Proc. Natt. Acad. Sci. USA
Vol. 83, pp. 1006-1010, February 1986
Cell Biology
A monoclonal antibody that cross-reacts with phosphorylated
epitopes on two microtubule-associated proteins and two
neurofilament polypeptides
(neuronal cytoskeleton/protein klnase/evolutdonary conservation)
FRANCIS C. LUCA, GEORGE S. BLOOM, AND RICHARD B. VALLEE*
Cell Biology Group, Worcester Foundation for Experimental Biology, 222 Maple Avenue, Shrewsbury, MA 01545
Communicated by Philip Siekevitz, October 3, 1985
ies. Reaction is with a phosphorylated epitope present on
both proteins. The antibody also shows an additional unique
feature: it cross-reacts with two of the three major polypeptide components of neuronal intermediate filaments (neurofilaments).
A monoclonal antibody is described that was
ABSTRACT
raised against bovine brain microtubule-associated protein
(MAP) 1B. Immunoblot analysis revealed that immunoreactivity was abolished by dephosphorylation of the antigen. The
antigen/antibody reaction was also directly inhibited by sodium phosphate. In whole brain tissue, MAP 1B was the primary
immunoreactive species. However, the antibody was also found
to react with MAP 1A as well as with the high and middle
molecular weight neurofilament polypeptides. No cross-reaction with MAP 2, which is known to be extensively phosphorylated, other MAPs, or the low molecular weight
neurofilament polypeptide was observed. This evidence suggests at least some sequence homology between these different
polypeptide components of the neuronal cytoskeleton and
points to a common mechanism for their phosphorylation.
The major nontubulin proteins isolated with purified brain
microtubules are large polypeptides known as the high
molecular weight microtubule-associated proteins (HMW
MAPs). These proteins represent substantial components of
the neuronal cytoskeleton (see, for example, ref. 1) and are
present in a wide variety of other cell types as well (2).
Recent work has indicated that the HMW MAPs are
complex in their molecular composition. Two HMW MAP
classes-termed MAP 1 and MAP 2 (3)-were originally
identified on the basis of their distinct electrophoretic mobility. MAP 2 has since proven to consist of two polypeptides-MAP 2A and MAP 2B-that show extensive structural and immunological similarity (2, 4-8). Our laboratory
has described three components of MAP 1-MAP 1A, MAP
1B, and MAP 1C-that are structurally and immunologically
distinct (5, 9, 10). Peptide mapping of the three MAP 1 species
revealed little apparent homology between these proteins (5,
9). We have also produced several monoclonal antibodies, all
of which were specific for either MAP 1A (9, 10) or MAP 1B
(5). We have observed differences in the binding efficiency of
MAP 1A and MAP 1B to microtubules and in their regional
distribution in brain tissue (5). Other laboratories have also
noted the complexity of the MAP 1 polypeptides (8, 11-15),
though the correspondence of proteins from laboratory to
laboratory has not been established.
In the course of characterizing antibodies prepared against
electrophoretically purified MAP 1B, we found one that
appeared to cross-react with MAP 1A. Because of the
potential usefulness of such an antibody in evaluating the
structural relationship between the MAPs, we have characterized its properties in detail.
We report here that this antibody, termed "MAP 1B-3,"
indeed recognizes MAP 1A and MAP 1B as defined by
immunoprecipitation with monospecific monoclonal antibod-
MATERIALS AND METHODS
Biochemical Reagents. Bovine casein (technical grade), egg
yolk phosvitin, calf thymus histone (type II-A), potato acid
phosphatase (type III), bovine intestinal alkaline phosphatase (type VII-N), Escherichia coli alkaline phosphatase
(type III), pepstatin A, leupeptin, and phenylmethylsulfonyl
fluoride were products of Sigma. a2-Macroglobulin was
obtained from Boehringer Mannheim. NaDodSO4 was obtained from British Drug House.
