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Journal of Neurochemistry
Raven Press, Ltd., New York
C 1994 International Society for Neurochemistry
Protein Kinase FA/Glycogen Synthase Kinase-3a After
Heparin Potentiation Phosphorylates Ton Sites Abnormally
Phosphorylated in Alzheimer's Disease Brain
Shiaw-Der Yang, Jau-Song Yu, Shine-Gwo Shiah, and Jun-Jae Huang
Institute of Biomedical Sciences, National Tsing Hua University, Hsinchu, and Institute of Basic Medicine,
Chang Gung Medical College, Tao-Yuan, Taiwan, R .O.C.
Abstract : Previously, we identified protein kinase FA/glycogen synthase kinase-3a (GSK-3a) as a brain microtubule-associated T kinase that phosphorylates Ser 235 and
Ser' o4 of T and causes its electrophoretic mobility shift
in gels, a unique property characteristic of paired helical
filament-associated pathological T (PHF-T) in Alzheimer's
disease brains . In this study, we found that the activity
of kinase F A/GSK-3a towards phosphorylation of brain -r
could be stimulated approximately fourfold by heparin .
The phosphorylation molar ratio was increased simultaneously up to 9 mol of phosphates/mol of T, resulting in
a reduced mobility of T with an apparent molecular mass
shift to -68 kDa in sodium dodecyl sulfate gels, which
is very similar to that observed in Alzheimer-T . Tryptic
digestion of 32 P-labelled T, followed by HPLC and twodimensional separation on TLC cellulose plates, revealed
eight major phosphopeptides. Phosphoamino acid analysis together with sequential manual Edman degradation
and protein sequence analysis further revealed that, in
addition to Ser 235 and Ser 404 , heparin generated Thr 2' 2 ,
Thr 231 , Ser 262 , Ser 324 , and Ser 35s , the five extra phosphorylation sites in T. As Ser 235 , Ser 262 , Ser 324 , Ser 356 , and
Ser 4o4 (particularly the site of Ser 262 ) have been identified
as five of the most potent sites in T responsible for reducing microtubule binding possibly involved in neuronal degeneration, and Thr 231 , Ser 235 , Ser 262 , and Ser 4o4 are four
of the most well documented sites abnormally phosphorylated in Alzheimer-T, the results provide initial evidence
that protein kinase F A/GSK-3a after heparin potentiation
may represent one of the most potent systems possibly
involved in the abnormal phosphorylation of PHF - T in
Alzheimer's disease brains . Key Words : Alzheimer's disease--r protein-Kinase F A/glycogen synthase kinase3a-Heparin -Abnormal sites .
J. Neurochem. 63,1416-1425 (1994) .
al ., 1988 ; Kondo et al ., 1988 ; Wischik et al ., 1988 ;
Lee et al ., 1991) . As compared with normal brain T
protein, the PHF-associated T appeared to be abnormally phosphorylated, which can cause an electrophoretic mobility shift in sodium dodecyl sulfate (SDS)
gels (Greenberg and Davies, 1990; Ksiezak-Reding
and Yen, 1991 ; Lee et al ., 1991) . The PHF-associated T
was found to be abnormally phosphorylated on several
235 ,
262
sites, including Thr"', Ser
and Ser , as demonstrated by Ihara and co-workers using protein sequence
and mass spectrometric analysis of T protein isolated
from Alzheimer's disease brain (Hasegawa et al .,
1992) . On the other hand, Mandelkow and co-workers
235
262
identified Ser , Ser , and Ser 4° (particularly the site
of Ser262) as three of the most potent sites in T responsible for reducing microtubule binding (Gustke et al .,
1992 ; Biernat et al ., 1993) . A reduction in microtubule
binding would lead to destabilization of microtubules
and, hence to a loss in axonal transport to cause neuronal degeneration (Kosik, 1990 ; Goedert et al ., 1991) .
Several protein kinases, such as mitogen-activated protein kinase (MAP kinase) (Drewes et al ., 1992 ; Lichtenberg-Kraag et al ., 1992), T protein kinase I glycogen
synthase kinase-3ß (GSK-3ß) (Ishiguro et al., 1992,
1993), T protein kinase II cyclin-dependent kinase-5
(cdk5) (Ishiguro et al ., 1991 ; Kobayashi et al ., 1993),
proline-directed protein kinase (Vulliet et al ., 1992 ;
Paudel et al ., 1993), and protein kinase FA/glycogen
synthase kinase-3a (GSK-3a) (Hanger et al ., 1992 ;
Mandelkow et al., 1992 ; Yang et al ., 1993b), have
been reported to be capable of phosphorylating some
Received January 14, 1994; revised manuscript received March
7, 1994 ; accepted March 8, 1994 .
Address correspondence and reprint requests to Dr . S.-D. Yang
at Institute of Biomedical Sciences, National Tsing Hua University,
Hsinchu, Taiwan, R.O .C .
Abbreviations used: cdk5, cyclin-dependent kinase-5 ; GSK-3, glycogen synthase kinase-3 ; MAP kinase, mitogen-activated protein
kinase ; PAGE, polyacrylamide gel electrophoresis; PEG, polyethylene glycol ; PHF, paired helical filament; PMSF, phenylmethylsulfonyl fluoride ; SDS, sodium dodecyl sulfate.
