(Received for publication, December 26, 1995)
From the
The phosphorylation of microtubule-associated proteins (MAPs) is
thought to be a key factor in the regulation of microtubule stability.
We have shown recently that a novel protein kinase, termed p110
microtubule-affinity regulating kinase (``MARK''),
phosphorylates microtubule-associated protein tau at the KXGS
motifs in the region of internal repeats and causes the detachment of
tau from microtubules (Drewes, G., Trinczek, B., Illenberger, S.,
Biernat, J., Schmitt-Ulms, G., Meyer, H. E., Mandelkow, E.-M., and
Mandelkow, E.(1995) J. Biol. Chem. 270, 7679-7688). Here
we show that p110 phosphorylates analogous
KXGS sites in the microtubule binding domains of the neuronal
MAP2 and the ubiquitous MAP4. Phosphorylation in vitro leads
to the dissociation of MAP2 and MAP4 from microtubules and to a
pronounced increase in dynamic instability. Thus the phosphorylation of
the repeated motifs in the microtubule binding domains of MAPs by
p110
might provide a mechanism for the
regulation of microtubule dynamics in cells.
In living cells, microtubules undergo transitions between stable
and dynamic states. They are organized into stable cytoskeletal
structures such as the processes of neuronal cells or the axonemes of
cilia and flagella, but are also key players in dynamic events during
cell morphogenesis or chromosome partitioning at mitosis. Microtubule
stability is thought to be modulated by a variety of post-translational
modifications of both tubulin and MAPs. ()Structural MAPs
are filamentous proteins which bind to microtubules in a
nucleotide-insensitive way, forming elongated projections from the
microtubule surface (for reviews, see Olmsted(1991), Hirokawa(1994),
Schoenfeld and Obar(1994), and Mandelkow and Mandelkow(1995)). MAPs can
control microtubule dynamics in vitro and in vivo (Drechsel et al., 1992; Pryer et al., 1992;
Umeyama et al., 1993; Gustke et al., 1994; Brandt et al., 1994; Dhamodharan and Wadsworth, 1995; Trinczek et
al., 1995). Tau and MAP2 are the most studied MAPs in the
vertebrate nervous system; tau is abundant in the axon, whereas MAP2 is
localized predominantly in dendrites (Binder et al., 1985;
Riederer and Matus, 1985). MAP4 is not limited to the nervous system
and is the predominant MAP in many types of cells and tissues (Bulinski
and Borisy, 1980; Parysek et al., 1984; Aizawa et
al., 1990). MAP2, tau, and MAP4 are grossly similar in domain
structure, having N-terminal projection domains and C-terminal
microtubule binding domains (Lee et al., 1988; Lewis et
al., 1988; Aizawa et al., 1991; West et al.,
1991; Chapin and Bulinski, 1991). The C-terminal part of these proteins
displays considerable homology in a repeated sequence motif. The
sequences in the C-terminal region are rich in basic amino acids which
probably interact with the acidic sequence in the C terminus of tubulin
(Littauer et al., 1986).
Several lines of evidence suggest
that the binding of MAPs to microtubules is regulated by
phosphorylation. MAPs isolated from tissue or cells are phosphoproteins
(Sloboda et al., 1975; Vallee, 1980; Burns et al.,
1984; Tsuyama et al., 1986; Brugg and Matus, 1991; Watanabe et al., 1993), MAPs are good substrates for many protein
kinases in vitro (Theurkauf and Vallee, 1983; Lindwall and
Cole, 1984; Mori et al., 1991; Drewes et al., 1992),
and phosphorylation interferes with their microtubule stabilizing
capacity (Brugg and Matus, 1991; Shiina et al., 1992; Drechsel et al., 1992; Biernat et al., 1993; Brandt et
al., 1994; Ookata et al., 1995; Trinczek et al.,
1995). In the case of tau protein, phosphorylation has been extensively
studied, because aberrantly phosphorylated tau is involved in the
neurofibrillar pathology of Alzheimer's disease (reviewed by
Goedert(1993), Mandelkow and Mandelkow(1993), and Trojanowski and
Lee(1994)). However, it has been difficult to establish the
relationship between protein kinases, phosphorylation sites, and their
effect on microtubule affinity, nucleation, and dynamic instability.
