Max Planck Institute for Biophysical Chemistry, Department of
Biochemistry, Am Fassberg 11, 37077 Goettingen, Germany
* Present address: Department of Molecular and Cell Biology, University of
California, Berkeley, CA 94720, USA
Author for correspondence (e-mail:
office.weber{at}mpibpc.gwdg.de)
Accepted 12 September 2002
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Summary |
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Key words: Crithidia, NIMA kinases, Polyglutamylation, Tubulin, Post-translational modification, Cell cycle
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Introduction |
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Polyglutamylation (Eddé et al.,
1990) and polyglycylation
(Redeker et al., 1994
) are two
unusual protein modifications consisting of the stepwise addition of glutamate
or glycine residues linked via isopeptide bonds to specific glutamate residues
in the carboxy termini of
and ß tubulin. Recently, Regnard et al.
showed that the nucleosome assembly proteins NAP-1 and -2 are also modified by
polyglutamylation (Regnard et al.,
2000
). Both PTMs are evolutionary conserved and already found in
tubulins from primitive protists such as Giardia lamblia
(Weber et al., 1997
), but they
are absent from yeast. While glutamylation is a general tubulin modification,
the occurrence of glycylation seems to be restricted to cells that have either
cilia or flagella. The functional importance of both polymodifications of
tubulin has recently been highlighted by a number of experiments (reviewed by
Rosenbaum, 2000
). Xia et al.,
using in vivo mutagenesis of the modified glutamate residues, showed that
polyglycylation of ß-tubulin is essential in Tetrahymena
(Xia et al., 2000
). The
lethality of the polyglycylation deficiency was due to a failure in assembling
functional axonemes and a defect in cytokinesis caused by an incomplete
severing of microtubules (Thazhath et al.,
2002
). Interestingly, trypanosomes have normal axonemes although
they completely lack polyglycylation and are instead highly modified by
tubulin polyglutamylation (Schneider et
al., 1997
).
In non-neuronal cells polyglutamylation is largely restricted to the
microtubules of the centrioles, the mitotic spindle and the midbody
(Bobinnec et al., 1998a).
Introduction of the glutamylation specific antibody GT335 into HeLa cells
caused a disassembly of centrioles and a transient disappearance of the
centrosome as a defined organelle (Bobinnec
et al., 1998b
). Tubulin polyglutamylase activity as well as the
overall level of glutamylated tubulin were shown to be under cell-cycle
control (Regnard et al., 1999
)
and a specific increase in ß-tubulin glutamylation was observed during
mitosis (Bobinnec et al.,
1998a
). The GT335 antibody also interfered with the motility of
reactivated sperm axonemes, suggesting a function for tubulin
polyglutamylation in flagellar motility
(Gagnon et al., 1996
). In
vitro assays with blot overlays indicate that the length of the polyglutamyl
side chain can differentially regulate the binding of microtubule associated
proteins (Bonnet et al., 2001
;
Boucher et al., 1994
).
Moreover, the processiviy of conventional and single-headed kinesins were
shown to be regulated by the interaction between conserved basic residues of
the motor proteins with the acidic C-terminus of tubulin
(Thorn et al., 2000
;
Okada and Hirokawa, 2000
).
Of the various enzymes involved in tubulin PTMs, so far only the tubulin
tyrosine ligase TTL (Ersfeld et al.,
1993) and the mirotubule associated deacetylase HDAC6
(Hubbert et al., 2002
) have
been cloned. While HDAC6 is a member of the histone deacetylase family, TTL
shares a fold with the glutathione synthetase ADP-forming family
(Dideberg and Bertrand, 1998
).
We have turned to trypanosomes as a starting material for the purification of
a tubulin polyglutamylase. The subpellicular and flagellar microtubules of
trypanosomatids are extensively glutamylated
(Schneider et al., 1997
) and
isolated cytoskeletons, obtained by detergent extraction, retain an enzymatic
activity that incorporates glutamic acid into tubulin in an ATP-dependent
manner (Westermann et al.,
1999a
). A tubulin polyglutamylase preparation from
Crithidia accepts mammalian brain tubulin as a substrate and is also
able to modify synthetic peptides representing the C-terminal residues of
and ß tubulin (Westermann et
al., 1999b
).