Neurofibrillary tangles are one of the major lesions
that accumulate in Alzheimer's disease brain . The
main components of neurofibrillary tangles are the
paired helical filaments (PHFs), which consist mainly
of the microtubule-associated protein T (Brion et al .,
1985 ; Kosik et al ., 1986 ; Yen et al ., 1987 ; Goedert et
1416
MODULATION OF ALZHEIMER-7 - KINASE
of these sites . However, none of the reported T kinases
is capable ofphosphorylating all of these sites (particularly the site of Ser 262) in PHF-T. The action mechanism for the abnormal phosphorylation of PHF-T,
therefore, remains to be established.
Protein kinase FA was identified originally as an
activating factor of type-1 protein phosphatase from
mammalian nonnervous tissues (Yang et al., 1980),
which is identical to GSK-3a (Vandenheede et al.,
1980; Hemmings and Cohen, 1983 ; Woodgett, 1991),
but has been demonstrated subsequently to be a multisubstrate protein kinase existing most abundantly in
the brain (Yang and Fong, 1985 ; Yu and Yang, 1993;
for review, see Yang, 1991) . Protein kinase FA/GSK3a was identified further as a brain microtubule-associated T protein kinase that is able to phosphorylate
Ser 23' and Ser er in T and reduces the electrophoretic
mobility shift of T on SDS-polyacrylamide gel electrophoresis (PAGE), a unique property characteristic of
PHF-T (Greenberg and Davies, 1990; Ksiezak-Reding
and Yen, 1991; Lee et al., 1991), suggesting a possible
involvement of kinase FA/GSK-3a in the abnormal
phosphorylation of pathological PHF-T in Alzheimer's
disease brain (Yang et al., 1991a, 1993b) . The phosphorylation of T by protein kinase FA/GSK-3a also
decreases T's affinity for microtubule and actin filament binding, suggesting a possible involvement in
the functional regulation of the neuronal cytoskeletal
system (Yang et al., 1993a) . Hanger et al . (1992) also
reported that kinase FA/GSK-3a reduced the mobility
of T on SDS-PAGE and generated several PHF epitopes. They localized kinase FA/GSK-3a within granular structures in pyramidal cells, particularly the relative abundance in the CAI subicular areas, illustrating
that kinase FA/GSK-3a may have a role in the pathogenesis of PHF, because these neurons are especially
vulnerable to tangle formation in Alzheimer's disease .
Mandelkow et al. (1992) also reported that kinase FA/
GSK-3a reduced the gel mobility shift of 7 -, generated
several PHF epitopes, and associated with normal brain
microtubules and with PHFs from Alzheimer brain,
suggesting that kinase FA/GSK-3a may have a role in
the induction of the Alzheimer-like state of T . However, as reported by Hanger et al. (1992) and Mandelkow et al. (1992), protein kinase FA/GSK-3a is present
in roughly equivalent amounts in both normal and Alzheimer's disease brains, and there is no obvious difference in the immunolabelling of sections of control
brain and Alzheimer brain . The role of kinase FA/GSK3a possibly involved in the abnormal phosphorylation
of pathological PHF-,r in Alzheimer's disease brain,
therefore, remains to be established .
Here we report that heparin, a polyanion substance,
can potentiate protein kinase FA/GSK-3a towards
phosphorylation of bovine brain T on at least seven
phosphorylation sites, including Thr2 ' 2, Thr23 ', Ser 23s,
Ser 262, Ser 324, Ser 356 and Ser 404, numbered according
to the longest human brain T isoform as described by
Goedert et al. (1989), and causes a reduced mobility
141 7
of T with an apparent molecular mass shift to -68
kDa in SDS gels, which is very similar to that observed
in PHF-T (Lee et al., 1991 ; Goedert et al., 1992), representing one of the most potent systems possibly involved in the abnormal phosphorylation of PHF-T. As
protein kinase FA/GSK-3a is present in roughly equivalent amounts in normal and in Alzheimer's disease
brain (Hanger et al., 1992; Mandelkow et al., 1992),
raising the possibility that deregulation, but not ectopic
expression, of kinase FA/GSK-3a may play an important role in Alzheimer disease pathogenesis, the
polyanion compound-mediated generation of Alzheimer-like -r by kinase FA/GSK-3a reported here presents a new approach to this pathogenic mechanism .
EXPERIMENTAL PROCEDURES
Materials
-32
[1' p]ATP was purchased from Amersham (Buckinghamshire, U.K.). ATP, ammonium bicarbonate, heparin
(grade 1, from porcine intestinal mucosa), and L-1-tosylamide 2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin were from Sigma (St. Louis, MO, U.S.A.). EGTA, 2mercaptoethanol, phenylmethylsulfonyl fluoride (PMSF),
methanol, benzamidinium chloride, trifluoroacetic acid, trichloroacetic acid, dithiothreitol, polyethylene glycol (PEG),
and ferric chloride were from Merck (Darmstadt, F.R.G.).
Phosphocellulose was from Whatman (Maidstone, U.K.). Ultrogel AcA-34 and chelating Sepharose CL 6B (fast flow)
were from LKB (Uppsala, Sweden) . C,$ reverse-phase column (Cosmosil 5C,$-AR, 4.6 x 250 mm) was from Nacalai
Tesque (Kyoto, Japan). Acetonitrile was from J. T. Baker
(Phillipsburg, NJ, U.S.A.). Edman degradation reaction
membrane (GEN 920033), phenylisothiocyanate, and coupling buffer (GEN 920020) were from Millipore (Bedford,
MA, U.S .A.) .
