Biochemical Society Transactions ( 1 995) 23
PIfOSPHORYIATION OF TACT RY GI,Y<'OGF&
SYNTHASE hlNASFr3P IN VI7'ROPRODLTCES SPECIES
WITXI SIMILAR ELECTROPHORETIC AND
UVLWINOGENTC PROPFRTIES TO PHF-TAU FROM
AIZHEIMERS DISFASE BRAIN
MulofS.F.C.,il~ughes,K.,zWoodgeqJ.R.,LAnderton,B.~l.1,and
Ilanger, I).P.'
'Department of Neuroscience, lnshtute of Psycluatry, I)e Crespigny Park
Denmark Hill, h n d o n SES 8AF. IJK and 'Ontano Cancer Institute, Pnncess
MarRaret Hospital. 500 Shmhoiune Street, Toronto, Ontano M4X IK9,
('LUYdda.
Neurofibrillary tangles and senile plaques are the two
histopathological hallmarks of Alzheimer's disease brain.
Neurofibrillary tangles consist of paired helical filaments
(PHF) a major constituent of which is the microtubuleassociated protein, tau (PHF-tau) [ 11. P€iE'-tau is composed
of several tau isoforms that are in a hyperphosphorylated
state compared to tau from control human brain [2-61. PHFtau usually separates into three major species on SDS-PAGE
with a fourth and less prominent, but often observable, band
possessing a lower mobility than the three main bands. GSK3a and GSK-3(3are present in neurones of both control and
Ali.heimer's disease brain [7] and have been reported to copurify with PHE' (81. Here, we show that PHF-like tau
comprising four electrophoretically distinct species can be
generated, after 24 h incubation with GSK-3a, from both
human brain tau isolated from normal brain and from a
mixture of recombinant human tau i s o f o m . Phosphorylation
of the individual recombinant isoforms of tau enabled us to
assign probable identities to the four PHF-tau species that are
different from previous assignments [3,5,61. Control human
brain tau and individual recombinant human tau i s o f o m
were incubated with GSK-3(3 (prepared as described in [9])
in 2OmM HEPES buffer, pH 7.5, containing 5mM MgCI,,
I O p M leupeptin, 1pM pepstatin, ImM PMSF, 0.5mM DIT,
5pM okadaic acid, 1 j ~ Msodium orthovanadate and 3mM
A",
for 24 h at 30°C. Appropriate controls containing heatinactivated enzyme were incubated in parallel. The reaction
was stopped by heating at 100°C for 5 min. Samples were
analysed by SDS-PAGE (loo/, gels) [lo] and Western
blotting (41. Following phosphorylation by GSK-3f3,
recombinant human tau and control human brain tau (Fig I ,
lanes 1-4) shifted to align with PHF-tau isolated from
Alzheimer's disease brain (Fig 1, lane 5 ) . Both types of
phosphorylated tau protein as well as PHF-tau appeared as
four bands when immunolabelled with the polyclonal
antibody to tau (Fig 1, lanes 3-5). Individual recombinant
human tau isoforms were incubated with GSK-3(3,
1 2 3 4 5
Figum 1: Western blot labelled with polyclonal antibody to
tau. Lane 1- all six recombinant human tau isofom$; lane 2purified control human brain tau; lane 3- the same sample as
in lane 1, phosphorylated by GSK-3(3; lane 4- the same
sample as in h e 2, phosphorylated by GSK-3p; lane 5PHF-tau. Molecular weight markers are indicated by
arrowheads and correspond to phosphorylase b (97.4 kDa),
catalase (58.1 kDa) and alcohol dehydrogenase (39.8 m a ) .
T a u 15ofOrn15
Figurp 2: Schematic representation 01 the six human tau
i s o f o m and PIIF-tau. CrSK-3!3-phosphorylated tau isoforms
align with each of the four PHF-tau species as indicated by
the arrows.
following phosphorylation, the smallest tau isofom, 3K.O
(Fig 2) migrated close to the lower band of PHF-tau.
Isoforms 4R,O and 3R29 aligned close to the second band of
PIG-tau, with 4R,O migrating slightly above 3R3,29.Isoforms
4R,29 and 3R,58 aligned with the third band of PHF-tau and
isofom 4R,58 aligned with the fourth, slowest migrating
band of PHF-tau. 'Ihus, with regard to electrophoretic
mobility, the PHF-tau pattern can be reproduced by GSK-3[\phosphorylated human brain tau and recombinant human tau
and the relationship between the individual tau isofoms and
the PHF-tau species is illustrated schematically in fig 2.
Extensive in vitro phosphorylation of a purified
preparation of control human brain tau consistently produces
four rather than, as previously believcd, three tau species on
SDS-PAGE. The species thus gtnerated are shifted on S I X PAGE to positions that match those of PHF-tau isolated from
Alzheimer's disease brain. In vitro phosphorylation of the
individual recombinant isoforms by GSK-3p showed that the
four bands of P€W-tau are likely to consist of isofoms 3K.0
alone; 4R,O with 3R,29; 4R,29 with 3R.58 and 4R58 alonc.
This research was funded by the European Union,
Glaxo Research and Development Limited, The Wellcomc
Trust and the Canadian MKC. We thank I
k M. Goedert for
providing the human tau cDNA. We are gratehi to I)r N.
Cairns (MRC Alzheimer's Disease Brain Bank, Institute of
Psychiatry, London, UK) for kindly providing us with the
brain tissue used in these studies.
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