Williamson, Biomedical Research Institute, Ninewells Medical School, University of Dundee, Dundee DD1 9SY, UK Fax: +44 1382 740 359 Tel: +44 1382 740 347 E-mail: R.Williamson@dundee.ac.u
Trang 1with Fyn SH2 domains, but not SH3 domains, altering the cellular localization of tau
Alessia Usardi1, Amy M Pooler1, Anjan Seereeram1, C Hugh Reynolds1, Pascal Derkinderen1, Brian Anderton1, Diane P Hanger1, Wendy Noble1,* and Ritchie Williamson1,2,*
1 Department of Neuroscience, MRC Centre for Neurodegeneration Research, Institute of Psychiatry, King’s College London, UK
2 Biomedical Research Institute, Ninewells Medical School, University of Dundee, UK
Keywords
Fyn-SH2; Fyn-SH3; phosphorylation; tau;
tyrosine
Correspondence
R Williamson, Biomedical Research
Institute, Ninewells Medical School,
University of Dundee, Dundee DD1 9SY, UK
Fax: +44 1382 740 359
Tel: +44 1382 740 347
E-mail: R.Williamson@dundee.ac.uk
*These authors contributed equally to this
work
(Received 19 April 2011, revised 20 May
2011, accepted 16 June 2011)
doi:10.1111/j.1742-4658.2011.08218.x
Recent reports have demonstrated that interactions between the microtu-bule-associated protein tau and the nonreceptor tyrosine kinase Fyn play a critical role in mediating synaptic toxicity and neuronal loss in response to b-amyloid (Ab) in models of Alzheimer’s disease Disruption of interactions between Fyn and tau may thus have the potential to protect neurons from Ab-induced neurotoxicity Here, we investigated tau and Fyn interactions and the potential implications for positioning of these proteins in membrane microdomains Tau is known to bind to Fyn via its Src-homology (SH)3 domain, an association regulated by phosphorylation of PXXP motifs in tau Here, we show that Pro216 within the PXXP(213–216) motif in tau plays an important role in mediating the interaction of tau with Fyn-SH3 We also show that tau interacts with the SH2 domain of Fyn, and that this associa-tion, unlike that of Fyn-SH3, is influenced by Fyn-mediated tyrosine phos-phorylation of tau In particular, phosphos-phorylation of tau at Tyr18, a reported target of Fyn, is important for mediating Fyn-SH2–tau interactions Finally,
we show that tyrosine phosphorylation influences the localization of tau to detergent-resistant membrane microdomains in primary cortical neurons, and that this trafficking is Fyn-dependent These findings may have implica-tions for the development of novel therapeutic strategies aimed at disrupting the tau⁄ Fyn-mediated synaptic dysfunction that occurs in response to ele-vated Ab levels in neurodegenerative disease
Structured digital abstract
l Fyn physically interacts with tau by pull down (View interaction)
l Fyn physically interacts with tau by pull down (View interaction)
Introduction
The microtubule-associated protein tau is a
predomi-nantly neuronal soluble phosphoprotein that is mainly
cytoplasmic, but is also present in nuclear [1,2]
and membrane [3–5] compartments of various cell
types Abnormalities in tau, including its aberrant
phosphorylation, truncation and aggregation, are causally associated with neuronal loss in a family of neurodegenerative disorders named the tauopathies, which include Alzheimer’s disease (AD), progressive supranuclear palsy and frontotemporal dementia with
Abbreviations
AD, Alzheimer’s disease; Ab, b-amyloid; CHO, Chinese hamster ovary; CNS, central nervous system; DRM, detergent-resistant
microdomain; GST, glutathione-S-transferase; NMDA, N-methyl- D -aspartate; PSD, postsynaptic density; SEM, standard error of the mean;
SH, Src homology.
