From the Science Applications International
Corporation-Frederick, NCI-Frederick Cancer Research and Development
Center, National Institutes of Health, Frederick, Maryland 21702, § Facility for Biotechnology Resources, Center for Biologics
Evaluation and Research, Food and Drug Administration, Bethesda,
Maryland 20892, and ¶ Laboratory of Drug Discovery Research and
Development, Developmental Therapeutics Program, Division of Cancer
Treatment and Diagnosis, National Cancer Institute-Frederick Cancer
Research and Development Center, National Institutes of Health,
Frederick, Maryland 21702
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ABSTRACT |
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Tubulin with [8-14C]GDP bound
in the exchangeable site was exposed to ultraviolet light, and
radiolabel was cross-linked to two peptide regions of the The To determine which amino acid(s) of We recently applied the boronate methodology to isolate a peptide of
Although we had no radiolabel to assist in optimization of the
photoreaction and peptide isolation, we had an ample supply of
[8-14C]GDP-tubulin (16) and used this material to define
conditions for the photoreaction and optimal use of the boronate
matrix. Unlike Shivanna et al. (7), who used high
performance liquid chromatography to purify further peptides retained
by the boronate matrix before sequencing, we used SDS-PAGE and blot
transfer methodologies for this purpose, and we usually observed
several radiolabeled bands derived from
[8-14C]GDP-tubulin. The most prominent yielded a sequence
consistent with a guanosine- Materials--
Preparation of [8-14C]GDP-tubulin
(16) and of GDP-tubulin (17) from bovine brain were described
previously. Affi-Gel 601 was from Bio-Rad, polyacrylamide gels and PVDF
membranes from Novex, sequencing grade EP-GC from Roche Molecular
Biochemicals, and CNBr, N-ethylmaleimide, and alkaline
phosphatase from Sigma.
Direct Photoaffinity Labeling--
As before (15), a 2-ml
mixture containing [8-14C]GDP-tubulin at 12 mg/ml, 0.2 M 4-morpholineethanesulfonate (pH 6.9, NaOH), and 2 mM each of MgCl2, EGTA, and dithiothreitol was
placed in a weighing boat on ice and irradiated at 254 nm for 15 min at 2750 microwatts/cm2. Tubulin was partially precipitated
with N-ethylmaleimide (2.5 mM, at 4 °C
overnight) and harvested by centrifugation. Residual protein was
precipitated with 50% trichloroacetic acid and collected by
centrifugation. The combined pellets were lyophilized and treated with
alkaline phosphatase for 3 h at 37 °C in 0.1 M
Tris-HCl (pH 9).
CNBr and EP-GC Digestion--
Irradiated
[8-14C]GDP-tubulin was treated with 20 mg/ml CNBr in 70%
formic acid for 24 h in the dark at 37 °C. CNBr and formic acid
were removed by repeated lyophilization. For EP-GC digestion, irradiated [8-14C]GDP-tubulin was treated at an enzyme to
substrate ratio of 1:50-100 in 0.1 M phosphate buffer (pH
7.8), and the peptide mixture was lyophilized.
Chromatography of Peptide Mixtures on Affi-Gel 601--
Peptides
were dissolved in 1.0 M Tris (pH unadjusted, about 10.5),
and the mixture was diluted with 2.0 ml of 0.05 M
glycine-NaOH (pH 10) containing 1 mM MgCl2. The
solution was applied to an Affi-Gel 601 column (1.5 × 25 cm),
which was washed with the same solution. Bound peptides were eluted
with 0.1 M formic acid.
Other Procedures--
SDS-PAGE, electrophoretic transfer from
gel to PVDF membrane, and peptide sequencing were performed as before
(15). Autoradiograms were prepared with Kodak Biomax MR film (24-48 h exposures).
Following exposure to UV light, [8-14C]GDP-tubulin
was digested with either CNBr or EP-GC. The lyophilized peptide
mixtures were applied to boronate matrix columns in glycine buffer at
pH 10, and bound peptides were eluted with formic acid. A typical experiment with EP-GC peptides is shown in Fig.
