(Received for publication, November 2, 1994; and in revised form, January 5, 1995)
From the
Aberrant phosphorylation of the microtubule-associated protein tau is one of the pathological features of neuronal degeneration in Alzheimer's disease. The phosphorylation of Ser-262 within the microtubule binding region of tau is of particular interest because so far it is observed only in Alzheimer's disease (Hasegawa, M., Morishima-Kawashima, M., Takio, K., Suzuki, M., Titani, K., and Ihara, Y.(1992) J. Biol. Chem. 26, 17047-17054) and because phosphorylation of this site alone dramatically reduces the affinity for microtubules in vitro (Biernat, J., Gustke, N., Drewes, G., Mandelkow, E.-M., and Mandelkow, E.(1993) Neuron 11, 153-163). Here we describe the purification and characterization of a protein-serine kinase from brain tissue with an apparent molecular mass of 110 kDa on SDS gels. This kinase specifically phosphorylates tau on its KIGS or KCGS motifs in the repeat domain, whereas no significant phosphorylation outside this region was detected. Phosphorylation occurs mainly on Ser-262 located in the first repeat. This largely abolishes tau's binding to microtubules and makes them dynamically unstable, in contrast to other protein kinases that phosphorylate tau at or near the repeat domain. The data suggest a role for this novel kinase in cellular events involving rearrangement of the microtubule-associated proteins/microtubule arrays and their pathological degeneration in Alzheimer's disease.
Microtubule-associated proteins (MAPs) ()regulate the
extensive dynamics and rearrangement of the microtubule network, which
is thought to drive neurite outgrowth (recently reviewed by
Hirokawa(1994) and Kosik and McConlogue(1994)). Several lines of
evidence suggest that the phosphorylation state of MAPs, balanced by
protein kinases and phosphatases in a hitherto unknown way, plays a
pivotal role in the modulation of these events. Tau protein, a class of
MAPs stabilizing microtubules in mammalian brain (Cleveland et
al., 1977; Drubin and Kirschner, 1986), is phosphorylated on
several sites in vivo (Butler and Shelanski 1986; Watanabe et al., 1993) and is a substrate for many protein kinases in vitro (for review, see Lee(1993), Goedert(1993), Mandelkow
and Mandelkow(1993), and Anderton(1993)). During neuronal degeneration
in Alzheimer's disease, tau protein aggregates into paired
helical filaments (PHFs), the principal fibrous components of the
characteristic neurofibrillary lesions (for review, see Lee and
Trojanowski(1992)). Tau isolated from these aggregates displays some
biochemical alterations, of which hyperphosphorylation is the most
striking (Grundke-Iqbal et al., 1986; Brion et al.,
1991; Ksiezak-Reding et al., 1992; Goedert et al.,
1992). Most of the reported aberrant phosphorylation sites are
Ser/Thr-Pro sequences (Lee et al., 1991; Biernat et
al., 1992; Lichtenberg-Kraag et al., 1992; Watanabe et al., 1993), suggesting a dysregulation of proline-directed
kinases (Ishiguro et al., 1991; Drewes et al., 1992;
Mandelkow et al., 1992; Hanger et al., 1992; Vulliet et al., 1992; Baumann et al., 1993; Paudel et
al., 1993, Kobayashi et al., 1993) or the corresponding
phosphatases (Drewes et al., 1993; Gong et al.,
1994). Phosphorylation-dependent antibodies, which discriminate between
``normal'' tau and the hyperphosphorylated,
``pathological'' forms, were prepared by several laboratories
(Kondo et al., 1988; Lee et al., 1991; Mercken et
al., 1992; Greenberg et al., 1992). All of these
antibodies were shown to be directed against epitopes of the
Ser/Thr-Pro type (Biernat et al., 1992; Lichtenberg-Kraag et al., 1992; Lang et al., 1992; Watanabe et
al., 1993).