Purification and characterization of kinase FA/
GSK-3cr and T protein
Protein kinase FA/GSK-3a was purified to homogeneity
from porcine brain basically as described in previous reports
(Yang, 1986; Yu and Yang, 1993) . The preparations of kinase FA phosphorylated myelin basic protein with a specific
activity of - 1,600 nmol/min/mg and with a specific activity
of 26,000 nmol/min/mg toward activation of inactive type1 protein phosphatase . When analyzed by SDS-PAGE and
Coomassie Blue staining, the purified kinase FA gave a single
major protein band at M, = 53,000 . Analysis of the radioactively autophosphorylated kinase FA on the autoradiogram
also revealed a single major phosphorylated protein band
at Mr = 53,000 . The enzyme preparations could also be
specifically immunoblotted and immunoprecipitated by an
anti-GSK-3a antibody produced from the peptide TETQTGQDWQAPDA, corresponding to the carboxyl terminal
regions from amino acids 462-475 of the sequence of GSK3a (Woodgett, 1990) . The antibody could specifically immunoblot GSK-3a from crude brain extracts and could not
cross-react with GSK-3ß, as described and demonstrated
in a previous report (Yu and Yang, 1993) . The kinase FA
preparations used in this report, therefore, belong to the category of GSK-3a according to the definition ofWoodgett and
co-workers (Woodgett, 1990; Hughes et al., 1991).
T protein was prepared from bovine brain following the
J. Neurochem., Vol. 63, No. 4, 1994
141 8
S.-D.
YANG ET AL.
procedure of Cleveland et al . (1977) with some modifications . The purification began with homogenization of 400 g
of brain in 2 volumes of solution A (40 mM Tris-HCl, pH
7 .4, 4 mM EDTA, 1 mM EGTA, 0 .1 % 2-mercaptoethanol,
1 mM PMSF, and 1 mM benzamidinium chloride) . The homogenate was centrifuged at 10,000 g at 4°C for 40 min .
The crude brain extract was boiled at 100°C for 6 min,
followed by centrifugation at 10,000 g at 4°C for 40 min .
The resulting supernatant was applied directly to a phosphocellulose column (2 .6 X 20 cm) and washed with 5 column
volumes of solution B (20 mM Tris-HCI, pH 7 .0, 1 mM
EDTA, 0 .1% 2-mercaptoethanol, 0 .5 mM PMSF, and 0 .5
mM benzamidinium chloride). T protein was then eluted
with solution B containing 0 .5 M NaCl . The crude T protein
fractions identified by 10% SDS-PAGE were pooled and
concentrated by dialysis against 30% PEG to a total volume
of less than 5 ml, and then further purified by an AcA-34Ultrogel filtration column (2 .5 X 90 cm) previously equilibrated with solution B containing 0.2 M NaCl and eluted
with the same solution . The pure T protein fractions on the
AcA-34 column identified by 10% SDS-PAGE were collected, concentrated by dialysis against 30% PEG, and stored
at -20°C for all the experiments mentioned throughout the
text. The purified T revealed four major and two minor protein bands with apparent molecular masses ranging from
approximately 55 to 62 kDa, basically as described by Cleveland et al . (1977) .
Phosphorylation of T protein
Standard phosphorylation of T protein was performed at
30°C in a 25-/cl reaction mixture containing 20 mM TrisHCI, pH 7 .0, 1 mM dithiothreitol, 0 .2 mM [,Y_32P]ATP (1
pmol = 1,000 cpm), 20 MM MgC1 2 , 360 hg/ml T protein,
and 5 pg/ml kinase F A/GSK-3a in the presence or absence
of 50 Fig/ml heparin . 32p incorporation into T protein was
determined by spotting 20 /d of the reaction mixture onto
Whatman P81 paper (1 X 2 cm), dropping into 75 mM
phosphoric acid, and processing as described by Reimann et
al . (1971) or by quantitation of the 32 P bands from SDSPAGE .
Preparations of immobilized metal (Fe3 ') affinity
chromatography column
An immobilized metal affinity column was prepared according to Andersson and Porath (1986) . The chelating Sepharose CL 6B column (1 .5 X 5 cm) was washed with deionized water and equilibrated with FeC13 solution . The column
was washed further with three column volumes of solution
C (0 .1 M acetic acid/NaOH, pH 3 .1) and ready for use .
Trypsin digestion and purification of tryptic
digests of [3Z P]-r phosphorylated by kinase
F A/GSK-3a
For preparative phosphorylation of T protein, the reaction
mixture, at a total volume of 0 .4 ml containing 0 .5 mg of
pure T protein ; 0 .2 mM [y3 2 P]ATP (1 pmol ATP = 2,000
cpm), 20 MM MgC12 , 20 mM Tris-HCI, pH 7 .0, 0 .5 mM
dithiothreitol, and 10 pg of pure kinase FA in the presence
and absence of 50 pg/ml heparin, was incubated at 30° C for
3 h . The 100% trichloroacetic acid at a volume of 0 .1 ml
was added next to stop the phosphorylation reaction . After
centrifugation at 13,000 g at 25° C for 5 min, the pellets were
washed twice with 0 .4 ml of 20% trichloroacetic acid and
twice with 0 .4 ml of acetone . The washed pellets were resuspended in 0 .15 ml of 50 mM ammonium bicarbonate buffer,
J. Neurochem., Vol. 63, No. 4, 1994
pH 8 .0, containing 6 wg of TPCK-treated trypsin and incubated at 37°C for 6 h . Another 6 Ng of trypsin was added
to the reaction mixture every 6 h . This continued for 24 h .