Here we describe the cloning of the major component of a highly purified tubulin polyglutamylase preparation. We identify a 54 kDa polypeptide copurifying with tubulin glutamylation activity from Crithidia as a novel member of the NIMA family of putative cell cycle regulators. This finding is especially intriguing since NIMA related kinases from different organisms have been implicated in various aspects of microtubule organisation yet their molecular mechanism of action has largely remained elusive.
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Materials and Methods |
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Purification of tubulin polyglutamylase
Isolation of Crithidia cytoskeletons, solubilisation of tubulin
glutamylation activity by salt extraction and subsequent purification steps
were as described by Westermann et al.
(Westermann et al., 1999a)
with certain modifications. The purification was scaled up so that 9 l of
Crithidia culture were typically used to generate the crude enzyme
fraction. For ATP-affinity chromatography on a 4 ml Sepharose-sebacic acid
ATP-column, the crude enzyme fractions obtained from three preparations (27 l
of Crithtidia culture) were pooled. As the final purification step,
glycerol gradient fractions containing glutamylation acitivity were pooled
(
5 ml) and dialyzed against 10 mM sodium phosphate pH 6.8, 1 mM
MgCl2, 1 mM DTT for 1 hour. The partially purified glutamylase was
applied on a 1 ml CHT-2 hydroxyapatite column (Bio-Rad, Munich, Germany) at a
flow rate of 0.6 ml/minute. The column was developed with a 16 ml linear
gradient from 10 mM to 400 mM sodium phosphate, 0.5 ml fractions were
collected and 10 µl aliquots were tested for glutamylation activity.
Fractions containing glutamylation activity were supplemented with 10%
glycerol and 0.1 mg/ml soybean trypsin inhibitor (Sigma, Deisenhofen,
Germany), quick frozen in liquid nitrogen and stored at -80°C.
Cloning of p54
Multiple preparations of tubulin polyglutamylase were used to excise the 54
kD band from SDS-polyacrylamide gels. The peptides resulting from an in situ
digest with endoproteinase LysC were separated by reversed phase HPLC and
sequenced by automated Edman degradation.
Total RNA was isolated from logarithmically growing Crithidia cells using the TRIzol reagent (Gibco-BRL, Karlsruhe, Germany). To obtain the corresponding cDNA, RT-PCR was performed using Superscript reverse transcriptase (Gibco-BRL) and an oligo-(dT) primer.
The sequence information from the p54 peptides was used to synthesise
degenerate antisense primers that were used in different combinations in PCRs
against a spliced leader primer (SL:
5'-CGCTATAAATAAGTATCAGTTTCTGTACTTTATTG-3') that is directed
against a common sequence at the 5' end of all Crithidia mRNAs
(Muhich et al., 1987). The
following combination gave a correct PCR product: In the first PCR round the
primer SW 46 (5'-ACYTCIGGIACIGGRAANACRTC-3' with R=A/G, Y=T/C,
N=A/G/T/C, based on peptide DVFPVPEV) was used against SL and the product was
reamplified using the nested Primer SW 42
(5'-TCYTTIGGIGGIATNGTYTCNGA-3' based on peptide SETIPPKD). The
resulting product was gel-purified, cloned into vector pCR2.1 (Invitrogen,
Groningen, The Netherlands) and custom-sequenced (MWG-Biotech, Ebersberg,
Germany). Based on the established 5' sequence, two gene-specific
primers, SW 47 (5'-AGCATGAAGGCGCTGCTGGACCCGC-3') and SW 48
(5'-CGACGCAGCAGCTGCTCCAGACAGAG-3') were synthesized and used in a
3'-RACE-PCR (Gibco-BRL) to obtain the 3' end of the p54 cDNA.
Expression of recombinant CfNek in Crithidia
The full length p54 cDNA was amplified using primers pNus1
(5'-CAGGGAAACATATGCCGACCAACAAGGATGACAAG-3') and pNus2
(5'-CCTCGGTACCT-CACATGCCACAGGCGCGGTGAAAC-3',
restricition sites for NdeI and KpnI underlined) and
inserted into the respective site of the pNUS-HnH expression vector
(Tetaud et al., 2002). The
vector contains an N-terminal His-tag sequence and a hygromycin resistance
gene for the selection of transfected parasites.