1, and the pattern obtained with CNBr
peptides was similar (data not shown). About 25% of the recovered
radiolabel was in the unbound fractions and 75% in the bound
fractions. Without UV irradiation no radiolabel was retained by the
boronate column. SDS-PAGE, with subsequent electrophoretic transfer of
the peptides to PVDF membranes for protein staining, autoradiography,
and sequencing was performed on total peptides, unbound peptides, and
bound peptides. Selected gel patterns for CNBr peptides are shown in
Fig. 2A, and for EP-GC peptides in Fig. 2, B and C.
-subunit.
Following enrichment for peptides cross-linked to guanosine by boronate
chromatography, we confirmed that the cysteine 12 residue was the major
site of cross-linking. However, significant radiolabel was also
incorporated into a peptide containing amino acid residues 206 through
224. Although every amino acid in this peptide except cysteine 211 was
identified by sequential Edman degradation, implying that this was the
amino acid residue cross-linked to guanosine, radiolabel at C-8 was
usually lost during peptide processing (probably during chromatography
at pH 10). Consequently, the radiolabeled amino acid could not be
unambiguously identified.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-
-tubulin heterodimer contains two tightly bound
molecules of guanosine nucleotide (1, 2), one bound to each subunit (3). Because the nucleotide on
-tubulin is readily replaced with
exogenous nucleotide, including radiolabeled compounds, its binding
site has been called the exchangeable site (E
site)1 (1, 4-7). GTP bound
in the E site and its hydrolysis is generally necessary for microtubule
assembly (8-10). The
-tubulin nucleotide is always in the form of
GTP, and it probably derives from the tissue of origin of the tubulin
because it has never been replaced with exogenous nucleotide (11, 12).
The GTP bound to
-tubulin is thus described as being in the
nonexchangeable site (N site).
-tubulin were near the E site,
Shivanna et al. (7) employed boronate column chromatography to isolate tryptic peptides enriched for guanosine after direct photoaffinity labeling of tubulin bearing E site [3H]GTP.
Shivanna et al. (7) found most of the radiolabel retained by
the boronate matrix had reacted with Cys-12 of
-tubulin. Earlier, Little and Ludueña (13) had demonstrated that tubulin depleted of
E site nucleotide was susceptible to cross-link formation between Cys-12 and either Cys-201 or Cys-211 by
N,N'-ethylenebis(iodoacetamide). The second amino
acid involved in the cross-link was not precisely determined. Formation
of this dicysteine cross-link was inhibited by guanine nucleotides and
by antimitotic drugs that themselves inhibit nucleotide exchange at the
E site (14).
-tubulin cross-linked to the N site GTP by direct photoaffinity labeling by replacing E site GDP with dGDP (15). Following exposure of
this tubulin to UV light, E site peptide(s) were not retained by the
boronate matrix, and the predominant retained peptide was derived from
-tubulin. Sequence analysis indicated that the modified amino acid
was
-Cys-295.
-Cys-12 cross-link, as described by
Shivanna et al. (7). This paper describes studies that
demonstrate that a second reactive amino acid is probably
-Cys-211.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Chromatography of EP-GC digest of UV
irradiated [8-14C]GDP-tubulin on Affi-Gel 601. The
digest obtained from 10 mg of tubulin was dissolved in 0.1 ml of 1 M Tris (pH unadjusted) and diluted with 2 ml of a solution
containing 50 mM glycine-NaOH buffer (pH 10) and 1 mM MgCl2. The sample was applied to a 1.5 × 25 cm column. The column was washed with 80 ml of the same solution,
followed by 0.1 M formic acid (arrow). Fraction
size, 2 ml. , A280;
, cpm in 0.1 ml.
View larger version (23K):
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Fig. 2.
SDS-PAGE patterns obtained with peptides
derived from UV irradiated [8-14C]GDP-tubulin.
