as determined by
FACS analysis, by any of the transfected constructs (data not shown). However,
when the cells were scored for neurite outgrowth (cells with neurite
extensions longer than twice the size of the cell body were scored positive),
we noticed that the K194R mutant induced neurite outgrowth in about 10 percent
of the transfected cells (Fig.
7A, lane 2). A similar, but less pronounced effect could also be
observed in cells transfected with wild-type PCTAIRE-1.
|
It is known that addition of forskolin to the medium tends to induce
neurite outgrowth in a neuroblastoma cell line
(Shea et al., 1992). Given the
apparent link between PCTAIRE-1 and PKA, we checked the combined effects of
forskolin and PCTAIRE-1 expression by adding the drug to cells 48 hours after
transfection. After a further 16 hours of incubation, 5-20% of the transfected
cells produced neurites, except for the cells transfected with the double
mutant S119A/S153A, which were refractory to the neurite-inducing effect of
forskolin (Fig. 7A, lane 12,
and 7B). However, cells
expressing the kinase-inactive S119A/S153A/K194R triple mutant were able to
produce neurites under these conditions, suggesting that the inhibition of
neurite outgrowth observed with the kinase active S119A/S153A form might be
attributed to PCTAIRE-1 kinase activity
(Fig. 7A, lanes 12,13).
In the Neuro-2A cells, PCTAIRE-1 is largely excluded from the nucleus, and
presumably exerts its effects on neurite outgrowth as a cytoplasmic kinase
[(Charrasse et al., 1999) (and
our own observations)]. We produced constructs to target PCTAIRE-1 to the
nucleus by adding the nuclear localisation signal of nucleoplasmin to the
N-terminus of wild-type PCTAIRE-1 and the S119A/S153A mutant form. The
PCTAIRE-1 constructs were co-transfected with a membrane-localised form of CD2
(Lassus and Hibner, 1998
), in
order to be able to determine the morphology of the transfected cells. After
48 hours, the cells were treated for 12 hours with forskolin to induce PKA and
neurite outgrowth. Fig. 8A,B
shows that cells transfected with wild-type, non-nuclear PCTAIRE-1 readily
produced extensions (lane 1), whereas the cells expressing the non-nuclear
S119A/S153A mutant produced about 70% fewer outgrowths, which were also
shorter (lane 2). When PCTAIRE-1 was targeted to the nucleus, however, the
difference between wild-type and S119A/S153A mutant forms was abolished
(Fig. 8A, lanes 3,4, and
8B), suggesting that the
activated form of PCTAIRE-1 must be located in the cytoplasm to exert its
inhibitory effect on neurite outgrowth.
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Discussion |
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When PCTAIRE-1 was expressed in mammalian cell lines, it displayed higher kinase activity than was observed in bacteria, which allowed partial characterisation of its function. Thus, the conserved lysine 194 in the ATP-binding pocket was essential for activity. Truncation of the N-terminus beyond residue 106 also produced an inactive form of the kinase, suggesting that a minimal 50-residue N-terminal extension, which is highly conserved amongst all three PCTAIREs (Fig. 9), was required for PCTAIRE-1 activity. We suspect that this N-terminal sequence may provide the activation that cyclins normally provide to the rest of the CDK family. Neither we nor others have ever been able to identify an activating binding partner, and size exclusion chromatography by gel filtration indicated that monomeric PCTAIRE-1 was active as a protein kinase, although (1) its partner might be small, and (2) perhaps, if a partner were to be found, the activity of PCTAIRE-1 as a protein kinase might be very much enhanced. However, we found that the activity we were able to detect was comparable with that of CDK5 when associated with its partner.
Two other sites, S110 and S119 were also phosphorylated when the
bacterially expressed protein was incubated in the extracts, and when purified
PKA was added. Phosphorylation of S119 generated a 14-3-3 binding site that is
functional in vitro when using purified proteins, as well as when the protein
is expressed in cells. However, as shown in
Fig. 6B, lane 5, mutation of
S153 to alanine also compromised the 14-3-3 binding capability of PCTAIRE-1,
suggesting an interaction of the two sites. In at least some cases, for full
functionality the dimer of 14-3-3 is essential (reviewed by
Tzivion and Avruch, 2002;
Aitken, 2002
) and this may also
lead to 14-3-3 isoform preference due to the interaction of residues further
away from the immediate phosphoserine (or -threonine) ligand-binding site. The
binding to a dimer of 14-3-3 through two sites on a protein that might be
individually of relatively low affinity could greatly increase target protein
specificity of recognition. For example, a double peptide containing a repeat
of a mode 1 phosphopeptide (Yaffe et al.,
1997
) linked by a hexa-repeat of 6-aminohexanoic acid has been
shown to bind across a 14-3-3 dimer and span the binding grooves, which
enhances binding by more than 30-fold because of cooperative binding. In
addition, the R18 (non-phosphorylated peptide motif linked by an 11-mer
peptide) has also been shown to bind very efficiently to the groove in 14-3-3
(Masters and Fu, 2001
).