Recently, we have used an approach which combined site-directed
mutagenesis of recombinant tau and in vitro phosphorylation by
a brain tissue extract to identify sites that are crucial for
microtubule binding (Gustke et al., 1992). We found that
phosphorylation of tau at a single serine residue, located within the
sequence KIGS)) in the first repeat of the binding
domain, strongly suppresses microtubule binding (Biernat et
al., 1993). The phosphorylation of sites outside the microtubule
binding domain, which occurred mostly on Ser/Thr-Pro motifs, had a
relatively weak effect. Subsequently, we characterized and purified
from brain tissue a novel kinase of molecular mass 110 kDa, which
effectively phosphorylated Ser
and displayed a pronounced
specificity for all four KXGS motifs in the repeat domain of
tau (Drewes et al., 1995). This kinase efficiently caused the
loss of tau's affinity for microtubules, resulting in high
dynamic instability, and was termed p110
(microtubule affinity regulating kinase). In this paper, we
show that p110
phosphorylates MAP2 and MAP4
efficiently on their microtubule binding domains in vitro, and
that the KXGS motifs within the conserved repeats are the
major phosphorylation sites. Both MAP2 and MAP4 become detached from
microtubules upon phosphorylation by the kinase, and the microtubules
become unstable. The data suggest that phosphorylation of MAPs by
p110
could be generally important in the
MAP-mediated regulation of the dynamics and rearrangement of the
microtubule network in cells.
MALDI-MS measurements were obtained using a Lasermat 2000 instrument (Finnigan).
Figure 1: Bar diagram of microtubule-associated proteins tau (human tau40, longest isoform), MAP2 (rat, juvenile isoform), and a C-terminal MAP4 fragment comprising the microtubule binding domain of murine MAP4. Acidic domains are shown in white, basic domains are shaded. For domain definitions see text (``Materials and Methods''). Locations of phosphorylation sites are indicated (see Table 1).
We delimit tau into an acidic N-terminal domain (Ala, residues
Met to Ala
, containing the two near
N-terminal inserts Glu
-Glu
and
Asp
-Thr
, exons 2 and 3, which may be absent
due to alternative splicing, see Goedert et al. (1989) and
Himmler et al.(1989)), the basic region (``B,''
``P,'' ``R,'' residues Gly
to
Ser
, Fig. 1), and the acidic tail
(``C,'' Gly
to Leu
). The basic
region contains the proline-rich domain (``P,''
Ile
-Leu
, containing a chymotryptic cleavage
site Tyr
which subdivides ``P'' into
``P1'' and ``P2,'' and separates the projection and
assembly domains), and the repeats (see below). In the ``big
tau'' isoform of peripheral nerves, there is an insert of about
254 residues between positions Gln
and Ala
(judging from the rat sequence, exon 4a, Couchie et al.,
1992); the nature of this insert is acidic so that the size of the
acidic region is expanded 3-fold (from 119 to 379 residues).
In
MAP2, the acidic N-terminal region extends from Met to
Ala
, the basic region is
Arg
-Ser
, and the neutral C-terminal tail
is from Ser
to Leu
. The basic region
contains a proline-rich domain (Leu
-Leu
)
and the repeats (Arg
-Ser
); it also
contains cleavage sites for trypsin (behind Lys
and
Arg
, Wille et al.(1992) and thrombin (behind
Arg
, Ainsztein and Purich(1994)) which roughly separate
projection and assembly domains. In MAP2c, the region
Asp
-Thr
(1363 residues) is spliced out.
Most of the insert has acidic character, except for the last 90
residues (Arg
-Thr
), and thus can be
regarded as a large extension of the acidic N-terminal region (from 151
residues in MAP2c to 1424 in MAP2).