Microtubule and Neurofilament Purification. Microtubules
were purified with the aid of taxol from calf brain gray and
white matter or whole rat brain as described (16). MAPs were
prepared from the calf brain white and gray matter microtubules by exposure to 0.35 M NaCl in the presence of taxol and
were used without further dialysis. Neurofilaments isolated
from bovine spinal cord (17, 18) were generously provided by
Anne Hitt and Robley Williams (Vanderbilt University).
Neurofilaments from rat brain (19) were generously supplied
by Ronald Liem (New York University School of Medicine).
Electrophoretic Techniques. NaDodSO4 gel electrophoresis
was performed according to the method of Laemmli (20). A
modification of this method was also used in which NaDodSO4 was omitted from the stacking and separating gels, and
2 M urea was added to the separating gel (5). Gels were
stained with Coomassie brilliant blue R250 (21) or ammoniacal silver (22).
Anti-MAP Antibodies. Monoclonal anti-MAP 1A, now
referred to as antibody "MAP lA-i," has been described (9,
10). Monoclonal antibody "MAP 1B-4" was one of four
anti-MAP 1B antibodies described in an earlier report (5).
Antibody MAP 1B-3 was obtained from the same hybridoma
fusion, which was performed by using spleen cells from a
mouse immunized with electrophoretically purified calf brain
white matter MAP 1B. Antibody MAP 1B-3 was found to be
of the IgM class using isotype-specific antibodies (9). Hybridoma cells were cloned twice. All positive wells from the
second cloning step showed cross-reaction with MAP 1A and
MAP 1B, indicating that this was an inherent property of
antibody MAP 1B-3 (see Results).
Immunological Techniques. Immunoblotting was performed as described (23). All washing steps were conducted
in 50 mM Tris (pH 7.4) containing 150 mM NaCl (Tris/NaCl).
Bovine serum albumin (0.25%) and Nonidet P-40 (NP-40)
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: HMW, high molecular weight; MAP, microtubuleassociated protein; NP-40, Nonidet P-40.
*To whom reprint requests should be addressed.
1006
Cell
Biology: Luca et al.
(0.05%) were added prior to the first antibody incubation and
prior to and during the second antibody incubation.
To evaluate the role of protein-bound phosphate in the
antigen/antibody reaction, nitrocellulose strips to which
electrophoretically separated polypeptides had been transferred were washed with Tris/NaCl, exposed to bovine
serum albumin and NP-40, and then incubated with protein
phosphatase. Conditions were based on those of Sternberger
and Sternberger (24). Exposure to phosphatase was for 2.5 hr
at 370C in 2 ml of 0.1 M Tris buffer containing leupeptin at 10
pg/ml, pepstatin A at 10 pg/ml, 2 mM phenylmethylsulfonyl
fluoride, and 1 trypsin-inhibitory unit of a2-macroglobulin.
Calf intestinal alkaline phosphatase was used at 10.5 units/ml
(pH 8.0), E. coli alkaline phosphatase was at 10 units/ml (pH
8.0), and potato acid phosphatase was at 5 units/ml (pH 6.0).
The nitrocellulose strips were subsequently rinsed three
times in Tris/NaCl, treated with bovine serum albumin and
NP-40, and then exposed to primary and secondary antibodies as usual.
Immunoprecipitation was performed as follows. MAPs (1
mg in 200 ,l) were incubated in a boiling water bath for 5 min
in the presence of NaDodSO4 (1%) and 2-mercaptoethanol
(5%). The samples were chilled and diluted 1:10 in Tris/NaCl
containing 1% NP-40. Primary antibody was added as ascites
fluid (50 ,ul) and the samples were incubated on ice for 2 hr.
Six hundred twenty-five microliters of goat anti-mouse
immunoglobulins coupled to agarose beads (Hyclone Immunochemical Reagents, Logan, UT) was added, and the
samples were incubated overnight with gentle mixing at 2°C.
The beads were washed with Tris/NaCl, taken up in 2 vol of
NaDodSO4 electrophoresis sample buffer, and centrifuged.