The microtubule binding region of tau (Fig. 1) includes three or four pseudorepeats of 31 residues each (Ennulat et al., 1989, Lee et al., 1989), depending on isoform type (Goedert et al., 1989; Himmler et al., 1989). This region probably forms the building block of the paired helical filaments (Kondo et al., 1988; Wischik et al., 1988; Ksiezak-Reding and Yen, 1991; Wille et al., 1992). It does not contain any of the 14-16 Ser/Thr-Pro motifs, which cluster in the regions flanking the repeats. However, it contains a conserved serine residue (Ser-262) within the sequence KIGS in the first repeat, which we found to be one of the predominant sites phosphorylated by a tissue extract from brain (Gustke et al., 1992). This site is also found to be phosphorylated in Alzheimer PHF-tau, but not in normal tau or fetal tau (Hasegawa et al., 1992). So far, it is the only pathological phosphorylation site found within the repeat domain of tau.
Figure 1: Bar diagram of human tau (isoform htau40, the largest one in central nervous tissue (Goedert et al., 1989), construct K18 containing the four repeats and several sites phosphorylated by the brain extract (Gustke et al., 1992). The hatchedboxes near the N terminus are inserts that may be absent because of differential splicing, the boxes labeled 1-4 represent the four repeats, of which repeat 2 may be absent. Most phosphorylated sites are in Ser-Pro or Thr-Pro motifs outside the repeats, but the brain kinase activity also phosphorylates two sites within the repeats, Ser-262 and Ser-356.
Recently, we used a site-directed
mutagenesis approach to show that phosphorylation of tau at this site
strongly decreases its microtubule binding capacity, whereas the
phosphorylation on Ser/Thr-Pro motifs had only a minor effect (Biernat et al., 1993). This initiated a search for protein kinases in
neuronal tissue with the ability to phosphorylate tau at Ser-262. In
our previous work, we had partially purified a kinase activity
consisting of two components with apparent molecular masses of 35 and
41 kDa. Here we report the characterization of a novel kinase of 110
kDa, termed p110 (for MAP/microtubule
affinity-regulating kinase). This kinase phosphorylates all four
KXGS motifs in the repeat domain of tau, particularly the
first one containing Ser-262, and thus efficiently causes the
detachment of tau from microtubules and the subsequent destabilization
of microtubules, as measured by their higher dynamic instability.
Because of this, p110
is a good candidate for
regulating the dynamics and rearrangements of microtubules in cells via
the phosphorylation of tau or other MAPs.
Six chromatographic steps were
used to achieve a =10,000 fold purification of a
Ser-262-phosphorylating activity from porcine brain. As shown in detail
in Fig. 2, we employed phosphocellulose (A), ion
exchange chromatography on Q- and SP-Sepharose and Mono Q (B-D), gel filtration (E), and, finally,
affinity chromatography using immobilized ATP. When using tau construct
K18 as a substrate (comprising only the repeat region), the activity of
kinase(s) in the tissue extract was approximately 0.2 milliunits/mg of
protein, the activity of the affinity-purified kinase was approximately
2 units/mg (1 unit transfers 1 µmol of phosphate/min at 37 °C,
measured with 0.5 mM substrate and 1 mM ATP). The
apparent molecular weight of the enzyme was around 90-100 kDa by
gel filtration, but the activity peak was broad and showed pronounced
tailing (Fig. 2E). On SDS gels, the apparent molecular
mass was approximately 110 kDa (Fig. 3). The enzyme could be
renatured in the gel; if tau was polymerized into the gel matrix as a
substrate and the gel was incubated with
[
-
P]ATP, the 110 kDa band became prominent
upon autoradiography (Fig. 3, lanes4-6), whereas some minor contaminations observed in
the silver-stained gel had no detectable activity. After
phosphorylation with the 110-kDa kinase, both whole tau and construct
K18 showed small but distinct mobility change in SDS-polyacrylamide gel
electrophoresis (Fig. 4, lanes1-4). The
final amount of incorporated phosphate is approximately 1.8-2.5
mol/mol of tau, depending somewhat on enzyme concentration and
activity; this level of phosphorylation could be achieved after
approximately 2 h.