In MAP4, there is an acidic
N-terminal domain (Met-Thr
, including the
acidic ``KDM'' domain Thr
-Lys
, a
basic proline-rich domain (Asn
-Arg
) which
can be subdivided into ``P''
(Asn
-Ala
, proline-rich) and
``SP'' (Thr
-Arg
, rich in Ser-Pro
motifs), the basic repeats (Ala
-Gly
), and
an acidic C-terminal tail (A1091-I1125). Repeats ``1a'' and
``2'' can be absent due to alternative splicing (Chapin et al., 1995).
The repeats are the most striking aspects of
the three MAPs. They are typically 31-32 residues long, and are
similar to one another within one MAP and between different MAPs. They
can be subdivided into about 13 residues of lower homology
(sometimes referred to as the linker region or inter-repeat region),
and the
18 C-terminal residues of higher homology, the repeats
proper, mostly ending with a PGGGX motif. The boundaries
between the repeats can be chosen in different ways; we prefer the
alignment shown in Table 1because in this case the
``second'' repeat of tau coincides exactly with one
alternatively spliced exon (number 10,
Val
-Ser
). The MAPs first cloned or
sequenced contained three repeats (for tau, see Lee et al. (1988), for MAP2, Lewis et al.(1988) and Kindler et
al.(1990), for MAP4, see Aizawa et al.(1989)). Later,
other isoforms were found which contained four repeats (tau, Goedert et al.(1989) and Himmler et al.(1989); MAP2, Doll et al.(1993); MAP4, West et al.(1991) and Chapin and
Bulinski(1991)). In addition it was realized that other stretches in
the repeat domain were repeat-like, albeit with even lower homology (e.g. the 38-residue repeat following repeat ``1''
in MAP4 and the 32-residue repeat following repeat ``4'' in
tau, Chapin and Bulinski(1992)). In order to unify the nomenclature we
will denote the ``classical'' repeats (containing the higher
degree of homology) as 1, 2, 3, and 4. One of these, 2, may be absent
due to alternative mRNA splicing. The low homology repeat of MAP4 will
be called 1a. The repeat following 4 will be ``4a.'' Thus Table 1shows 6 repeats, 5 of which are common to all three MAPs
(1-4 and 4a), and 1a is specific for MAP4 (note that 4a of MAP4
shows less homology than the corresponding tau and MAP2 sequences).
These repeats correspond to repeats 1-6 in Chapin and
Bulinski(1992), and 4a was called R` in Gustke et al.(1994).
The four classical repeats all contain a motif KXGS in repeats
1-4, with X = Ile, Cys, or Val (the minor
exception is KCVS in repeat 2 of MAP4). In repeat 1a of MAP4 a similar
motif is KAAGS. Repeat 4a contains motifs KTDH (tau), RVDH (MAP2), or
AGEE (MAP4) in equivalent positions. Although formally not homologous
to KXGS, they have the character of ``constitutively
phosphorylated'' KXGS.
Figure 2:
Phosphorylation of microtubule-associated
proteins with p110. A, 5
µg of each MAP were phosphorylated with 0.1 microunits of
p110
and 1 mM [
-
P]ATP (100 Ci/mol) for 4 h at 37
°C. Phosphorylated proteins were analyzed by SDS-gradient PAGE
(4-15%) and autoradiography. Lane 1, molecular weight
markers; Lane 2, MAP2c, expressed in E. coli; Lane 3, MAP2c, phosphorylated; Lane 4, MAP2 from
porcine brain; Lane 5, MAP2, phosphorylated; Lane 6,
MAP4-BDC, expressed in E. coli; Lane 7, MAP4-BDC,
phosphorylated; Lane 8, MAP4 from mouse; Lane 9, MAP4
from mouse, phosphorylated; Lane 10, tau, human isoforms,
expressed in E. coli; Lane 11, tau, human isoforms,
phosphorylated (B). Time course of phosphorylation of MAP4-BDC (triangles), MAP2c (squares), and tau (circles). C, double reciprocal plot showing initial
rates of phosphorylation of MAP4-BDC (triangles), MAP2c (squares), and tau (circles) as dependent on their
concentration. The inset shows the derived K
and V
values.
As reported previously,
p110 phosphorylated tau exclusively on serine residues.