The tryptic digests of [32p]T were diluted with 3 ml of solution C and then applied to the Fe 3' affinity column prepared
as described above . After absorption, the nonphosphopeptides were washed away by solution C, and the phosphopeptides of [ 32 P] r could be eluted with solution D (0 .1 M TrisHCI, pH 8 .5) . After concentration with a Speed-Vac concentrator (Savant), the phosphopeptides of the tryptic digests of
[32P] -r resuspended in 0 .1 ml of deionized water, followed
by syringe membrane filtration, were applied to a C, g reverse-phase column (0.8 X 10 cm) and eluted with a linear
gradient of 0-35% acetonitrile in 0 .1% trifluoroacetic acid
using a model 6200A HPLC System (Hitachi) . The flow
rate was 0 .5% acetonitrile/min, and 0 .2-ml fractions were
collected every 0 .25 min . The phosphopeptide peaks were
localized by counting 0 .01-ml aliquots from each collected
fraction in a liquid scintillation counter (model 1600TR,
Packard) .
Purity analysis of isolated phosphopeptides
The purity of each isolated phosphopeptide was analyzed
by the method used in two-dimensional peptide mapping as
described by Boyle et al . (1991) . In brief, the phosphopeptide
peaks resolved from HPLC as described above were collected separately and concentrated to dryness with a SpeedVac . The dried samples were resuspended in a minimal
volume of solution E (formic acid/acetic acid/H 2 0,
50 :156 :1,794, pH 1 .9) and subjected to high-voltage electrophoresis in the first dimension on TLC cellulose plates
(Merck) in the same buffer at 1 kV at 20°C for 40 min .
Ascending chromatography in the second dimension was
carried out in solution F (n-butanol/pyridine/acetic acid/H20,
15 :10 :3 :12) at 20°C for 6-8 h. After being air-dried, the
TLC cellulose plates were exposed to x-ray films and autoradiographed to localize the 32 P-phosphopeptides. The 32P
spots removed from TLC cellulose plates were extracted
with 200 [1 of solution E at 30°C for 10 min . After centrifugation at 10,000 g for 10 min to spin down the fine cellulose
powder, the extracted 32 p-phosphopeptides were dried and
subjected to phosphoamino acid analysis, Edman degradation, and amino acid sequence analysis .
Amino acid sequence analysis and determination
of phosphorylation site sequences
The amino acid sequencing analysis of the phosphopeptides isolated from tryptic digests of [32p] -r and eluted from
TLC cellulose plates, as described above, was performed on
a MilliGen/Bioresearch model 6600 sequencer . Radiosequencing of the phosphorylation site of the phosphopeptides isolated from tryptic digests of [32p] -r, as described
above, was performed by sequential manual Edman degradation essentially according to Laursen (1966) and Laursen and
Machleidt (1980) . In brief, the 32P-phosphopeptide fractions
eluted from TLC cellulose plates and resuspended in 30%
acetonitrile were incubated with reaction membrane at 56°C
for 20 min and then washed with methanol and deionized
water. The membrane was next incubated with 50 /1 of
phenylisothiocyanate at 56 ° C for 5 min, followed by incubation with 50 11 of coupling buffer at 56°C for 15 min . The
protected N-terminal amino acid was then cleaved from the
membrane-linked peptides by 50 p1 of trifluoroacetic acid at
56°C for 10 min . At each reaction cycle, the trifluoroacetic
acid extracts were subjected to 32p counting in a liquid scin-
MODULATION OF ALZHEIMER--r KINASE
FIG . 1 . Dose effect of heparin on the phosphorylation of T protein by kinase FA/GSK-3a. T protein (360 wg/ml) was phosphorylated by 5 wg/ml kinase FA/GSK-3a in the presence of various
concentrations of heparin, as indicated, in a total volume of 25
pl at 30°C for 10 min. Detailed phosphorylation conditions and
determination of 3ZP incorporation into T protein were as described in Experimental Procedures .
tillation counter for determination of the phosphorylation
site .
Phosphoamino acid analysis
Phosphoamino acid analysis was performed as described
by Boyle et al. (1991) . The phosphopeptides isolated from
tryptic digests of ["P]T and eluted from TLC cellulose
plates, as described above, were hydrolyzed in 5 .7 M HCl
under N 2 at 110 °C for 1 h . The hydrolysate was dried with
a Speed-Vac concentrator, resuspended in solution E, and
subjected to high-voltage electrophoresis in the first dimension on a cellulose-coated TLC plate in solution E at 1 .5 kV
at 20°C for 20 min . The plate was air-dried and then subjected to the second dimensional high-voltage electrophoresis in solution G (acetic acid/pyridine/water, 10 :1 :189, pH
3 .5) at 1 .3 kV for 16 min, followed by autoradiography .
The positions of the phosphoamino acids on the plates were
localized by ninhydrin stain of the phosphorylated amino
acid standards .