Figure 2:
Isolation of p110 from porcine brain. A, the tissue extract was
loaded onto phosphocellulose and eluted stepwise with 0.15-1 M NaCl. The shadedbars show the total
protein concentration of the eluted material; openbars show the activity as measured with tau construct K18 as substrate. B, the material eluted with 0.35-0.5 M NaCl was
submitted to ammonium sulfate precipitation, and the precipitate was
dialyzed and loaded onto a Q-Sepharose column. The closedsymbols show the protein concentration, and opensymbols show the activity profile. The gradient
composition is indicated on the rightaxis. C, fractions 8-15 from Q-Sepharose were dialyzed and
loaded onto a SP-Sepharose column. D, fractions 12-16
from SP-Sepharose were dialyzed and loaded onto a Mono Q HR 5/5 column. E, fractions 9-11 from Mono Q were loaded onto a
Superdex 200 gel filtration column. The elution positions of molecular
weight markers are indicated on the rightaxis.
Figure 3:
Final purification of p110 by affinity chromatography on ATP-Sepharose and
characterization by in-gel phosphorylation. The most active fractions
from the gel filtration column (lane1) were loaded
onto an ATP affinity column. The kinase was eluted specifically with 5
mM ATP (lanes2 and 3). The
silver-stained gel shows a fuzzy band with an apparent molecular mass
of approximately 110 kDa and a second, sharp band of approximately 95
kDa. Lanes4-6 show autoradiograms of the
in-gel phosphorylation of the samples in lanes1-3. As a substrate, tau (5 µM) was
polymerized into a 8.5% acrylamide gel matrix. After renaturation and
incubation with [
-
P]ATP, it is clearly
shown that only the 110 kDa band displays kinase activity toward
tau.
Figure 4:
Phosphorylation of wild-type tau and
construct K18 (microtubule binding domain) by
p110. Htau40 (10 µM, lanes1 and 2) and K18 (20 µM, lanes3 and 4) were phosphorylated with 5
milliunits/ml of p110
and 2 mM [
-
P]ATP at 37 °C for 2 h. Aliquots
were electrophoresed on a 7-20% SDS gradient gel. Lanes1 and 2, htau40 before and after
phosphorylation; lanes3 and 4, K18 before
and after phosphorylation. Note the small molecular weight shift upon
phosphorylation in lanes2 and 4. The rightpanel shows an autoradiograph of the same gel;
phosphorylated htau40 and K18 are seen in lanes2 and 4.
The specificity of the novel kinase for tau was
examined by tryptic digestion of phosphorylated protein and subsequent
two-dimensional high voltage thin layer electrophoresis/thin layer
chromatography (Fig. 5, A-D). If one compares the
phosphorylation patterns obtained from recombinant full-length 4-repeat
tau (Fig. 5A) and the 4-repeat fragment K18 (Fig. 5B), it is apparent that most phosphorylated
peptides are generated from the repeat domain. This was confirmed by
analysis of a mixture of both samples (Fig. 5D). In a
second approach, the tryptic digest was resolved by HPLC (Fig. 5E). This yielded several labeled peptides that
were analyzed by phosphopeptide sequencing. The results are compiled in Table 1. Most of the radioactivity was found in a peptide
containing phosphorylated Ser-262. Ser-356 (in the KIGS motif of the
fourth repeat) and Ser-324 (from the KCGS motif of the third repeat)
were also found. Two-dimensional analysis of these purified peptides
lead to the identification of spots shown in Fig. 5C.
This clearly shows that Ser-262 (spot1) is the main
target site of p110 on tau, followed by Ser-356 (spot2). Spot3 was identified as
the peptide containing Ser-305, spot4 as Ser-324 (in
the KCGS motif of the third repeat), and spot5 as
Ser-293 (in the KCGS motif of the second repeat).