While phosphoamino acid analysis showed that the same is true for
MAP2c, we found that MAP4 is also phosphorylated on threonine ( Table 2and Table 3).
Figure 3:
Identification of phosphorylation sites on
MAP2c and MAP2. 200 µg of MAP2c or full-length MAP2 were
phosphorylated with 0.5 microunits of p110 for
2 h at 37 °C. After performic acid oxidation and trypsin digestion,
peptides were analyzed by TLE/TLC (A-D) and HPLC (E). A, the juvenile MAP2 isoform, MAP2c, expressed in E.
coli. B, full-length MAP2 (isolated from porcine brain). C, diagram of the more prominent spots with identification of
the HPLC-purified and sequenced phosphopeptides (see Table 2). Spot 2 contains Ser
(KXGS in repeat
3), spot 3 Ser
(KXGS in repeat 1). D, mixture of A and B showing that the major
sites on MAP2 and MAP2c are identical. E, separation of the
tryptic digest of phosphorylated MAP2c by reversed phase HPLC
(C
). Radioactive fractions were purified by a second HPLC
run (not shown) and sequenced. The identification of phosphorylated
peptides is compiled in Table 2, e.g. spot 2 contains
the CGS motif of repeat 3, spot 3 contains the IGS motif of
repeat 1, spots 1 and 4 are peptides outside the
repeats.
Figure 4:
Identification of phosphorylation sites on
MAP4-BDC and MAP4. 200 µg of MAP4-BDC and 20 µg of full-length
MAP4 were phosphorylated with 0.5 microunits of p110 for 2 h at 37° C. After performic acid oxidation and
trypsin digestion, peptides were analyzed by TLE/TLC (A-D) and
HPLC (E). A, MAP4 fragment MAP4-BDC, expressed in E. coli. B, full-length MAP4 (isolated from mouse
tissue). C, diagram of the more prominent spots with
identification of the HPLC-purified and sequenced phosphopeptides (see Table 3). Spot 3 contains Ser
(KXGS in repeat 1), spot 7, Ser
(KXGS in repeat 4). D, a mixture of A and B showing that the major sites on MAP4 are localized
within the MAP4-BDC construct. E, separation of the tryptic
digest of phosphorylated MAP4-BDC by reversed phase HPLC
(C
). Radioactive fractions were purified by a second HPLC
run (not shown) and sequenced. The identification of phosphorylated
peptides is compiled in Table 3, e.g. spot 3 contains
the VGS motif in repeat 1, spot 7 contains the VGS motif of
repeat 4.
In the experiment shown
in Fig. 5the concentration of tubulin was 10 µM to
ensure that microtubules did not self-assemble. However, microtubules
nucleated and grew upon addition of native MAP4 prepared from brain (Fig. 5A, open circles), native MAP2 (Fig. 5B, open circles), or recombinant MAP2c (Fig. 5C, open circles). In these control experiments,
p110 was added together with the MAPs but without ATP so
that phosphorylation could not proceed. In a parallel experiment under
otherwise identical conditions, 1 mM MgATP was added together
with the kinase. The effect of the phosphorylation by p110
appeared rapidly (Fig. 5, A-C, closed circles).
After about 5 min, growth is largely inhibited. Some microtubule
nucleation initially took place while the MAPs were not yet
phosphorylated, but polymerization after this time was suppressed due
to progressive phosphorylation of the MAPs. In another type of
experiment, the MAPs were phosphorylated by p110
for 30
min prior to their addition to tubulin (Fig. 5, A-C,
triangles). In this case, nucleation was also abolished. However,
tubulin could still form polymers, as short microtubules of about 2
µm length could be observed when axonemes were added to promote
nucleation. Analysis of mutant forms of MAP2c shows that the loss of
binding capacity depends on the phosphorylation of both Ser
in repeat 1 and Ser
in repeat 3 (Fig. 5D). If only one of these sites is mutated,
microtubule growth is still induced by the phosphorylated mutants.