A, peptides obtained by CNBr digestion; B and
C, peptides obtained by EP-GC digestion. Panel A,
lane I shows the autoradiogram of the PVDF electroblot
obtained following electrophoresis of an aliquot of the entire CNBr
digest of irradiated [8-14C]GDP-tubulin; lanes
II and III show, respectively, the Coomassie Blue
stained electroblot and its autoradiogram of the peptide fraction bound
to the boronate matrix (formic acid eluate). The band numbers are
described in the text. Panel B, lanes I and
II show, respectively, the Coomassie Blue stained
electroblot and its autoradiogram of the peptide fraction bound to the
boronate matrix (formic acid eluate). Sample pH not adjusted before
electrophoresis. Panel C, lanes I and
II show, respectively, the Coomassie Blue stained
electroblot and its autoradiogram of the peptide fraction bound to the
boronate matrix (formic acid eluate). The band numbers are described in
the text. Sample pH adjusted with NaOH as described in the text before
SDS-PAGE.
Gel pattern I in Fig. 2A is an autoradiogram of a total CNBr
digest of irradiated [8-14C]GDP-tubulin (an autoradiogram
of unbound peptides was similar, data not shown). There were three
prominent peptides, but the relative amounts varied in different
digests. Even though these were not pure peptides (Coomassie Blue
staining revealed a broad streak of peptides), sequence analysis was
performed. These studies indicated that the upper band was the CNBr
165-233 peptide and the two lower bands were the
2-73 peptide
(sequence of Krauhs et al., Ref. 18). The
165-233
peptide includes
-Cys-201 and
-Cys-211, identified by Little and
Ludueña (13) as the two possibilities for the residue linked to
-Cys-12, and the
2-73 peptide includes the
-Cys-12 residue
itself, identified by Shivanna et al. (7) as the residue
that cross-links to [3H]GTP during direct photoaffinity labeling.
When the bound peptides were analyzed, we obtained a different gel pattern (Fig. 2A, II, Coomassie Blue stained, and III, autoradiogram). We cannot explain the faster mobility of the bound radiolabeled peptides, but this did not interfere either with resolution or subsequent sequencing.
The stained gel II (Fig. 2A) showed three major bands and
one minor band. All except the upper band 1 were radiolabeled (Fig. 2A, III). Note that the specific activity of the upper
radiolabeled band 2 is substantially lower than that of the lower
radiolabeled bands 3a and 3b. Band 1 yielded a sequence through 19 cycles of Edman degradation (Table I)
consistent with the CNBr peptide 204-302. Note that
-Cys-213 was
not positively identified because cysteine residues are destroyed
during the Edman procedure. This is the same peptide we obtained
previously from dGDP-tubulin (15) following UV irradiation, CNBr
digestion, and boronate chromatography, and we therefore identified it
as being derived from the N site.
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The upper radiolabeled band 2 yielded a sequence through 16 cycles of
Edman degradation consistent with the CNBr 165-233 peptide (Table
I), and it thus corresponds to the upper radiolabeled band in gel I.
Bands 3a and 3b both yielded sequences through 12 cycles of Edman
degradation consistent with the CNBr 2-73 peptide (Table I), and
these bands thus correspond to the lower radiolabeled bands in gel I.
To more precisely define the positions of the radiolabel in
-tubulin, we turned to enzymatic digestion to obtain smaller peptides. EP-GC yielded the best results (studies also performed with
endoproteinase Lys-C and trypsin). It should be noted that EP-GC will
cleave after aspartate as well as glutamate residues.
Initially we obtained puzzling results. Fig. 2B presents a
Coomassie Blue pattern (I) together with its autoradiogram (II) of the
peptide fraction bound to and eluted from the boronate column. The most
heavily stained band in the electroblot was not radiolabeled.