Figure 5:
Tryptic phosphopeptide maps obtained by
two-dimensional thin layer electrophoresis/thin layer chromatography of
wild-type tau and construct K18 phosphorylated with
p110. 30 µg of tau were phosphorylated with
0.5 milliunits of p110
for 2 h at 37 °C. A, full-length 4-repeat tau (htau40); B, construct
K18 (MT binding region, residues 244-372 of full-length tau); C, diagram of the more prominent spots (spot1 on upperleft contains Ser-262, spot2 on upperright Ser-356, spot3 (below 1) Ser-305, spot4 (always part of an overlapping doublet) contained Ser-324, spot5 Ser-293; D, mixture of identical
amounts of counts (10,000 cpm) derived from phosphopeptides shown in A and B; E, HPLC run of the tryptic digest.
Incorporated radioactivity is shown below the elution profile, and the
phosphorylated residues obtained by sequencing are indicated. The
identification of phosphorylation sites shown in C was
performed by two-dimensional analysis of the HPLC-purified and
sequenced peptides (Table 1). 10,000 cpm of the peptides each
were mixed with a 5000-cpm aliquot of the digest shown in A and analyzed by thin layer electrophoresis/thin layer
chromatography in order to allow unambiguous
identification.
Figure 6:
Phosphorylation of Ser-262 abolishes the
binding of tau to microtubules. A, binding of tau to
taxol-stabilized microtubules (30 µM) was measured in a
cosedimentation assay as described under ``Materials and
Methods.'' Full-length wild-type tau (wt, htau40) and a
Ser-262 Ala mutant (A262) (10 µM) were
previously phosphorylated with p110
(final
concentration 8.5 milliunits/ml) for 2 h at 37 °C. Curves were
obtained by nonlinear regression (Biernat et al., 1993). The
binding of wild-type tau is completely abolished by phosphorylation (closedcircles), whereas the A262 mutant still
binds, although with lower affinity (triangles). For
comparison, the binding of unphosphorylated tau is also shown (opencircles). B, microtubule-bound tau comes off
during phosphorylation by p110
. htau 40 (10
µM) was incubated with taxol-stabilized microtubules (30
µM). At t = 0, p110
was added to a final concentration of 10 milliunits/ml, and
aliquots were withdrawn at time intervals from 1 to 20 h and pelleted.
Tau was measured in the pellets and supernatants by densitometry of the
SDS gels (closedcircles). Incorporated phosphate was
measured by Cerenkov counting of gel pieces (opencircles) and is indicated on the rightaxis. Phosphate incorporation in tau without microtubules
is shown to proceed faster (squares).
In order to verify
this result, Ser-262 was mutated into Ala. In this case, incubation
with p110 for 2 h left the microtubule binding capacity
largely intact, although there was some decrease in affinity and
stoichiometry (
25%, Fig. 6A), probably due to
phosphorylation of Ser-356 (see Fig. 5). This confirms two
points of our previous study, (i) phosphorylation of Ser-262 is the
major switch controlling tau's affinity for microtubules, (ii)
other sites phosphorylated by the kinase have a small but measurable
effect on the binding (i.e. mainly the equivalent Ser-356 in
repeat 4).
Our next question was do microtubules protect tau from
being phosphorylated by p110? If this were the case,
then tau (once bound to microtubules) might retain its high affinity
for microtubules. To answer this point, taxol-stabilized microtubules
were first saturated with tau and then incubated with
p110
. As illustrated in Fig. 6B, tau
gradually dissociates from microtubules, concomitant with
phosphorylation. Thus microtubules retard phosphorylation of tau by the
kinase but cannot prevent it.