Figure 5:
Effects of unphosphorylated and
p110-phosphorylated MAP4 (A), MAP2 (B), MAP2c (C), and MAP2c point mutants (D)
on the length of self-nucleated microtubules measured by dark field
microscopy. For each condition 500-800 microtubules were
analyzed, and the mean length were plotted against time. Tubulin
concentration was 10 µM in all cases, the concentration of
MAP4 and MAP2 was 1 µM, that of MAP2c, 2 µM.
In control experiments, ATP was omitted (-ATP). Open
circles in A-C: the MAPs were preincubated for 30 min
with 2.5 milliunits/ml p110
(final
concentration), but without ATP. By adding 10 µM tubulin,
microtubules were nucleated and the mean microtubule length increased
up to about 20 µm within 30 min. If ATP was present no
self-nucleation occurred, showing that the phosphorylation of the MAPs
prevented microtubule formation. Short microtubules of about 2 µm
length could only be observed by adding axonemes (10-100 fmol) to
promote seeded nucleation (open triangles in A-C). Closed circles in A-C: tubulin and MAP were mixed at
4 °C with 2.5 milliunits/ml of p110
(final
concentration) and 1 mM ATP, and the temperature was shifted
immediately to 37 °C (so that initially the MAPs were
unphosphorylated). Microtubule growth was promoted in all three cases,
but the final mean microtubule length was only about half of that
observed for the unphosphorylated MAPs (compare to open
circles). D, the effect of phosphorylation site point
mutations of MAP2c. All proteins were preincubated with kinase and ATP
as described above. Triangles, wild type MAP2c; closed
circles, MAP2cA319 (KXGS in repeat 1 mutated to
KXGA); squares, MAP2cA350 (KXGS in repeat 3
mutated to KXGA); closed squares, MAP2cA319/A350
(KXGS in both repeats mutated to
KXGA).
The length histograms show the distribution of microtubule lengths at 5 min (Fig. 6, A-C) and 30 min (Fig. 6, D-F) after the addition of MAP and kinase. At 5 min, where the microtubules are still in the growing phase, the length distributions, peaking around 10 µm, are comparable in the presence or absence of ATP (kinase active or inactive, Fig. 6, A-C, open and closed circles). After 30 min the distribution of the control microtubules (no ATP) has become broader (Fig. 6, D-F, closed circles). However, incubation with ATP strongly suppresses long microtubule and shifts the distribution to short lengths (open circles in Fig. 6, D-F).
Figure 6:
Microtubule length histograms obtained at
5 min (A-C) and 30 min (D-F) derived from the
experiments shown in Fig. 5(open and closed
circles). Each sample shows a pronounced peak at around 10 µm
after 5 min (closed circles in A-C). If Mg-ATP was
absent (closed circles in D-F), the distribution
became broader and shifted to greater length at 30 min. By contrast,
phosphorylation of the MAPs with p110successfully decreased the mean microtubule length within 30 min
of incubation (open circles in D-F). n,
number of microtubules analyzed.
In summary, the
results show that phosphorylation by p110 has similar
dramatic effects on the function of MAP2c, MAP2, and MAP4. Microtubule
stabilization is progressively impaired when the kinase and ATP are
added together with the MAP to the tubulin sample. Moreover,
pre-phosphorylated MAPs are not able to support microtubule growth or
even nucleation. These effects are comparable to the previously
reported effects of p110
phosphorylation on tau (Drewes et al., 1995).
Figure 7:
Effect of the phosphorylation by
p110 on the binding of recombinant wild type MAP2c and
MAP2c point mutants to taxol stabilized microtubules (30 µM tubulin dimers). The mutations are Ser to Ala at positions 319
and/or 350 of MAP2c, corresponding to 1682 and 1713 in the full MAP2
sequence (Table 1). Open circles, non-phosphorylated
wild-type MAP2c. The binding is tight (K
about 0.25 µM) and saturates around 17
µM ligand (
1 MAP2c molecule per 2 tubulin dimers). Closed circles, wild-type MAP2c, phosphorylated previously
with p110
(2.5 milliunits/ml) for 2 h. Note
that there is essentially no binding. Closed and open
squares, MAP2cA319 and MAP2cA350, phosphorylated previously with
p110
(2.5 milliunits/ml) for 2 h. The affinity
to microtubules has decreased markedly (K
7 µM) although the stoichiometry remains
similar to the wild type MAP2c. Triangles, MAP2cA319/A350,
phosphorylated previously with p110
(2.5
milliunits/ml) for 2 h. The binding is similar to the unphosphorylated
protein, showing that the sensitivity to phosphorylation has
disappeared.