The supernate was used for subsequent electrophoretic and
immunoblot analysis.
RESULTS
Reaction of Antibody MAP 1B-3 with MAP 1A and MAP 1B.
Fig. 1 shows immunoblot data for antibody MAP 1B-3 in
brain tissue and purified microtubules. The antibody showed
a limited reaction with a HMW polypeptide species in whole
cerebral cortex (Fig. 1, lanes A and B). In purified microtubules, however, reaction with two bands corresponding to
both MAP 1A and MAP 1B was observed (Fig. 1, lanes C and
D). Reaction with a band below MAP 1B, presumed to
represent a fragment of MAP 1A or MAP 1B, was also seen
sometimes (compare Figs. 1 and 3). Reaction with MAP 1C
or with MAP 2 was not observed.
The structural relationship between the HMW MAPs is still
incompletely defined. These proteins are electrophoretically
complex (Fig. 1; refs. 5, 9, 11-15). Most of them are very
sensitive to proteolysis (9, 25, 26), which might further
contribute to the observed complexity. We, therefore, considered it important to establish whether the result shown in
Fig. 1, lane B, reflected true cross-reaction between MAP 1A
and MAP 1B as we have defined those species in our earlier
work.
To this end, MAP 1A and MAP 1B were independently
immunoprecipitated with specific monoclonal antibodies.
The immunoprecipitated proteins were then subjected to
immunoblotting (Fig. 2). Immunoprecipitation was performed with MAPs that had been incubated in 1% NaDodSO4
to disrupt protein/protein interactions that might complicate
interpretation of the experiment. White matter MAPs were
used as starting material for the isolation of MAP 1B because
of the greater abundance of this protein in white matter
preparations, whereas gray matter MAPs were used as
starting material for the isolation of MAP 1A. The immunoprecipitated MAPs were devoid of detectable contamination with other MAP species as shown by total protein
staining. Immunoblotting with monospecific anti-MAP 1A
Proc. Natl. Acad. Sci. USA 83 (1986)
A
B
C
.4
_-1
1007
D
-
~~~ A
1A_
I
Bz
iC-
2A2B-
.4
FIG. 1. Electrophoretic and immunoblot analysis of antibody
MAP 1B-3. Lanes A and B, whole calf brain cerebral cortex was
dissolved in sample buffer and subjected to NaDodSO4/polyacrylamide gel electrophoresis (7% acrylamide). Lane A, Coomassie
blue-stained gel; lane B, equivalent sample transferred to nitrocellulose and allowed to react with antibody. Lanes C and D, similar
analysis of purified microtubules from calf brain white matter
subjected to NaDodSO4/urea/polyacrylamide gel electrophoresis
(4% acrylamide). Lane C, Coomassie blue-stained sample; lane D,
nitrocellulose strip allowed to react with antibody. Arrowheads
denote positions of top and dye front of gel.
and anti-MAP 1B antibodies showed reaction only with their
corresponding immunoprecipitated species. In contrast, antibody MAP 1B-3 recognized polypeptides immunoprecipitated by the MAP 1B- and MAP lA-specific antibodies.
In further experiments, three additional anti-MAP lB
antibodies (5) and one additional anti-MAP 1A antibody
("MAP 1A-2"; F.C.L. and R.B.V., unpublished results)
reacted only with MAP 1B or MAP 1A, respectively, in this
type of analysis. None of the antibodies, including MAP
1B-3, reacted with immunoprecipitated MAP 2.
The unique behavior of antibody MAP 1B-3 raised the
possibility that it recognized an epitope with unusual properties. Several antibodies have been described that recognize
phosphorylated epitopes (24, 27-33). To determine whether
this were the case for MAP 1B-3, microtubule proteins were
separated by NaDodSO4/polyacrylamide electrophoresis,
blotted to nitrocellulose paper, and then exposed to calf
intestinal alkaline phosphatase (Fig. 3). This ttattnent completely abolished the immunoreactivity of MAP 1A and MAP
1B (Fig. 3, lane B). Potato acid phosphatwe azd E. -coli
alkaline phosphatase also reduced immunoreactivityj though
not as effectively as the calf enzyme.