Electroporation of Crithidia cells was performed as described
(Tetaud et al., 2002):
Parasites were washed twice in cold BHI-Medium and resuspended at a density of
2x107 cells/ml in a 0.4 cm electroporation cuvette containing
30 µg plasmid-DNA. The gene pulser apparatus (Bio-Rad, Munich, Germany) was
set to 450 V and 500 µF capacitance. The mixture was subjected to a single
pulse, and after 10 minutes on ice the cells were transferred to 5 ml
BHI-medium containing 10% FCS and incubated 5 hours at 26°C to allow for
recovery. Hygromycin B (Gibco-BRL, Karlsruhe, Germany) was added at a final
concentration of 50 µg/ml and after one week the cells were subcultured
into BHI-medium containing 200 µg/ml hygromycin.
Purification of His-tagged CfNek
Hygromycin resistant cells were used to prepare a crude enzyme fraction as
described previously (Westermann et al.,
1999a). The 0.25 M salt extract was diluted 1:1 with 20 mM Tris pH
8.5, 500 mM KCl, 20 mM imidazole, 5 mM 2-mercaptoethanol, 10% (v/v) glycerol
and applied at a flow rate of 0.5 ml/minute on to a 0.5 ml Ni-NTA (Qiagen,
Hilden, Germany) column equilibrated with the same buffer. The column was
washed with 20 mM Tris-HCl pH 8.5, 1 M KCl, 5 mM 2-mercaptoethanol, 10%
glycerol. Finally, the His-tagged protein was cleaved from the column by
incubating the resin overnight at 4°C with 20 U thrombin (Amersham
Pharmacia, Freiburg, Germany) in 20 mM Tris-HCl pH 8.5, 1 mM MgCl2,
1 mM 2-mercaptoethanol, 10% glycerol.
Tubulin polyglutamylase and kinase assays
Tubulin polyglutamylase activity was measured as described
(Westermann et al., 1999a)
using the filter disc method and brain tubulin as a substrate.
Kinase reactions (25 µl) were carried out in a standard kinase buffer
containing 20 mM Tris-HCl pH 7.5, 50 mM KCl, 10 mM MgCl2 and 5
µCi [-32P]ATP (3000 Ci/mmol, Hartmann Analytic,
Braunschweig, Germany). As an artificial substrate ß-casein (Sigma,
Deisenhofen, Germany) was used at a final concentration of 1 mg/ml. The kinase
reactions were incubated at 30°C for 30 minutes, stopped by the addition
of SDS sample buffer and loaded on a 10% SDS polyacrylamide gel. The gel was
dried and exposed overnight for autoradiograpy.
Immunofluorescence of Crithidia cells
Crithidia cells were pelleted, washed twice with PBS and spotted
(25 µl, 5x105 cells) on poly-L-lysine coated cover slips.
Cells were allowed to adhere for 10 minutes and then fixed in methanol at
-20°C for 1 hour. Slides were rehydrated in PBS and incubated with primary
antibody (Penta-His Antibody, Qiagen, Hilden, Germany, diluted 1:33 in 0.5
mg/ml BSA in PBS) for 1 hour at 37°C. Slides were washed three times with
PBS and then incubated with secondary antibody (goat anti mouse
rhodamine-conjugated, DAKO, diluted 1:80) for 45 minutes. Slides were given
three 5 minute washes in PBS and DNA was stained with Hoechst 33342. Finally,
the slides were mounted in Mowiol and examined with a Zeiss Axiophot
microscope.
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Results |
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Cloning of the p54-cDNA
We obtained amino acid sequences of internal p54 peptides and used them to
design degenerate antisense oligonucleotide primers. To clone the 5' end
of the p54-cDNA these primers were used in combination with a spliced leader
primer in a RT-PCR with total RNA as a template
(Fig. 2A). The established
nucleotide sequence was then used to design specific sense primers for a
conventional 3'RACE-PCR to obtain the full-length cDNA. The p54 cDNA
structure is shown in Fig. 2A:
the spliced leader sequence is followed by a 423 bp long 5' UTR which
contains 3 stop codons in the same reading frame as the starting methionine.