Our results imply that the role of MARK in regulating MAP
interactions with microtubules may be more general than expected.
Because tau, localized primarily to axons, and MAP2, distributed in
dendrites, are both substrates, MARK or related kinases could be active
in different neuronal cell compartments. An even more general role is
implied by the results with MAP4, since this ubiquitous MAP has been
inferred to affect microtubule stability in dividing cells (Bulinski
and Borisy, 1980; Parysek et al., 1984; Chapin and Bulinski,
1994; Olson et al., 1995). Thus far it has been difficult to
determine what combination of MAPs, phosphorylation sites, kinases, and
other factors are responsible for the pronounced increase in
microtubule dynamics during mitosis. MAP4 was considered a likely
candidate, as well as other related ones (e.g. XMAP from Xenopus eggs, Faruki and Karsenti(1994)). Regarding kinases,
cdc2 and MAP kinases were suggested as potential triggers of
microtubule reorganization (Gotoh et al., 1991; Verde et
al., 1992; Lieuvin et al., 1994; Ookata et al.,
1995). However, it remains to be seen whether these kinases act
directly or via other intermediate steps. The weak effect of
proline-directed phosphorylation on microtubule dynamics makes us
believe that other kinases, such as MARK, may be involved. In this
regard it is interesting that MARK is itself activated by
phosphorylation, pointing to other kinase(s) upstream in the signaling
pathway. ()
The phosphorylation of MAPs has been studied
by a number of authors, and it is pertinent to ask how the results
compare with ours. In most cases it was concluded that phosphorylated
MAPs bound less tightly to microtubules and supported their assembly
less efficiently (although exceptions were also noted, see Brugg and
Matus, 1991). However, in the majority of studies, the phosphorylation
sites involved in the regulation were not known, and indirect
information, such as kinase consensus motifs, are not reliable (as
illustrated for tau and CaM kinase by Steiner et al.(1990), or
for MAP4 and cdc2 by Ookata et al., 1995). There are, however,
a few cases where phosphorylation sites have been determined directly.
Examples include the sites in MAP2 altered by PKC (Ainsztein and
Purich, 1994), or the sites on tau phosphorylated by several kinases
(PKA, PKC, Ca/calmodulin dependent kinase II, and the proline-directed
kinases MAPK, GSK-3, cdc2, and related kinases, see below). While
additional parameters need to be measured for the influence of
site-specific modifications on microtubule dynamics to be rigorously
assessed, the observations with tau (the MAP studied most
comprehensively) allows a distinction to be made between the sites
within and outside the repeat domain. The sites outside the repeats
examined so far have either no effect on microtubule binding and
dynamics, or only a moderate effect, reducing the stabilizing power of
tau from ``high'' to ``medium'' (in the
classification of Trinczek et al.(1995)). This includes the
many Ser-Pro or Thr-Pro sites (phosphorylated by proline-directed
kinases), as well as PKA or Ca/calmodulin dependent kinase II sites
(Steiner et al., 1990; Scott et al., 1993; Brandt et al., 1994). Inside the repeats there are the KXGS
motifs affected by MARK. One of these (Ser in repeat 1)
eliminates the stabilizing power of tau, the others have only a
modulatory influence. The KXGS motifs of tau can also be
phosphorylated to some extent by PKC (Ser
in repeat 3,
Correas et al., 1992), PKA (mostly Ser
and
Ser
in repeats 3 and 4, Scott et al.(1993) and
Drewes et al.(1995)), and GSK-3 when activated by heparin
(Ser
in repeat 1, Song and Yang(1995)). (
)In vivo the phosphorylation at KXGS
motifs is normally low (Seubert et al., 1995), consistent with
its tight association with microtubules. This implies that the kinases
affecting KXGS motifs are normally down-regulated.