One important function of tau is to
stabilize microtubules and suppress their dynamic instability (Drechsel et al., 1992). Thus, if tau loses its binding to microtubules,
one would expect stable microtubules to become dynamic. This effect can
be illustrated by video dark-field microscopy of individual
microtubules seeded onto flagellar axonemes (Fig. 7). In the
experiment of Fig. 8A, the concentration of tubulin (5
µM) was chosen such that microtubules did not assemble by
themselves but grew upon the addition of (unphosphorylated) tau (Fig. 8A, opencircles). Tau
phosphorylated with p110 did not support growth, whereas
the phosphorylated Ser-262
Ala mutant did. Even more dramatic is
the conversion of microtubules from undynamic to dynamic behavior under
the influence of the kinase. In the experiment of Fig. 8B, we allowed microtubules to grow off axonemes
in the presence of tau and recorded their mean length, which increased
to approximately 50 µm over 20 min (similar to Fig. 8A, opencircles). When
p110
and ATP were added together with tau, the mean
length increased only to 20 µm and then dropped again, due to the
gradual phosphorylation of tau and concomitant increase in microtubule
dynamics (filledcircles). When we used the point
mutant, tau-A262, microtubules grew normally even when the kinase and
ATP were present (triangles). Only after prolonged incubation
with the kinase or with prephosphorylated tau-A262 (as in Fig. 8A), a small increase in dynamics is observed,
comparable with the small decrease in binding, which is seen with
A262-tau in cosedimentation assays (see Fig. 6A). Both
can be explained by an increase with time of phosphorylation at other
KXGS motifs. These results are summarized in the length
histograms of Fig. 8, C-D. At early times after
initiation of assembly, microtubules are short and rather homogeneous
in length (peaks of opencircles at 5 min), at later
times of uninterrupted growth the microtubules become long and show a
broad length distribution (Fig. 8, C and E, filledcircles). However, when p110
is
allowed to phosphorylate tau at Ser-262, microtubules remain short (Fig. 8D, filledcircles).
Figure 7:
Dark-field video microscopy of
microtubules and effect of Ser-262 phosphorylation on tau. Microtubules
(5 µM tubulin) were nucleated on sea urchin sperm axonemes
in the presence of 2.5 µM tau (isoform htau40) and 10
milliunits/ml of p110. a, 20 min
without ATP; b with ATP. In a, the microtubules grow
continuously; in b, Ser-262 can be phosphorylated, leading to
a destabilization and shortening of microtubules. Bar, 10
µm.
Figure 8:
Effect of the unphosphorylated and
p110-phosphorylated tau on the length of
axoneme nucleated microtubules measured by dark-field microscopy. For
each condition 500-600 microtubule plus ends were measured; the
mean length was plotted against time. For half-widths of length
distribution, see C-E. Tubulin concentration
was 5 µM; note that without added tau, no microtubules are
observed at this concentration. Tau was 2.5 µM in all
cases. A, microtubule assembly is initiated by the addition of
unphosphorylated wild-type tau (opencircles). Tau
pre-phosphorylated by p110
for 2 h does not
promote microtubule growth (filledcircles), but the
prephosphorylated point mutant tau-A262 does (triangles), in
accordance with time resolved binding assay in B. B,
tubulin and wild-type tau (circles) or tau-A262 (triangles) were mixed at 4 °C with 10 milliunits/ml of
p110
and 2 mM MgATP (final
concentrations). At t = 0, the temperature was raised
to 37 °C. With wild-type tau, initial growth but subsequent
shrinkage of microtubules is observed, whereas with the point mutant
Ser-262
Ala, microtubules grew continuously. C-E, microtubule length histograms at 5 min and
30 min of the corresponding curves in B. Each sample shows a
pronounced peak around 20 µm after 5 min (emptycircles). If MgATP was omitted (C) or Ser-262
was mutated into Ala (E), the distribution became broader and
shifted to greater lengths at 30 min. By contrast, phosphorylation of
tau successfully decreased the mean microtubule length within 30 min of
incubation (D).
Figure 9:
Tryptic phosphopeptide maps of wild-type
tau (htau 40) and construct K18 phosphorylated with (A) brain
extract, (B) p110, (C) protein kinase
C, or (D) protein kinase A, respectively. The numbering of the
spots is analogous to Fig. 5(spot1, Ser-262; spot2, Ser-356; spot3, Ser-305; spot4, Ser-324; spot5, Ser-293).
The panels on the right show the corresponding
two-dimensional phosphoamino acid analysis of full-length tau for each
kinase.