The open reading frame spans 1437 nucleotides and is followed by a 1.7 kb long
3' UTR and the polyA-tail. Conceptual translation of the open reading
frame predicts a protein of 479 amino acid residues with a calculated
molecular weight of 54.9 kDa which is in good agreement with the observed size
of the protein in SDS-PAGE. The isoelectric point of the protein is pI=6.9
(Fig. 2B) and the amino acid
sequence contains all 10 peptide sequences originally obtained from p54. The
cDNA sequence and the amino acid sequence of p54 are deposited in GenBank
under Acc.No. AJ494838.
|
p54 is a NIMA related kinase
A Blast search of the Crithidia p54 amino acid sequence revealed a
high homology to a predicted protein sequence obtained during the genome
project of the related trypanosomatid Leishmania major (acc. no.
QKT3). This sequence, annotated as Leishmania-NIMA related-Kinase 1
(LNK-1), showed 88.6% identity to Crithidia p54 over the entire
polypeptide. Both proteins contain a serine/threonine kinase domain (residues
70 to 325 of p54) related to the catalytic domain of the NIMA protein, which
was originally identified as a mitotic regulator necessary for G2/M transition
in Aspergillus nidulans (Osmani
et al., 1988). The domain structure of Crithidia p54
(which we named CfNek for Crithidia fasciculata
NIMA-related kinase) and Aspergillus
NIMA is shown in Fig. 2C. The
similarity between both proteins is largely restricted to the catalytic domain
(36% identity), but both proteins contain PEST-sequences commonly found in
proteins targeted for rapid degradation
(Rechsteiner and Rogers,
1996
). The PEST sequence of CfNek is located within the
aminoterminal domain of the protein (residues 54 to 65) while the two PEST
sequences of NIMA reside in the carboxyterminal extension. Both proteins share
basic C-terminal domains (residues 325-479 of CfNek have a pI of 9.6) which
are a structural feature of all NIMA-related kinases. The C-terminal domain of
CfNek also harbours a pleckstrin homology (PH) domain which is found in a
number of proteins implicated in intracellular signalling.
Next to the Leishmania kinase, p54 is most closely related to
NrkA, a NIMA related kinase from Trypanosoma brucei
(Gale and Parsons, 1993). The
two proteins display 46% sequence identity over the catalytic domain, both
contain the PH domain at the C-terminal end, but NrkA lacks a PEST-sequence.
Fig. 3A shows an alignment of
the catalytic domain of CfNek with NIMA related kinases from different
species. Strikingly, the Crithidia and the Leishmania
proteins lack one of the characteristic features of protein kinases, namely
the glycine rich loop GXGXXG in kinase subdomain I. This sequence is involved
in the binding and orientiation of ATP
(Hanks and Hunter, 1995
). The
catalytic loop RDXXXXN in subdomain VI, on the other hand, is completely
conserved in all Neks. While Neks normally have their catalytic domain at the
extreme N-terminus, the catalytic domains of CfNek and LNK-1 start 70 residues
downstream of the N-terminus. A phylogenetic analysis of the kinase domains
shows that the three trypanosomatid kinases cluster into a subfamily within
the NIMA group (Fig. 3B).
|
Native glutamylase fractions display phosphorylation activity
The finding that p54 is a NIMA-related kinase prompted us to investigate
the kinase activity of our tubulin polyglutamylase preparation. While few
physiological targets of Neks are known, ß-casein is a good artificicial
substrate for several NIMA related kinases
(Lu et al., 1993). Native
glutamylase fractions from the final hydroxyapaptite chromatography were
assayed for kinase activity. Fig.
4 shows that these fractions displayed ß-casein
phosphorylation activity. Moreover, the kinase activity profile exactly
mirrored the glutamylation activity profile of the corresponding fractions
from the hydroxyapatite column.
|
Expression and localization of His-tagged CfNek in
Crithidia
The complete CfNek cDNA was expressed in E. coli as well as in SF9
cells using a recombinant baculovirus. The recombinant proteins were purified
and assayed for glutamylation and phosphorylation activity. In contrast to the
native glutamylase fraction from Crithidia, neither bacterially nor
baculovirus expressed recombinant CfNek displayed casein-phosphorylation or
tubulin glutamylation activity (data not shown). We therefore turned to a
recently described expression vector for Crithidia fasciculata
(Tetaud et al., 2002). The
pNusHnH vector drives the expression of introduced genes while it is
maintained as an extrachromosomal plasmid (episome) which confers resistance
to the antibiotic hygromycin.