The
results on the other MAPs echo those of tau. Phosphorylation sites
outside the repeats may reduce the interaction with microtubules, but
they do not eliminate it. For MAP4, this includes the sites Ser and Ser
in the proline-rich domain (our numbering,
see Table 1) which are potential targets of cdc2 (Ookata et
al., 1995). Sites inside the repeats include the KGXS
motifs which cooperate to eliminate the interaction with microtubules
(see Fig. 7). They also include reported PKC sites in MAP2 at
serines 1705, 1713, and 1730 (our numbering, Fig. 1; see
Ainsztein and Purich, 1994). The second of these is in the
KXGS motif of repeat 3. The example illustrates how PKC could
exert a modulatory effect by phosphorylating one KXGS motif,
while MARK would eliminate microtubule interactions by phosphorylating
two motifs (in 1 and 3). In the case of MAP4, point mutants of
KXGS motifs are not available, but in analogy with MAP2 and
tau we expect that the full inhibition of microtubule binding by MARK
resides in repeats 1, 4, or both. Other phosphorylation sites of MAP4
have not been determined thus far.
Most studies on MAPs emphasize
their role as microtubule stabilizers, but it is worth noting that they
have at least two additional functions. One is their role as
``spacers'' between microtubules and other cellular
components (Chen et al., 1992). This is achieved mainly by the
acidic N-terminal domain which may be short (as in MAP2c or tau) or
long (as in MAP2 or MAP4). A third function is that of a docking site
for cellular enzymes, including kinases and phosphatases or their
cofactors (PKA, cdc2, PKC-, MAP kinase, PP-1, see Obar et
al.(1989), Mandelkow et al.(1992), Baumann et
al.(1993), Ookata et al.(1995), Lehrich and
Forrest(1994), Reszka et al.(1995), and Sontag et
al.(1995)). It is intriguing to speculate that the docked kinases
may be activated by some signaling cascade and then phosphorylate their
host protein or others nearby, thus modulating their association with
the microtubule cytoskeleton.
It still needs to be explained why certain sites in the KXGS motifs can have a major effect (e.g. in repeat 1 of tau, or in 1 and 3 of MAP2), independently of whether other KXGS motifs are phosphorylated as well. This conceptual difficulty could be overcome if one abandons the traditional models of MAP-microtubule interactions. These models have assumed that the repetitive elements in the MAP sequence correspond to the repetitive nature of the microtubule lattice, i.e. each repeat was thought to interact with a different tubulin subunit, but in an equivalent manner. In this picture one would expect that the phosphorylation of one repeat might release it from its subunit but leave the other repeats in place, and consequently one would expect that several phosphorylation sites would have to be combined before the MAP detaches from the microtubule.
There is, however, an alternative view: the repeats could be folded into a coherent structure, or ``buttoned up,'' such that they could link tubulin subunits in adjacent protofilaments on the microtubule surface. This interaction could be disrupted if the folding of the repeat domain were destroyed. Imagine a folded structure formed by several repeats of the MAPs in which the ``button'' was formed by several unphosphorylated KXGS motifs which could be ``unbuttoned'' by phosphorylation. Within this model, the positions of the most critical residues might be variable and depend on details of the surrounding sequence. This might explain the predominant role of the phosphorylation in repeat 1 of tau, compared with the cooperation between 1 and 3 in MAP2 or between 1 and 4 in MAP4. There is increasing evidence for a folded structure in the repeats: the two cysteines in repeats 2 and 3 of tau are in close proximity (Schweers et al., 1995), and the reaction of certain antibodies can only be explained by discontinuous epitopes involving a folded repeat domain (Lichtenberg-Kraag et al., 1992). Interestingly, the accessibility of the core of Alzheimer PHFs to proteases is also explained if one assumes that the repeats of tau are folded up; the result is that the resistant core is formed by peptides roughly equivalent to three repeats, but in different combinations (end of 1 plus 3, 4, 4a, or end of 1 plus 2, 3, 4, Jakes et al. (1991)). The details of this structure are not yet known, but it will be crucial for an understanding of the MAP-microtubule interaction in molecular terms.