Nevertheless, it yielded the sequence of the EP-GC 206-224 peptide,
which is a sequence within that of the radiolabeled CNBr peptide
represented by band 2 of the gels shown in Fig. 2A. This
peptide should not have been retained by the boronate matrix unless it
possessed the ribose cis-diol, but not necessarily the radiolabel at the C-8 position in guanine. The heavily radiolabeled bands yielded a sequence consistent with the EP-GC
4-22 peptide, which would contain the known target Cys-12.
We continued to investigate the peptides generated by EP-GC digestion. We noted that before SDS-PAGE the bromphenol blue tracking dye added to the peptide sample had a green color. Small amounts of NaOH added to the sample restored the blue color of the dye. Sometimes this resulted in an additional radiolabeled band, and the most dramatic example of this change is shown in Fig. 2C (Coomassie Blue pattern, I, compared with the autoradiogram, II). The Coomassie Blue stained sample showed two minor bands (1a and 1b) and three major bands (2, 3a, and 3b), with only the latter being radiolabeled.
The nonradiolabeled bands, 1a and 1b, yielded sequences for 16 and 12 cycles, respectively, of sequential Edman degradation consistent with
the EP-GC peptide 291-306 (Table I), the same peptide identified
earlier from dGDP-tubulin (15) that led us to propose
-Cys-295 as
the reactive amino acid at the N site (note that
-Cys-305 is not
included in the large CNBr peptide derived from
-tubulin, which
should terminate at
-Met-302).
Radiolabeled band 2 yielded a sequence for 19 degradation cycles
consistent with the EP-GC peptide 206-224 (Table I). The mobility
of this band does not differ substantially from the analogous band
shown in Fig. 2B. Because all residues were positively
identified except Cys-211, this suggests that the radiolabeled amino
acid is in fact Cys-211. If the modification had occurred at a
different residue, that amino acid should have either been
unidentifiable or misidentified following the Edman procedure (but see
"Discussion"). The microsequencing technique does not permit
specific localization of the radiolabel in the peptides purified by
SDS-PAGE, because specific activity is too low.
Radiolabeled bands 3a and 3b yielded sequences for 17 degradation
cycles consistent with the EP-GC peptide 4-22 (lacking only Trp-21,
which would have been destroyed during the Edman procedure, and the
terminal Glu-22). These bands, also, have similar mobility to the
analogous bands shown in Fig. 2B. Only Cys-12 was not
identified in this sequence, consistent with its bearing the
radiolabeled guanosine fragment shown directly by Shivanna et
al. (7). Note that there is much less difference in the specific
activities of the band 2 and band 3 EP-GC peptides than in the
analogous CNBr peptides (Fig. 2A).
Our finding of a second peptide cross-linked to radiolabeled E site
nucleotide by direct photoaffinity labeling differs from the result of
Shivanna et al. (7), although in our studies -Cys-12-containing peptides were always more heavily labeled than
those containing
-Cys-211 (see "Discussion"). Besides
differences in reaction conditions, our tubulin contained
[8-14C]GDP and theirs contained [3H]GTP in
the E site. It was thus possible that the difference in results was due
to a subtle conformational change in the tubulin as a consequence of
the change in nucleotide. However, using CNBr digestion followed by
SDS-PAGE as our assay, we could find no condition that caused
significant reduction in the upper radiolabeled band (Fig.
2A, gel I).
The sole remaining possibility was that the lability of the link from
the nucleotide to the Cys-211 peptide caused substantial loss of
radiolabel at some point during sample processing by Shivanna et
al. (7). The experiment presented in Fig.
3 demonstrates that this is a reasonable
explanation, and, in addition, explains the erratic retention of
radiolabel we observed. A CNBr digest was prepared and dialyzed against
0.05 M glycine-NaOH (pH 10), the application buffer for the
boronate column. SDS-PAGE was performed with the original digest and
with samples that had been dialyzed for different times. The radiolabel
initially present in the 165-233 peptide (gel I) was reduced after
2 h and nearly completely gone after 12 h (gel II) of
dialysis.