Enzyme purifications and assays
Phosphorylase b (Fischer and Krebs, 1958), phosphorylase b kinase (Cohen, 1973), and [3ZP]phosphorylase a (Krebs
et al ., 1958) were purified and prepared from rabbit skeletal
muscle . The inactive ATP-Mg-dependent type-1 protein
phosphatase (Yang and Fong, 1985) and myelin basic protein
(Yang et al ., 1987) were purified from porcine brain . The
assay conditions for the measurement of kinase F A/GSK-3a
as a myelin basic protein kinase and as an activating factor
of inactive ATP-Mg-dependent type-1 protein phosphatase
were as described in a previous report (Yang, 1986) .
Analytic methods
Protein concentrations were determined by the method of
Lowry et al. (1951) using bovine serum albumin as the standard . SDS-PAGE was performed essentially according to
the method of Laemmli (1970) . Autoradiography was carried
out with a Fuji RX x-ray film using a Kodak X-Omatic
cassette with intensifying screens .
RESULTS
The activity of protein kinase FA /GSK-3a towards
phosphorylation of brain microtubule-associated pro-
141 9
tein T could be stimulated approximately fourfold by
heparin in a concentration-dependent manner (Fig . 1).
The 32 p-labelled T phosphorylated by kinase FA/GSK3a in the absence and presence of 50 /.cg/ml heparin
at 30°C for 3 h was next subjected to 10% SDSPAGE, followed by autoradiography. In agreement
with the previous reports (Yang et al., 1991a, 1993b;
Hanger et al ., 1992 ; Mandelkow et al ., 1992), kinase
FA/GSK-3a reduced the mobility of T on SDS-PAGE
(Fig . 2A, lanes 1 and 2) . However, to our surprise,
kinase FA/GSK-3a after heparin potentiation generated a reduced mobility of T with an apparent molecular mass shift to -68 kDa in SDS gels (Fig . 2A and
B, lanes 2 and 3), which is very similar to that observed in PHF-T (Lee et al ., 1991 ; Goedert et al .,
1992). The further reduction in the gel mobility shift
of T was not due to heparin itself, because heparin
alone had no effect on T's mobility on SDS-PAGE
(data not shown). By quantification of the 3Z P bands
as shown in Fig . 2, we found that kinase F A/GSK-3a
after heparin potentiation could phosphorylate T up
to 9 mol of phosphates/mol of T. Furthermore, the
12 p-phosphates were incorporated rather equally into
the different bands (Fig . 2), indicating that the in vitro
phosphorylation described here was independent of
the isoforms, as in Alzheimer's disease (Goedert et
al ., 1992). Moreover, as normal human brain T contains 2-3 mol of phosphates/mol of T, whereas Alz-
FIG. 2. SDS-PAGE and autoradiography of [ 3Z P]T phosphorylated by kinase FA/GSK-3a in the presence and absence of heparin. T protein (360 l,tg/ml) was 3ZP-phosphorylated with or without
5 IZg/ml kinase FA/GSK-3a in the absence and presence of 50
Mg/ml heparin in a total volume of 25 yI at 30°C for 3 h. The
reaction mixture was quenched in Laemmli's sample buffer and
analyzed by 10% SDS-PAGE, followed by autoradiography .
Lane M, marker proteins, i.e., bovine serum albumin (68 kDa),
glutamate dehydrogenase (55.6 kDa), and glyceraldehyde-3phosphate dehydrogenase (36 kDa) ; lanes 1, T protein alone;
lanes 2, T protein plus kinase FA/GSK-3a; lanes 3, T protein plus
kinase FA/GSK-3a and heparin. A: Coomassie Blue stain. B:
Autoradiogram of the same gel .
J. Neurochem ., Vol. 63, No. 4, 1994
1420
S . -D.
YANG ET AL.
FIG. 3 . C18 reverse-phase HPLC of the tryptic di-
gests of [32p]T phosphorylated by kinase FA /GSK3a in the absence and presence of heparin. The
tryptic digests of 0.5 mg of [32 P]T phosphorylated
by 10 leg of kinase FA /GSK-3a in the absence (A)
and presence (B) of 50 1cg/ml heparin in a total
volume of 400 N,I at 30°C for 3 h were applied to a
Fe" affinity column to remove nonphosphopeptides and then purified by C 18 reverse-phase HPLC .
Detailed conditions were as described in Experimental Procedures . Fractions of 0.2 ml were collected every 0.25 min, and a 0.01-ml aliquot from
each fraction was counted in a liquid scintillation
counter to localize the phosphopeptide peaks.
Fractions 33/34, 108/109, 128/129, 159-162, and
185 were taken as peaks I, II, III, IV, and V, respectively .
heimer brain T contains 5-9 mol of phosphates/mol
of T (Gong et al ., 1993), kinase FA/GSK-3a after
heparin potentiation may generate an Alzheimer-like
state of T. In an attempt to answer this question, the
32P-labelled T phosphorylated by kinase FA /GSK-3a
in the presence of 50 wg/ml heparin up to 9 mol of
phosphates/mol of T, was subjected subsequently to
complete trypsin digestion, followed by C18 reversephase HPLC . The tryptic digests of [32p]T could be
resolved into five major phosphopeptide peaks designated as peaks I, II, III, IV, and V on C, 8 reversephase HPLC when heparin and kinase FA/GSK-3a
were used (Fig . 3B); in the absence of heparin, however, only peaks I and II could be phosphorylated
significantly by kinase FA/GSK-3a (Fig . 3A) .