To determine whether the inhibition of immunoreactivity
was due specifically to the phosphatase actiity of the
enzyme preparation, 50 mM sodium phosphate w"a included
in the preparation as a competitive inhibitor during the
phosphatase treatment (33, 34). Under these conditions
immunoreactivity was preserved (Fig. 3, lane C). Thus, the
epitope recognized by MAP 1B-3 was judged to include an
essential phosphate group.
1008
Proc. Natl. Acad. Sci. USA 83 (1986)
Cell Biology: Luca et al.
A B
A
B
A
B
A
A
B WG
B
C
1A
--
-7B
.2B
MAP 1AMAP JB-
MAP 1B-4 MAP 1B-3 MAP lA-l
BLOT
SI LVER
FIG. 2. Reaction of antibody MAP 1B-3 with MAP 1A and MAP
1B. MAP 1A and MAP 1B were individually immunoprecipitated
with monospecific monoclonal antibodies. MAP 1A was immunoprecipitated from gray matter MAPs with antibody MAP lA-1
(9), and MAP 1B was immunoprecipitated from white matter MAPs
with antibody MAP lB-4 (5). The immunoprecipitates were subjected to NaDodSO4/polyacrylamide gel electrophoresis (6% acrylamide) and visualized by silver staining (Right). The immunoprecipitates were also transferred to nitrocellulose and allowed to react
with antibodies MAP 1B-4, MAP 1B-3, and MAP lA-1 (Left). Lanes:
A, immunoprecipitated MAP 1A; B, immunoprecipitated MAP 1B;
W, white matter microtubules; G, gray matter microtubules. MAP
1A appears to split in this preparation, possibly due to proteolysis.
The heavy bands at low molecular weight in the silver-stained
material in lanes A and B are the antibody heavy chains. Arrowheads
denote positions of top and dye front of gel.
We also observed that inorganic phosphate directly inhibited the antibody/antigen reaction. Inclusion of sodium
phosphate at levels as low as 50 mM during the primary
antibody incubation almost abolished immunoreactivity.
That this was not simply an ionic strength effect was shown
by control experiments in which levels of NaCl as high as 1
M had no effect on the reaction. We noted that the effect of
phosphate was most dramatic at short times of antibody
incubation. After 1 hr in the presence of 50 mM phosphate,
almost no antibody reaction was observed. However, after 3
hr, substantial reaction was seen.
Reaction of Antibody MAP 1B-3 with Neurofilaments.
Recognition of a phosphorylated epitope raised the question
of how broad the cross-reactivity observed with antibody
MAP 1B-3 might be. Presumably, most protein phosphates
were not recognized because of the very limited reaction
observed with whole brain tissue or purified microtubules
(Fig. 1). In the latter case, in particular, MAP 2 is known to
be highly phosphorylated (35-37), and tubulin (see Fig. 4)
also contains close to 1 mol of phosphate per mol of protein
(38). Neither protein showed detectable reaction with antibody MAP 1B-3.
Nonetheless, we did notice in calf brain tissue, particularly
in white matter, weak reaction with two polypeptides of
lower molecular weight than MAP 1B. In rat brain, the
antibody recognized a major polypeptide of Mr -200,000
(Fig. 4). This species was completely removed by centrifugation of the tissue homogenate, in contrast to the immunoreactivity observed at the position of the MAP 1 polypeptides. Though the cross-reactive polypeptide was prominent
in adult brain tissue, it was absent in young rat brain. In view
of these properties, and reports of the high frequency with
which antibodies to phosphorylated neurofilament epitopes
could be obtained (24), we examined the reaction of MAP
1B-3 with purified neurofilaments (Fig. 5).