The CfNek cDNA was cloned into pNusHnH in frame with a N-terminal poly His-sequence. Parasites were transfected by electroporation and selected in liquid culture with 200 µg/ml hygromycine. His6-CfNek expressing cells, as well as control cells transfected with vector only, were examined by immunofluorescence microscopy using a monoclonal His-tag antibody. Fig. 5 shows that only CfNek expressing parasites displayed a characteristic staining with the anti-His antibody. The cells were labelled strongly at the point where the flagellum is attached to the cell body. In some cells the staining ran along the length of the flagellum but was always stronger at the base than at the tip. An immunblot performed on crude extracts showed that the antibody detected a protein of of the expected size only in the cells transfected with CfNek (Fig. 5C). We conclude that, as judged by immunofluorescence, the recombinant CfNek expressed in Crithidia seems to localise to the flagellar attachment zone and presumably to the basal body.
|
Purification of His6-CfNek from Crithidia
We used the introduced His-tag to purify recombinant CfNek from
Crithidia cells. A crude enzyme fraction obtained from 6 1 of CfNek
expressing cells was applied on to a Ni2+ column. The resin was
washed with 1 M NaCl and the recombinant protein was eluted by thrombin
cleavage, which leaves the His-tag bound to the column while the remainder of
the protein is found in the eluate. Fig.
6A shows a silver stained gel of the thrombin eluate from CfNek
expressing and wild type cells. While a contaminating triple band of about 98
kDa was found in both preparations, only the CfNek eluate displayed a protein
with the expected size of 55 kDa. Both eluates were assayed for kinase
activity and only the CfNek eluate showed significant ß-casein
phosphorylation activity (Fig.
6B). We also investigated the tubulin polyglutamylation activity
of both fractions. Only the CfNek containing eluate catalyzed the
incorporation of glutamic acid into TCA-precipitable tubulin
(Fig. 6C). To confirm that the
observed incorporation of radioactivity was due to tubulin polyglutamylation a
series of assays were conducted in which different components of the reaction
mixture were omitted. As expected the incorporation of glutamic acid was
dependent on the presence of the enzyme fraction, tubulin and ATP
(Fig. 6D).
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Discussion |
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The candidate glutamylase is a NIMA related kinase
Cloning of the p54 cDNA clearly identified the enzyme as a novel
trypanosomal member of the NIMA family of mitotic kinases (CfNek). The
original NIMA enzyme from Aspergillus was identified as a factor
critical for G2/M transition (Osmani et
al., 1988). Detailed analysis of NIMA function in
Aspergillus revealed that elevated NIMA activity, dependent on
phosphorylation by p34cdc2/cyclinB
(Ye et al., 1995
) is necessary
for the correct organization of spindle microtubules and for the integrity of
the nuclear envelope (Osmani et al.,
1991
). Recent evidence suggests that NIMA is a mitotic histone H3
kinase, which also localises to spindle microtubules and spindle pole bodies
(DeSouza et al., 2000
). The
fact that overexpression of NIMA in mammalian cells caused premature mitotic
events such as chromosome condensation (Lu
and Hunter, 1995
) suggested the existence of a NIMA like pathway
in vertebrates. The relationship between NIMA and several mammlian Neks,
however, has remained unclear, as the only protein that could complement the
original nimA-mutation, was nim-1 from the related fungus
Neurospora crassa (Pu et al.,
1995
). Of the various mammalian Neks, Nek2 is most closely related
to NIMA (Fig. 3B) and part of
its function affects the centrosome cycle as overexpression causes premature
splitting of the centrosome while expression of dominant negative Nek2 leads
to loss of centrosome integrity (Fry et
al., 1998
). A centrosome-related function has also been reported
for the Xenopus Nek2 homolog (Uto
and Sagata, 2000
; Fry et al.,
2000
) and another related kinase is involved in the formation of
microtubule organizing centres in Dictyostelium
(Gräf, 2002
).