Analysis of these phosphopeptide peaks phosphorylated by kinase FA/GSK-3a in heparin by high-voltage
electrophoresis/TLC and autoradiography on TLC cellulose plates revealed that peak I contained three spots,
peaks II, III, and V contained only one spot each, and
peak IV contained two spots ; in the absence of heparin,
peak I contained only two spots instead of the three
spots generated by heparin, and peak II contained one
spot (data not shown) . Amino acid sequence analysis
of each phosphopeptide spot eluted from the TLC cellulose plates further revealed that the peak I fraction
obtained from FA/GSK-3a in heparin contained three
peptide fragments, with TPPKSPSAAK (la), TTPTPK
(lb), and TPPKSPSAAK (Ic) as the amino acid sequences, whereas the peak I fraction obtained from FA/
GSK-3a in the absence of heparin contained only two
peptide fragments, with TPPKSPSAAK (Ia) and TTPTPK (lb) as the amino acid sequences . It is noted that
J. Neurochem., Vol. 63, No. 4, 1994
the two phosphopeptides (peaks la and Ic) appeared to
have exactly the same sequence, but they could be
differentiated from each other by the degree of phosphorylation (see Fig. 5) . On the other hand, peak IV
contained two peptide fragments, with SRTPSLPTPPTR and CGSLGNIHHK as the amino acid sequences,
and peaks II, III, and V were found to be homogeneous
and contained only one peptide each, with SPVVSGDTSPR, IGSTENLK, and IGSLDNITHVPGGGNK as
their respective amino acid sequences, as summarized
in Fig . 5 . By taking together the results obtained from
phosphoamino acid analysis (Fig . 4) and sequential
manual Edman degradation (Fig . 5) of each phosphopeptide fraction eluted from the TLC cellulose plates
as described above, we finally demonstrated TPPKS(p )PSAAK, TTPT(p) PK, T(p )PPKS( p)PSAAK, SPVVSGDTS (p )PR, IGS (p)TENLK, SRT(p )PSLPTPPTR,
CGS (p )LGNIHHK, and IGS( p)LDNITHVPGGGNK as
the phosphorylation site sequences in T phosphorylated
by kinase FA/GSK-3a after heparin potentiation, as
depicted in Fig . 5, whereas in the absence of heparin,
FA/GSK-3a phosphorylates only TPPKS (p) PSAAK,
TTPT(p)PK, and SPVVSGDTS (p)PR obtained from
peaks I and II resolved from HPLC (Fig . 3A); these
data are consistent with those of the previous report
(Yang et al ., 1993b ; data not shown) . When mapping
the human brain T sequence, we found that kinase FA/
GSK-3a after heparin potentiation phosphorylated T
on Thr 212 , Thr 231 , Ser 23s Ser 262 Ser 324 Ser 3s6 , and
Ser 404 , whereas in the absence of heparin, FA /GSK-3a
phosphorylated only Ser 23s and Ser 4°4, according to
the numbering of the longest T isoform isolated from
human brain (Fig . 6 ; Goedert et al., 1989).
Mnn»rATInw OF ALZHEIMER-T KINASE
1421
phosphorylating at least seven sites in T, including
Thr 2 ` 2 , Thr 2", Ser 235 Ser 262, Ser 324, Ser 3s6 , and Ser 4°4 .
Mandelkow and co-workers reported that among the
11 sites of T exhaustively phosphorylated by their unidentified T kinase(s) from brain extracts, Ser 235,
Ser 262, and Ser 4°4 (in particular, the site of Ser 262) were
found to be the most potent sites for most effectively
reducing the microtubule binding that is possibly involved in the neuronal degeneration (Gustke et al .,
1992; Biernat et al., 1993) . The hyperphosphorylation
of T may constitute one of the major causes of decreased microtubule binding, leading to destabilization
of microtubules and, hence, to a loss in axonal transport to generate neuronal degeneration (Kosik, 1990;
Goedert et al ., 1991) . Ihara and co-workers also reported Ser 4°4 as the site hyperphosphorylated in both
PHF-T and fetal T as compared with adult T, which is
only moderately phosphorylated on Ser 404 (Kanemaru
et al., 1992; Watanabe et al., 1993). On the other hand,
Ihara and co-workers, using protein sequence and mass
spectrometric analysis of T from Alzheimer's disease
brain, demonstrated Thr 231 , Ser 235 , and Ser 262 as three
of the abnormal phosphorylation sites in PHF-T as
compared with normal T (Hasegawa et al., 1992) . As
summarized in Table 1, none of the reported T kinases,
including MAP kinase (Drewes et al., 1992; Lichtenberg-Kraag et al ., 1992), T protein kinase I/GSK-3ß
(Ishiguro et al ., 1992), T protein kinase II/cdk 5 (Ishiguro et al ., 1991 ; Kobayashi et al., 1993), prolinedirected brain kinase/cdk5 (Paudel et al ., 1993), and
protein kinase F/GSK-3a (Mandelkow et al., 1992 ;
FIG. 4. Phosphoamino acid analysis of the phosphopeptide
peaks resolved from C, 8 reverse-phase HPLC of the tryptic digests of ]s2P]T protein phosphorylated by kinase FA/GSK-3a . The
phosphopeptide peak fractions as indicated in Fig. 3 were first
subjected to high-voltage electrophoresis/TLC, followed by autoradiography to localize the 32 P spots. The 32 P spots on the
TLC cellulose plates were eluted and then subjected to phosphoamino acid analysis, as described in Experimental Procedures. The positions of the phosphorylated amino acid standards
were visualized by ninhydrin staining . PS, phosphoserine; PT,
phosphothreonine ; PY, phosphotyrosine; P,, inorganic phosphate.