FIG. 3. Effect of phosphatase on MAP 1B-3 immunoreactivity.
Calf brain white matter microtubules were subjected to NaDodS04/urea/polyacrylamide gel electrophoresis (4% acrylamide) followed by immunoblotting as described in the legends to Figs. 1 and
2. The nitrocellulose paper was incubated with Tris buffer alone (pH
8.0) (lane A), calf intestinal alkaline phosphatase (lane B), and calf
intestinal alkaline phosphatase in the presence of 50 mM sodium
phosphate (lane C). The nitrocellulose strips were subsequently
incubated with antibody MAP 1B-3, and the immunoblot procedure
was completed as usual.
It may be seen that two of the three subunits of neurofilaments were recognized by the antibody. Reaction was most
intense with the HMW component but was also detectable
with the middle molecular weight species. A trace reaction
could also be seen at the position of MAP 1B. No reaction
with the low molecular weight neurofilament polypeptide was
detected. Similar results were obtained with a preparation of
rat brain neurofilaments (data not shown).
In separate experiments, it was found that immunoreactivity of the neurofilament bands was abolished when rat
brain samples were exposed to phosphatase, as was described in the legend to Fig. 3 for microtubule proteins, or
when phosphate was present during incubation of purified
calf brain neurofilaments with antibody MAP 1B-3.
To examine the specificity of the antibody further, immunoblotting was conducted with several proteins known to
contain high levels of covalently bound phosphate. No
reaction was observed with 5 ,ug of phosvitin or histone.
Occasional weak reaction was observed with a single casein
polypeptide at a total protein loading of 5 ,g. No reaction was
observed at 2 ,ug of protein.
DISCUSSION
We have found that one of a series of five antibodies raised
against MAP 1B, a recently identified MAP in brain tissue (5,
9), cross-reacts with an additional microtubule protein, MAP
1A (9). Reaction was with a phosphorylated epitope, indicating that MAP 1A and MAP 1B are phosphoproteins.
Further cross-reaction was observed with two of the three
neurofilament polypeptides. It is known that all three neurofilament polypeptides contain phosphate and that the high
and middle molecular weight species are extensively
phosphorylated (17, 34). This raises the possibility that the
H
E
H
P
E
.<
Proc. Natl. Acad. Sci. USA 83 (1986)
Cell Biology: Luca et al.
p
S -M AP IA
MAP 2AB
-4P.
- 1_W
A
VW
_
1009
B
_4 -H
_ -M
q
-
L
-TUB
C BB
BLOT
FIG. 4. Immunoblot analysis of rat brain subcellular fractions.
Microtubules were prepared from whole adult rat brain tissue, and
the preparative fractions were subjected to NaDodSO4/polyacrylamide gel electrophoresis (7% acrylamide) and immunoblotting.
Lanes: H, tissue homogenate; E, cytosolic extract; P, microtubule
pellet. CBB, Coomassie blue-stained gel; BLOT, immunoblot; TUB,
tubulin. Arrow indicates immunoreactive species present in
homogenate but absent from extract and microtubule pellet. Only a
single MAP band was detected in the extract and microtubule lanes
due to poor resolution of MAP 1A and MAP 1B on higher percent
acrylamide gels. Arrowheads denote positions of top and dye front
of gel.
observed cross-reaction is a simple function of total phosphate content. This seems unlikely in view of the limited
cross-reactivity of the antibody in whole brain tissue (Figs. 1
and 4) and the failure to observe cross-reactivity with MAP
2, which is itself extensively phosphorylated (35-37) and very
abundant in microtubule preparations (Figs. 1 and 4), with
brain tubulin, which contains close to 1 mol/mol of phosphate (38), or with high levels of phosvitin, histone, or casein.