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DISCUSSION |
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Fig. 4 presents a summary of the
sequence data for the two regions of -tubulin (18) described above.
The cleavage sites by CNBr (CB) and EP-GC (GC)
are shown above the amino acid sequences with the presumptive carboxyl
cleavage sites in parentheses. Our data agree (7) that
-Cys-12 is
the primary reactive amino acid with E site nucleotide when tubulin is
exposed to UV light. We, however, also find significant radiolabel
bound to a second region of
-tubulin. The CNBr digestion data
establish that the secondary site is within the
165-233 sequence,
and the EP-GC data narrow the location of the reactive residue to the
206-224 sequence. The likely specific amino acid is
-Cys-211.
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Little and Ludueña (13) found that the cross-link formed by
N,N'-ethylenebis(iodoacetamide) in -tubulin
depleted of E site nucleotide was between
-Cys-12 and either
-Cys-201 or
-Cys-211. Nogales et al. (3) recently
presented a three-dimensional structure of tubulin in zinc-induced
sheets and noted that the E site GDP was located between
-Cys-12 and
-Cys-211 and that it was closer to Cys-12 than to Cys-211. This is
in accord with our observation of more extensive incorporation of
radiolabel in Cys-12-containing peptides than Cys-211 peptides.
However, we have not actually demonstrated that the labeled position in
either the radiolabeled CNBr peptide or the radiolabeled EP-GC peptide
is -Cys-211. In the microsequencing technique, it is the sliced PVDF
membrane electroblot that is placed in the sequencing apparatus, with
the Edman degradation performed on the membrane fragment, and generally
fewer than 5-10 pmol of amino acid derivative is recovered at even the
initial position of the sequenced peptide. The radiolabel in the EP-GC
peptide (
206-224) is labile, even though the peptide is clearly
bound by the boronate matrix. This suggests that the cross-linked
peptide undergoes some sort of "depurination" reaction, with
retention of the C2'-C3'-cis-diol responsible for binding of
the peptide to the boronate column but loss of the radiolabeled C-8
atom in the guanine residue.
Although the thus far uncontrollable loss of radiolabel in the EP-GC
206-224 peptide probably accounts for the difference in our results
from those of Shivanna et al. (7), it limits our confidence
in assigning the position of the cross-link to Cys-211. Specific
identification of all residues except
-Cys-211 in the EP-GC peptide
206-224 favors Cys-211 as being the site of the cross-link, but
this requires that the peptide modification causing retention of the
peptide on the boronate column is stable throughout the sequential
Edman degradation. The ready loss of C-8 (the radiolabel) from the
EP-GC peptide is worrisome in this regard, but the cross-link to the
C2'-C3'-cis-diol segment appears to be more stable
chemically. Because Cys residues are destroyed during the Edman
procedure, failure to identify them cannot be taken as positive
evidence that they are the site of the cross-link. As a minimum, it is
unlikely that the photoreactions between Cys-12 and Cys-211 proceed by
the same chemical mechanism (cf. Refs. 7 and 15). Thus it
remains possible that another amino acid residue in the
206-224
peptide is cross-linked to the C2'-C3'-cis-diol derived from
[8-14C]GDP and that the chemical bond is hydrolyzed
during sequential Edman degradation, regenerating an unaltered amino
acid residue.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Laboratory of Drug
Discovery Research and Development, National Institutes of Health,
Bldg. 37, Rm. 5D02, 37 Convent Dr., Bethesda, Maryland 20892-4255. Tel.: 301-496-4855; Fax: 301-402-0752.
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ABBREVIATIONS |
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The abbreviations used are: E site, exchangeable nucleotide binding site of tubulin; N site, nonexchangeable nucleotide binding site of tubulin; [8-14C]GDP-, GDP-, and dGDP-tubulin, tubulin with the indicated nucleotide bound in the exchangeable site; EP-GC, endoproteinase Glu-C; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride.
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REFERENCES |
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