Trang 2Parkinsonism associated with tau mutations on
chro-mosome 17 [6,7] In AD, tau is believed to act in
syn-ergy with b-amyloid (Ab) to mediate neuronal loss [8]
Recently, we and others have highlighted the
impor-tance of tau interactions with the membrane-anchored
nonreceptor tyrosine kinase Fyn during Ab-mediated
neurodegeneration in cell and animal models of AD
[9–12]
Ab-induced neurotoxicity in primary cultured
neu-rons is dependent upon both Fyn and tau [9,13] This
toxicity is associated with the accumulation of Ab on
plasma membranes and the recruitment of tau into
lipid rafts [9], where tau is phosphorylated on the
putative Fyn residue Tyr 18 [14] In addition,
interac-tions between Fyn and tau are important for the
den-dritic positioning of these proteins, a localization that
has been shown to be a critical factor for Ab-induced
toxicity [11] In a transgenic mouse model of AD
over-expressing mutant human amyloid precursor protein,
tau-dependent positioning of Fyn in dendrites appears
to regulate Fyn activation in response to Ab [11]
Subsequent Fyn-dependent stabilization of
N-methyl-D-aspartate (NMDA) receptor interaction with the
postsynaptic density (PSD) protein PSD-95 results in
Ab-induced excitotoxicity [11] Furthermore, Fyn
sensitizes mice to the toxic effects of Ab [15,16], and
tau is required for the Fyn-mediated and Ab-mediated
synaptic deficits and network impairments observed in
other mouse models of AD [12], further supporting the
idea that disruption of Fyn–tau interactions may have
therapeutic utility in AD
Fyn can phosphorylate tau directly on Tyr18, one of
five tyrosines present on tau [17–19], and tau also
interacts with Fyn via its Src homology (SH)3 domain
[20,21] The SH3 domain of Fyn binds to proline-rich
motifs within the sequence PXXP in interacting
pro-teins Seven such motifs are present in the proline-rich
domain of the longest isoform of tau in the human
central nervous system (CNS) Two distinct PXXP
motifs in tau, residing within residues 213–219 and
233–236, have been suggested to mediate its
associa-tion with Fyn-SH3 [20,21], and this interacassocia-tion is
regu-lated by the serine⁄ threonine phosphorylation status of
tau [21]
Fyn also interacts with proteins through its SH2
domain, which recognizes phosphorylated tyrosines on
target proteins [22,23] Such interactions regulate the
induction of several signal transduction pathways [24]
that could play a role in Ab-induced neuronal loss
Tau is known to be tyrosine phosphorylated in
post-mortem AD brain [17,18,25] as well as in transgenic
mouse models of tauopathy, in which tyrosine
phos-phorylated tau is associated with the development of
tau pathology and neuronal loss [26] Thus, it is important to establish whether or not tau interacts with Fyn-SH2, as this would probably reveal that the tyrosine phosphorylation status of tau is important for mediating the association of these two proteins Here, we used Chinese hamster ovary (CHO) cells to characterize the interaction between exogenously expressed human wild-type tau and Fyn Previous studies using truncated tau constructs have indicated that Fyn-SH3 interactions are mediated by either Pro216 or Pro233 on tau [20,21] Using mutant forms
of full-length tau, we show that direct interaction with Fyn-SH3 is mediated predominantly by Pro216 in tau
In addition, we show that tau interacts with Fyn-SH2 and that tyrosine phosphorylation of tau, particularly
on Tyr18, mediates this binding Furthermore, we show that Fyn-mediated tyrosine phosphorylation of tau is important for its recruitment to detergent-resis-tant microdomains (DRMs) on primary neurons These findings suggest that the tyrosine phosphoryla-tion-dependent interactions of tau and Fyn may play
an important role in regulating the cellular localization
of Fyn and tau
Results
Tau interacts with Fyn-SH2 Because tau contains tyrosines that could be targeted
by Fyn, and tyrosine phosphorylation influences Fyn-SH2 binding to target proteins, we set out to deter-mine whether or not tau binds to Fyn-SH2
CHO cells were transiently cotransfected with plas-mids expressing V5-tagged human 2N4R tau, the lon-gest isoform of tau present in the adult human CNS, and Fyn Following transfection, CHO cells were trea-ted either with pervanadate, to prevent the dephos-phorylation of tyrosines, or with catalase as a control Pervanadate is a cell-permeable inhibitor of protein tyrosine phosphatases that acts by irreversible oxida-tion of the catalytic site [27] We have previously dem-onstrated that pervanadate increases tyrosine phosphorylation of several proteins, including tau, in cell lines [25] CHO cell lysates were then incubated with glutathione-S-transferase (GST)–Fyn-SH2 and GST–Fyn-SH3 fusion proteins linked to glutathione Sepharose beads, and bound proteins were collected
An antibody directed against total (phosphorylated and nonphosphorylated) tau revealed a primary band
of 64 kDa, corresponding to V5-tagged tau, on western blots of cell lysates (Fig 1A) Tau was also detected in the GST–Fyn-SH2-bound fraction, but not
in the GST-only-bound fraction pulldowns (Fig 1A)