The patterns shown in Fig. 9A were obtained by phosphorylating full-length tau and K18 with brain extract. With full-length tau, only spot 1 (Ser-262) is clearly seen; spot 2 (Ser-356) is barely visible. This is even more prominent in the phosphorylation pattern of K18. The data are in agreement with our earlier results (Gustke et al., 1992).
When we examine the
phosphorylation of K18 by p110, we find a peptide
pattern similar to that generated by the brain extract (compare Fig. 9, A and B); the most prominent spots are
again 1 and 2, containing Ser-262 and Ser-356. This confirms the role
of p110
as the major Ser-262 kinase in brain extracts.
By contrast, reinvestigation of PK35 has so far yielded inhomogeneous
results. Although it phosphorylates the same serines as
p110
, the weighting is different, and the activity of
the kinase in brain extracts is at least 10-fold lower (data not
shown). This explains why even prolonged incubations of tau with this
kinase activity lead to only partial suppression of tau's binding
to microtubules, as described earlier.
As seen in Fig. 9C, protein kinase C only phosphorylated Ser-305 (spot3), Ser-324 (spot4) and Ser-293 (spot5) to a significant extent in K18, but not Ser-262, which is in agreement with Correas et al.(1992). The smear and outermost spot to the left (arrow) are not phosphopeptides derived from tau since they also occurred in control experiments where no tau had been added (not shown). The remaining two spots could not be identified; the spot on the upper right (asterisk) did not colocalize with either Ser-262 (spot1) or Ser-356 (spot2). Comparison of this pattern with one obtained from full-length tau revealed that the major phosphorylation sites of protein kinase C are outside the repeat domain. Only Ser-305 (spot3) was faintly visible in this pattern (note that the spot on the upper right does not correspond to the upper right spot from K18 (asterisk), as confirmed by control experiments (not shown)).
When using purified protein kinase A to phosphorylate full-length tau and construct K18 (Fig. 9D), we find mainly Ser-356 (spot2), Ser-305 (spot3), Ser-324 (spot4), and Ser-293 (spot5). Spot1 is present but barely visible, showing that Ser-262 is only a very minor phosphorylation site. Phosphorylation of full-length tau (Fig. 9D, leftpanel) yielded similar spots, plus additional sites outside of the repeat region of tau. These result are in general agreement with earlier data (Scott et al., 1993; Steiner, 1993). Some of these sites had also been seen with the 35/41-kDa kinases (Biernat et al., 1993). In subsequent experiments, we have now determined that the 41-kDa component is the catalytic subunit of protein kinase A (using an antibody against protein kinase A obtained from H. Hilz, Hamburg, data not shown); this explains in part the overlap in the data.
Neurons affected by Alzheimer's disease are characterized by a decrease in the number of microtubules and an increased amount of tau protein in a hyperphosphorylated and aggregated state (paired helical filaments). In order to establish the relationship between these observations, it is necessary to identify the phosphorylation sites on tau, the kinases and phosphatases acting on them, and the functional consequences of phosphorylation, such as microtubule binding or aggregation. A number of phosphorylation sites have now been mapped. They include Ser/Thr-Pro motifs that are of diagnostic value because their phosphorylation state can be monitored by antibodies that discriminate between normal and Alzheimer tau (Lee et al., 1991; Lichtenberg-Kraag et al., 1992). These sites are the targets of certain proline-directed kinases and can be cleared by at least two phosphatases (PP-2A and PP-2B) (Drewes et al., 1993; Gong et al., 1994); they are phosphorylated not only in Alzheimer PHFs but also to some extent in fetal tau (Kanemaru et al., 1992; Bramblett et al., 1993; Watanabe et al., 1993). However, from a functional point of view, the importance of these sites is less clear since a major effect on tau-microtubule binding is not observed (Biernat et al., 1993). Since these sites lie mostly in the regions flanking the repeat domains, this would be consistent with the view that the repeats (but not the flanking regions) determine the interactions with microtubules (but see discussion below).