There is a lot of flexibility in these loops and so the exact length may
not be important, but it is interesting to note that the spacing between the
phosphorylated serines in PCTAIRE-1 (residues S119 and S153) is similar to
that in the above double peptides (Fig.
9B). This hypothesis is also supported by the recent elucidation
of the arylamine N-acetyl transferase (AANAT) structure in complex with
14-3-3, where two molecules of AANAT bind to a dimeric 14-3-3
(Ganguly et al., 2001)
resulting in enhanced enzymic activity. It may also be noteworthy that both
the PCTAIRE-1 phosphoserines are in LSL and VSL sequences, which resemble the
high affinity non-phospho-interaction motif DALDL in ExoS
(Hallberg, 2002
) and WLDL in
peptide R18 (Masters and Fu,
2001
).
What is the function of PCTAIRE-1 in the brain? And is its expression in
immortalized cell lines of any significance? In contrast to CDK5, which is
expressed in many cell lines without its activating partner and is therefore
inactive, we believe (as argued above) that PCTAIRE-1 is potentially active.
Although Charrasse et al. suggested a cell-cycle-dependent regulation of
PCTAIRE-1 kinase activity (Charrasse et
al., 1999), we were unable to detect any significant differences
in the cell cycle distribution attributable to either wild-type or mutant
PCTAIRE-1, using a variety of cell lines expressing wild-type or kinase-dead
PCTAIRE-1. We also tested the effects of expression of the PKA-site mutants,
but again there was no detectable effect on the cell cycle. The related
PITSLRE protein kinases were shown to be activated during the process of
apoptosis (Lahti et al.,
1995a
). When 293 cell lines expressing various forms of PCTAIRE-1
were deprived of serum, they underwent apoptosis irrespective of the PCTAIRE
levels and there was no evidence to support a role of PCTAIRE-1 in the
induction or execution of apoptosis. The protein kinase activity of PCTAIRE
did not change upon induction of apoptosis (data not shown).
We next examined the effects of manipulating PCTAIRE levels in neuronal cells, which are the natural source of PCTAIRE. The induction of neurite outgrowth in neuronal cell lines is normally preceded by an exit of the cells from the cell cycle. When Neuro-2A cells were transfected with cDNAs encoding either wild-type, kinase-dead or activated forms of PCTAIRE, there was no effect on the FACS profile of the cell cycle distribution; that is, cells were neither inhibited from cell cycle exit, nor did any form of PCTAIRE promote such exit. By contrast, reproducible effects on neurite outgrowth were observed, although their interpretation is complex. In Neuro-2A cells, addition of forskolin to the medium tends to promote neurite outgrowth, presumably as a result of protein kinase A activation. This stimulation was almost completely abolished by introduction of an activated form of PCTAIRE-1 that contains mutations of S119 and S153 to alanine. S119 lies in a 14-3-3-binding site, and its mutation to alanine seriously compromised the ability of PCTAIRE-1 to bind 14-3-3 proteins. We also showed that PKA could phosphorylate S119 and thereby generate a strong 14-3-3 protein-binding site. PKA also phosphorylated S153, which decreased the kinase activity of PCTAIRE-1. Thus, the kinase activity of the doubly mutant S119A/S153A form of PCTAIRE-1 should be unaffected by cAMP levels in the cell. Whether the inhibition of neurite outgrowth by this form of PCTAIRE is significant or not is difficult to say. We also observed a reproducible stimulation of neurite outgrowth by forms of PCTAIRE-1 that contained active 14-3-3-binding sites and, significantly, kinase dead forms of PCTAIRE-1 were somewhat more active than the wild-type in this respect. The simplest interpretation of the effects of PCTAIRE-1 on Neuro-2A cells would be that sequestration of 14-3-3 proteins tends to promote neurite outgrowth, whereas active PCTAIRE-1 tends to inhibit it. One way to think about this would be to propose that these cells contain a repressor of neurite outgrowth that is active when bound to 14-3-3 proteins. Raising PCTAIRE levels would sequester some of the 14-3-3 proteins, and reduce the activity of this repressor. But PCTAIRE kinase activity must also act, either to activate the repressor independently of the effects on 14-3-3 proteins or to inhibit the (implied) activator of neurite outgrowth. Until more is understood about the underlying control of neurite outgrowth, however, these models only help us understand the rather complex data.
It is intriguing that PCTAIRE-1 is normally found in cells that contain stable microtubules, neurons and sperm. In the Neuro-2A cells expressing high levels of activated PCTAIRE, time-lapse movies of the cells suggested that they were `trying' to build neurites, but were unable to stabilise the processes they put out, as though PCTAIRE-1 antagonised the formation of stable microtubules. At first sight, these are contradictory observations, but it may be that the presence of the correct level of PCTAIRE permits the remodelling of stable microtubules that probably occurs during their assembly. What is required, of course, is a mouse lacking PCTAIRE, but although we were able to generate (male) ES cells lacking PCTAIRE-1, they never showed germline transmission (data not shown). One explanation for this failure could be that the enzyme is required for the formation of viable sperm, although other explanations are feasible.
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