Crithidia p54 has a clear homolog in the predicted Leishmania
major protein LNK-1 (Fig.
3A), while the similarity to Trypanosoma brucei NrkA is
significantly reduced, leaving the question open whether these proteins are
true homologs. All three trypanosomatid proteins, however, share the
pleckstrin homology domain at the C-terminus. The unusual catalytic domain
which lacks the glycine-rich loop in subdomain I is restricted to the
Crithidia and the Leishmania protein
(Fig. 3A). We do not know
whether this unique sequence has consequences for the catalytic activity, but
we note that a special ATP-affinity resin with a long spacer had to be used
for the purification of the glutamylation activity
(Westermann et al.,
1999a).
Properties of recombinant CfNek expressed in Crithidia
When expressed in E. coli or insect cells we did not observe
phosphorylation or glutamylation activity of the purified recombinant CfNek.
The failure to obtain an active enzyme by expression in a heterologous system
could be due to a misfolded protein or to the lack of some activation step.
NIMA for example has been shown to depend on the phosphorylation by
p34cdc2/cyclinB for full enzymatic activity
(Ye et al., 1995). As judged
by immunofluorescence, only upon expression in Crithidia was a
specific localisation of the recombinant protein to the basal body/flagellar
attachment zone of the parasite observed. Future studies, involving the
generation of antibodies and the use of immuno-electronmicroscopy on extracted
cytoskeletons, will have to establish the precise localisation of the
endogenous CfNek. When the enzyme was expressed in HeLa cells only a
cytoplasmic staining was seen in immunofluorescence (our unpublished
observations). More importantly, basal bodies are structures homologus to
centrioles and are known to contain highly glutamylated microtubules
(Geimer et al., 1997
). We also
observed CfNek staining along the flagellum which is interesting in light of
the fact that a polyglycylation deficient mutant in Tetrahymena shows
defects in axonemal architecture with large gaps in the flagellar transition
zone (Thazhath et al., 2002
).
As trypanosomes lack glycylated tubulins
(Schneider et al., 1997
) but
have functional axonemes, it seems likely that they compensate for the
essential function of tubulin glycylation by generating a high level of
tubulin glutamylation.
We did not observe any obvious effects on growth rate or motility of the
Crithidia cells when expressing the recombinant CfNek. The pNus
expression vector lacks promotor sequences and therefore the introduced gene
is expressed only at a moderate level
(Tetaud et al., 2002). With
the future development of novel vectors allowing overexpression of genes in
Crithidia it will be possible to investigate the consequences of a
highly increased CfNek concentration.
Possible relationship between glutamylation and NIMA related
kinases
Surprisingly, the amino acid sequence identifies the p54 polypeptide
copurifying with glutamylation activity as a phosphotransferase and
recombinant CfNek displays casein-phosphorylation activity. As we were unable
to obtain an active enzyme preparation by expression in a heterologous system,
we cannot rule out the possibility that CfNek does not directly catalyse the
glutamylation reaction but that the actual glutamylase is instead associated
with CfNek and possibly regulated through phosphorylation. On the other hand,
it is tempting to speculate that the glutamylation reaction could directly
require the phosphotransferase activity of CfNek as the generation of a
peptide bond is likely to proceed via the generation of an intermediary
acylphosphate. NIMA related kinases comprise a group of biochemically distinct
kinases that can transfer a phosphate group within an acidic environment. We
noted previously that upon incubation with partially purified polyglutamylase,
synthetic C-terminal tubulin peptides became both glutamylated and
serine-phosphorylated (Westermann et al.,
1999a; Westermann et al.,
1999b
). Thus, a definite decision whether CfNek and tubulin
polyglutamylase are identical or associated will need further experimentation,
for example the knock-out of the homologous Leishmania protein. As
centrosome stability depends upon tubulin polyglutamylation
(Bobinnec et al., 1998b
) and
Nek2 kinases from different organisms are involved in centrosome maturation
and integrity (Fry et al.,
1998
; Uto and Sagata,
2000
) these enzymes appear good candidates to be tested for
glutamylation activity in the future.
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Conclusions |
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Acknowledgments |
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References |
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