Perhaps the most interesting phosphorylation sites are those that strongly affect tau-microtubule interactions. Thus far, only one such site is known, Ser-262 (Biernat et al., 1993). The significance of this finding is enhanced by the fact that Ser-262 is also uniquely phosphorylated in Alzheimer tau, as shown by Hasegawa et al.(1992) and confirmed in our laboratory (Gross et al., 1994). This prompted our search for kinases and phosphatases that regulate the phosphorylation at Ser-262. The phosphatase part of this search turned out to be relatively simple. Both PP-2A and PP-2B are capable of clearing in vitro all phosphorylation sites of tau we have studied so far, including Ser-262 (Drewes et al., 1993). However, the kinase part of the search was more protracted.
First, it was clear from the beginning that the kinase(s) must be present and active in extracts obtained from normal mammalian brain. When tau is phosphorylated by the extract in the presence of phosphatase inhibitors (such as okadaic acid), the prominent target sites are several Ser/Thr-Pro motifs and the two KIGS motifs in repeats 1 and 4 (Ser-262 and Ser-356; Gustke et al., 1992). Second, some of these motifs or the corresponding KCGS motifs in repeats 2 and 3 can be phosphorylated by protein kinase A or protein kinase C, respectively (Scott et al., 1993; Correas et al., 1992; Steiner, 1993). However, the efficiency of phosphorylation at these sites is low and thus, not surprisingly, the effect of these kinases on tau-microtubule interactions is only minor. Another surprising feature was that in spite of the similarity of these four motifs, only the first one (Ser-262) has the pronounced effect on microtubule interactions (Biernat et al., 1993), a result that we have now confirmed in the present study.
Our previous purification of the Ser-262 kinase had yielded a doublet of proteins at 35 and 41 kDa. We now know that the 41-kDa component is the catalytic subunit of protein kinase A. It phosphorylates Ser-214 (in the N-terminal flanking region); Ser-293, Ser-324, and Ser-356 (the KXGS motifs in repeats 2-4); Ser-409 and Ser416 (in the C-terminal flanking region; Ser-416 is also the main target of CaM kinase); however, Ser-262 is only a minor site (Table 2).
During the continued efforts to improve the purification procedure
and to analyze phosphorylation sites, we detected a novel kinase of
higher M, now termed p110
, which
effectively phosphorylated the microtubule binding domain of tau and
turned out to be of much higher activity toward Ser-262. While the
35/41-kDa kinases required a prolonged incubation time (16 h) to get
appreciable phosphorylation, p110
affected tau binding
already after incubations of 30 min to 2 h. Because of the low yield of
purification, it has not yet been possible to characterize the 35-kDa
component in detail; we note, however, that the M
is just slightly larger than the kinase catalytical domain (Hanks
and Quinn. 1991), leaving the possibility that it is a degradation
product of p110
, which is, in fact, easily degraded
during the purification procedure (data not shown). p110
phosphorylates all four KXGS motifs, the first and
fourth (Ser-262 and Ser-356) being the most pronounced sites. In this
regard, the kinase mimics our earlier observations with the brain
tissue extract (Gustke et al. (1992) and see Fig. 9).
The most dramatic effects of the kinase are that it virtually
eliminates tau's binding to microtubules (Fig. 6B), it causes the release of tau from
microtubules, and it turns stable microtubules into dynamically
unstable ones, as seen by video microscopy. These effects are mainly
dependent on the phosphorylation of Ser-262, as shown by the point
mutant Ser-262
Ala. These features make p110
a
candidate enzyme for controlling the state of assembly of microtubules
in neurons. They are also consistent with the ``Tau Hypothesis of
Alzheimer's Disease,'' which assumes that tau's
failure to bind to and stabilize microtubules leads to their breakdown
and cessation of axonal transport. This could occur either by the
detachment of tau from microtubules or by the inhibition of newly
synthesized tau to bind to microtubules, in both cases resulting from
phosphorylation. According to this scheme, an intervention that would
slow down p110
or turn off its potential activating
cascade would be suitable for a treatment of Alzheimer's disease.
p110 can be renatured in SDS gels, showing that its
active form is a single polypeptide with an molecular mass of
approximately 110 kDa, which is relatively large for a protein kinase.