It should be mentioned in this context that little is known
about the extent of MAP 1 phosphorylation. Phosphate
incorporation in vivo into some component of the MAP 1
complex has been reported (3, 11, 39). The correspondence
of these species to those characterized in our laboratory is not
certain, and the extent and mechanism of MAP 1 phosphorylation are unknown. The present study has served to
demonstrate that MAP 1A and MAP 1B as defined in our
laboratory are both phosphorylated as isolated. Since
immunoreactivity was observed in tissue directly dissolved in
electrophoresis sample buffer, we conclude that MAP 1A and
MAP 1B were phosphorylated in vivo. Whether phosphorylation of these proteins occurs to an extent comparable to that
of the neurofilament polypeptides must await further work.
Until the present study, the accumulating evidence on the
properties of the MAP 1 polypeptides had shown more
differences than similarities other than their obvious similarity in molecular weight. The two proteins had largely distinct
peptide patterns (5). In addition, four monospecific antibodies to MAP 1B (5) and one to MAP 1A (9) have been
produced. Six additional monospecific monoclonal antibod-
FIG. 5. Immunoblot analysis of purified neurofilaments. Calf
brain neurofilaments (6 Iug per lane) were subjected to NaDodS04/polyacrylamide gel electrophoresis (7.5% acrylamide). Lane A,
immunoblot; lane B, Coomassie blue staining. H, high; M, middle;
and L, low molecular weight neurofilament polypeptide components.
Arrowheads denote positions of top and dye front of gel. Arrow
indicates position of very high molecular weight species present in
neurofilaments at the position of MAP 1B, which showed barely
detectable reaction with MAP 1B-3.
ies to MAP 1A have also been produced recently in our
laboratory (F.C.L., unpublished results). In addition to this
structural evidence, we have noted reciprocal developmental
changes in the two MAP 1 polypeptide species (40) as well as
differences in their microtubule binding properties (5). Indeed, MAP 1B had remained unnoticed until recently because of its low efficiency of copurification with microtubules
relative to MAP 1A and other MAPs.
Despite these observations, the evidence presented here
indicates that at least some structural homology does exist
between the two proteins. Since inorganic phosphate inhibited antibody binding, the antibody must recognize a phosphorylation site directly rather than a separate site that is
indirectly affected by phosphorylation state. However, since
most protein phosphates are not recognized by MAP 1B-3,
the antibody must also recognize a specific sequence of
amino acids. Thus, MAP 1A and MAP 1B must have some
sequence homology, at least in the immediate vicinity of the
immunoreactive phosphate group.
It is too soon to conclude whether the common presence of
this epitope in neurofilaments and the MAP 1 polypeptides is
indicative of some fundamental relationship between these
proteins, either in their function or in their evolutionary
development. One of the more intriguing possibilities raised
by this finding is that the epitope represents a common
sequence recognized as substrate by some normal cellular
constituent, such as a protein kinase or phosphatase. Perhaps
phosphorylation of neurofilaments and the MAP 1 polypeptides, which are all components of the neuronal cytoskeleton,
occurs by means of a common mechanism.
So far as we know, cross-reaction of neurofilaments with
microtubule proteins has not been reported previously. It has
been observed that the components of the neurofilament
triplet polypeptides are particularly immunogenic (24, 41) and
that antibodies to phosphorylated epitopes can be obtained
with considerable frequency (24). Whether these antibodies
react with other proteins, such as the MAPs, has not been
investigated. Perhaps among these antibodies, some with the
properties described here will prove to occur with significant
frequency as well.
1010
Proc. Natl. Acad. Sci. USA 83 (1986)
Cell Biology: Luca' et al.
We thank Anne Hitt and Dr. Robley Williams, Jr., for their
generous gift of calf spinal cord neurQfilaments; Dr. Ronald Liem for
his generous gift of rat brain neurofilaments; Dr. Christine Collins for
preparation of microtubules; and Dr. Collins and Dr. Jeremy Hyams
for their critical reading of the manuscript. This study was supported
by National Institutes of Health Grant GM26701 and March of Dimes
Grant 5-388 to R.B.V. and by the Mimi Aaron Greenberg Fund.
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