DISCUSSION
In this report, we present further evidence that protein kinase F A/GSK-3a after heparin potentiation may
generate an even more Alzheimer-like state of T . First,
heparin can stimulate approximately fourfold the activity of kinase F/GSK-3a towards phosphorylation of
T and increase the phosphorylation molar ratio up to
9 mol of phosphates/mol of T. Second, T phosphorylated by kinase F,,/GSK-3a and heparin appears to
cause a reduced electrophoretic mobility of T with an
apparent molecular mass shift to ^-68 kDa in SDS
gels, which is very similar to that observed in PHF-T
(Lee et al., 1991 ; Goedert et al., 1992) . Finally, kinase
F/GSK-3a after heparin potentiation is capable of
FIG. 5. Phosphorylation site sequences of the phosphopeptides
resolved from C, 3 reverse-phase HPLC of the tryptic digests of
[32p]T phosphorylated by kinase FA/GSK-3a in the presence of
heparin. The 32 P spots eluted from TLC cellulose plates, as described in the legend to Fig. 4, were subjected to sequential
manual Edman degradation and amino acid sequence analysis
for the phosphorylation site sequence determination, as described in Experimental Procedures . The phosphorylated amino
acids are indicated by asterisks in each peptide sequence . The
experiments were performed in the presence of heparin .
J. Neurochem., Vol. 63, No. 4, 1994
142 2
S. -D. YANG ET AL.
r
FIG. 6. Peptide mapping of the phosphorylated peptide fragments of 2P]-r by kinase F A/GSK-3a in the presence of heparin with the
human brain r sequence . Amino acids were numbered according to the longest isoform of human T, as described by Goedert et al .
(1989) . The kinase FA/GSK-3a-phosphorylated peptide fragments as depicted in Fig. 5 were mapped with the human T sequence and
localized at residues 210-221, 231-240, 260-267, 322-331, 354-369, and 396-406, which are underlined . The phosphorylation
sites, indicated by asterisks, were identified as Thr 2 ' 2, Thr231 , Ser 235 , Ser 262 , Ser 32° , Ser 356 and Ser°°^, respectively, in human T . The
peak Ib sequence specifically exists in bovine brain T . The new consensus sequence -S-K-I(C)-G-S- in phosphopeptides of peaks III,
IVb, and V are double-underlined. The experiments were performed in the presence of heparin.
Yang et al., 1993b), were capable of phosphorylating
all of these important sites, in particular, the site of
Ser 262 . Protein kinase FA/GSK-3a after heparin potentiation turned out to be the only system currently available that could phosphorylate all of these important
sites in T, as presented in this report . Taken together,
the results provide initial evidence that protein kinase
F/GSK-3a after heparin potentiation may represent
one of the most potent systems possibly involved in the
abnormal phosphorylation of PHF-T in Alzheimer's
disease brain . Furthermore, as kinase F/GSK-3a appears to be present in roughly equal amounts in both
normal and Alzheimer brain (Hanger et al., 1992; Mandelkow et al., 1992), this suggests that if the generation
of PHF-7' in Alzheimer brain is indeed partly due to
abnormal phosphorylation by kinase FA/GSK-3a as
proposed, it may not be due to ectopic expression of
kinase FA/GSK-3a, but due to the modulation of the
kinase itself. The results presented here support this
notion . It is possible that the abnormal phosphorylation
of PHF-T by kinase FA/GSK-3a is due to the deregulation of kinase FA/GSK-3a via accumulation of a kinase
J. Neurochem., Vol. 63, No. 4. 1994
stimulator, such as heparin and/or a heparin-like substance (a polyanion compound), as well as possibly
a lower phosphatase activity, which can be potently
inhibited by heparin (Yang et al., 1991b) in Alzheimer's disease brain (Gong et al ., 1993). This obviously
presents a new approach for elucidating the pathogenic
mechanism of Alzheimer's disease .
In addition to Thr23 ', Ser 235 , Ser 262 , and Ser 404 as
important sites possibly involved in the abnormal phosphorylation of PHF-T, both Tau-1 sites (Ser" and
Ser e°2 ) (Kosik et al ., 1988; Biernat et al ., 1992 ; Goedert
et al., 1993) and a T3-P site (Ser 396) (Lee et al., 1991 ;
Bramblett et al., 1993) have also been identified as
important sites abnormally phosphorylated in PHF-r.