Its preference for KXGS motifs is reminiscent of protein
kinase C or calcium/calmodulin-dependent kinase II, whose consensus
sequence is (K/R)XXS, and protein kinase A (consensus
RRXS) (Kemp and Pierson, 1990). However, tau possesses several
consensus motifs that do not get phosphorylated by these kinases and
other nonconsensus motifs that do (a case in point being
calcium/calmodulin-dependent kinase II, see Steiner et al. (1990)), so that predictions based on consensus motifs seem of
little value here. We also note that the motif KXGS is
conserved not only within the tau repeats but also within other MAPs
such as the neuronal MAP2 and the ubiquitous MAP4 (for review, see
Chapin and Bulinski(1992)). It is therefore possible that p110
has a more general role, affecting different MAPs and/or other
substrates. Preliminary experiments point in this direction.
We conclude by commenting on two problems that are currently not resolved, the questions of microtubule binding and tau aggregation. The repeat domain of tau is usually considered as the microtubule binding domain (Butner and Kirschner, 1991; Goode and Feinstein, 1994); however, it is also known that the repeats alone bind poorly to microtubules (Ennulat et al., 1989; Joly and Purich, 1990), and several reports pointed to the importance of the flanking regions (e.g. Lee and Rook(1992) and Chen et al. (1992)). We have recently studied a set of tau constructs with different combinations of domains and confirmed that the repeats alone (such as construct K18) bind rather poorly, whereas repeatless tau binds strongly, albeit nonproductively in terms of microtubule polymerization (Gustke et al., 1994). These results can be rationalized by considering the flanking regions as ``jaws'' that position tau in the proper binding conformation. In this context it is surprising that the many phosphorylatable Ser/Thr-Pro motifs that lie in the strongly binding flanking regions of the repeats have such a weak influence on microtubule binding or assembly, whereas the single Ser-262, which lies in the weakly binding repeat domain, has a uniquely strong influence on microtubule binding. It is possible that phosphorylated Ser-262 prevents the approach of tau into its binding site on the microtubule surface (as illustrated in Fig. 10). Alternatively, Ser-262 might control a conformation of tau that is important for binding, and is acquired only upon docking onto the microtubule surface. This would be consistent with the fact that certain ``conformation-sensitive'' antibodies require the repeat domain plus phosphorylation on either one of the two flanking regions (Lichtenberg-Kraag et al., 1992).
Figure 10: Diagram representing the influence of different phosphorylation sites on tau-microtubule interactions. The majority of Ser/Thr-Pro motifs are in the flanking regions of the repeat domain, they have only a small influence on the binding of tau. The repeat domain contains several phosphorylatable non-Ser-Pro sites, especially the four KXGS motifs. Of these, Ser-262 in the first KIGS motif has by far the greatest influence on microtubule binding.
The second comment is that tau has an ill-defined structure, unlike a typical folded protein but comparable to a denatured random coil (Schweers et al., 1994). This makes it difficult to imagine how tau could bind specifically to microtubules, and how tau aggregates into the periodic PHF fibers. In particular, the self-assembly of tau into PHF-like fibers can be achieved in vitro with the repeat domain of tau alone (Wille et al., 1992), consistent with the fact that the Pronase-resistant core of PHFs also consists of the repeats (Kondo et al., 1988; Wischik et al., 1988; Ksiezak-Reding and Yen, 1991). PHFs containing full-length tau are hyperphosphorylated, whereas the selfassembly of the repeat domain in vitro does not require phosphorylation. It is therefore currently not possible to relate tau's self-assembly to its phosphorylation (in contrast to the clear relationship between tau's microtubule binding and phosphorylation described here). It is even possible that the hyperphosphorylation of tau observed in PHFs is only of secondary importance for the pathological process. This question can only be solved by testing the self-assembly of tau domains in defined states of phosphorylation.