Mandelkow and co-workers found four sites phosphorylated by kinase FA/GSK-3a ; in particular, they demonstrated the phosphorylation of Tau-1, SM131,
SMI33, and SM134 sites, corresponding to the phos
phorylation of Ser' 99 , Ser e°2 , Ser 235 , Ser 396 , and Ser 404
(Mandelkow et al., 1992), in which recombinant T and
specific antibodies were used to establish these phosphorylation sites . In this report, on the other hand,
1423
MODULATION OF ALZHEIMER-T KINASE
TABLE 1. In vitro phosphorylation of T protein by various protein kinases
Kinase
Cat '/calmodulin kinase II
Phosphorylation site"
Ser
T protein kinase II/cdk5
Ser2°2, Thr2°5 , Set 235, Ser404
Kinase C
Ser324
,r protein kinase I/GSK-3ß
MAP kinase
References
Steiner et al ., 1990
Ishiguro et al., 1991
Kobayashi et al ., 1993
Ser' yy , Thr23 1 , Ser"', Set' 13
Set", Ser' yy, Ser22, Ser 235 , Ser396 , Ser°°°, Ser422
Kinase FA/GSK-3a
Ser202 , Thr205 , Thr23 ', Ser235
Ser2 ", Ser324 , Ser'56, Set.409, Ser416
Ser' 15 , Set202, Thr205 , Thr23 ', Set 215, Ser3y6 , Ser104
Ser' yy G, Ser2°2 b, Ser235 , Ser"", Ser4°4
Kinase FA/GSK-3a + heparin
Thr2 ' 2 , Thr23 ', Ser235 , Ser262, Ser324 , Ser356, SerAO4
p58`Y` °° A/p34°d" kinase from FM3A cells
Cyclic AMP kinase
Proline-directed brain kinase/cdk5
Correas et al., 1992
Ishiguro et al ., 1992
Drewes et al ., 1992
Lichtenberg- Kraag et al ., 1992
Vulliet et al ., 1992
Scott et al ., 1993
Paudel et al ., 1993
Mandelkow et al ., 1992
Yang et al ., 1993b
Present study
"The amino acid residue numbers are based on that of the longest human T isoform, as described by Goedert et al . (1989) .
b Determined by antibody, not by peptide sequencing.
we used purified brain T to determine them by direct
sequencing. Whether the discrepancy on the phosphorylation of the Tau-1 and T3-P sites by FA/GSK-3a is
due to nonspecific recognition of antibody (Poulter et
al ., 1993) or due to the disadvantage of direct peptide
sequencing obviously presents an intriguing problem
that remains to be solved .
In comparison with the similarities in T phosphorylation in fetal brain and in Alzheimer's disease brain,
the sites of Thr 23 ', Ser 235 and Ser 4' appeared to be
hyperphosphorylated in both fetal and Alzheimer's disease brains as compared with the normal adult brain
T (Kanemaru et al., 1992; Hasegawa et al., 1993;
Poulter et al., 1993 ; Watanabe et al., 1993) . The abnormal phosphorylation of T at Thr 23 ', Ser 235 , and Ser 4oa
in Alzheimer disease, thus, recapitulates normal phosphorylation during development and can be regulated
developmentally, suggesting a possible involvement of
kinase FA/GSK-3a in the development of the brain .
On the other hand, although the recognition determinants for kinase FA/GSK-3a have not been clearly established, two important features of the consensus sequence motif for kinase FA/GSK-3a have been proposed : (a) proline residues are usually in the vicinity
of the phosphorylation sites, and kinase FA/GSK-3a is
a Ser/Thr-Pro motif-directed protein kinase (Hemmings and Cohen, 1983 ; Vulliet et al., 1989; Ramakrishna et al., 1990; Woodgett, 1991) ; and (b) a prephosphorylated residue is essential for the subsequential action, and Ser-Arg-X-X-Ser may represent
another mode of consensus sequence motif for kinase
FA/GSK-3a (Fiol et al., 1987, 1990 ; Dent et al., 1989;
Ramakrishna et al., 1990). In this study, we found
that kinase FA/GSK-3a after heparin modification may
recognize a new mode of consensus sequence motif,
i.e., -S-K-I(C)-G-S- in the phosphopeptides of peaks
III, IVb, and V, as presented in Fig . 5 and doubleunderlined in Fig . 6. Whether this new sequence motif
can be recognized in other substrates is currently under
investigation in this laboratory . Moreover, the new
consensus sequence motifs were all located in the internal repeated sequences near the highly conserved carboxyl-terminal domain of T, which represents the tubulin-binding motifs (Himmler et al., 1989) . For instance, kinase FA/GSK-3a after heparin potentiation
can phosphorylate Ser 262, Ser 324 , and Ser 356 , the three
serine residues conserved in the repetitive tubulinbinding motifs (Fig. 6), which have been reported to
be required for the in vivo colocalization of T protein
to microtubules (Vulliet et al., 1992) . Phosphorylation
of the three-repeat microtubule-binding domain may
constitute an important mechanism for weakening the
interactions of T with tubulin, thus destabilizing microtubules (Correas et al ., 1992). This further supports the
notion that heparin may potentiate kinase FA/GSK-3a
to cause hyperphosphorylation of T, with a subsequent
destabilization of the microtubule cytoskeleton possibly involved in the neuronal degeneration in Alzheimer's disease (Kosik, 1990; Goedert et al., 1991) .
Acknowledgment: This work was supported by grants
NSC 81-0203-BO07-509 and NSC 82-0203-BO07-029 from
the National Science Council of Taiwan, R.O.C . The work
was also supported by grant CMRP-263 from Chang Gung
Medical College and Memorial Hospitals of Taiwan, R.O .C.
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