Trang 3This indicates that a proportion of tau interacts with
Fyn-SH2 and that this binding is specific, as it is not
related to the presence of GST Western blotting with
a polyclonal antibody against GST confirmed that an
equal amount of GST–Fyn-SH2 beads was used in
each pulldown Pervanadate treatment of CHO cells
coexpressing tau and Fyn resulted in an increased
amount of tau bound to Fyn-SH2, as compared with
cells treated with catalase (Fig 1A) Furthermore, a
tau species of 68 kDa was apparent in SH2
pull-downs from pervanadate-treated cells that had been
transfected with tau This may represent a more highly
phosphorylated tau species or might indicate a
differ-ent conformation of tau with reduced electrophoretic
mobility Densitometric analysis of
GST–Fyn-SH2-bound tau, as a proportion of total tau in each cell
lysate, revealed that pervanadate increased Fyn-SH2–
tau binding by approximately six-fold as compared
with controls (Fig 1B) These results show that
inhibi-tion of tyrosine phosphatases with pervanadate results
in significantly increased binding of tau to Fyn-SH2,
thus suggesting that the interaction between tau and
Fyn-SH2 is enhanced by increased tyrosine
phosphory-lation in cells
Tyr18 of tau plays an important role in the interaction of tau with Fyn-SH2
To determine whether the tyrosine phosphorylation status of tau itself is important for its interaction with Fyn-SH2, CHO cells were transiently cotransfected with wild-type tau or a mutant construct in which all five tyrosines in tau (Tyr18, Tyr29, Tyr197, Tyr310 and Tyr394) were replaced with phenylalanine, gener-ating YallF tau This mutant tau species is therefore unable to be phosphorylated on tyrosines In addition,
to determine whether phosphorylation at individual tyrosines in tau is important for its interaction with Fyn-SH2, CHO cells were transiently cotransfected with Fyn together with one of five mutant tau con-structs in which single tyrosines were mutated to phen-ylalanine (Y18F, Y29F, Y197F, Y310F or Y394F) Transfected CHO cells were treated with pervanadate
or catalase, as above, prior to pulldown with GST– Fyn-SH2 and determination of bound proteins on western blots
Immunolabelling with an antibody directed against tau confirmed previous findings that pervanadate treat-ment increased the association of wild-type tau with GST–Fyn-SH2 In addition, although a 64-kDa YallF tau band was apparent in lysates from cells cotrans-fected with the Fyn construct, there were only trace amounts of YallF tau bound to GST–Fyn-SH2 (Fig 2A) Moreover, the association of YallF tau with Fyn-SH2 was not influenced by pervanadate These results show that prevention of tau tyrosine phosphor-ylation almost completely ablates the ability of tau to bind to Fyn-SH2, indicating that this interaction is dependent on tyrosine phosphorylation of tau A small proportion of expressed Y18F, Y29F, Y197F, Y310F and Y394F tau each bound to Fyn-SH2 under control conditions, and this binding was elevated with per-vanadate (Fig 2A) Notably, Y18F tau appeared to be less able than Y29F, Y197F, Y310F and Y394F tau to bind GST–Fyn-SH2, suggesting that phosphorylation
of tau at Tyr18 may be particularly important for its interaction with Fyn-SH2 (Fig 2B) Western blotting with a polyclonal antibody against GST confirmed that the same amount of GST–Fyn-SH2 beads was used in each pulldown
The amount of tau bound to GST–Fyn-SH2 follow-ing pervanadate treatment was quantified as a propor-tion of tau in cell lysates (Fig 2B) These results revealed that tau binding to Fyn-SH2 was almost com-pletely ablated when all tyrosines on tau were substi-tuted (YallF) In contrast, all of the tau mutants with substitutions of individual tyrosines were able to bind GST–Fyn-SH2 to some extent Y18F tau showed
Fig 1 Tyrosine phosphorylation of tau increases its association
with Fyn-SH2 CHO cells were cotransfected with plasmids
expressing Fyn and V5-tagged wild-type tau Cells were treated
with 100 l M pervanadate (P) or catalase (C) (A) CHO cell lysates
and proteins pulled down by GST or GST–Fyn-SH2 beads on
wes-tern blots labelled with antibodies against tau (V5) or GST
Num-bers on the left indicate molecular masses (kDa) (B) Bar chart
showing the proportion of total tau bound to Fyn-SH2 in CHO cells
treated with pervanadate or catalase as mean ± SEM N = 4.
***P < 0.005.
Trang 4significantly reduced association with GST–Fyn-SH2,
as compared with wild-type tau (P < 0.01), whereas
phenylalanine substitutions of Tyr29, Tyr197, Tyr310
or Tyr394 in tau did not significantly influence the
interaction with Fyn-SH2 These results suggest that
Tyr18, the putative Fyn kinase site on tau, plays a
major role in mediating interactions between tau and
Fyn-SH2 However, a contribution from other
tyro-sines cannot be excluded, as a greater proportion of
Y18F than of YallF mutant tau cosedimented with
GST–Fyn-SH2
Interestingly, whereas pervanadate induced the
appearance of an 68-kDa band in wild-type, Y18F,
Y29F, Y197F and Y310F tau, this band was not
apparent on western blots of lysates of Y394F tau
(Fig 2A) The absence of this larger species could
indi-cate that phosphorylation of tau by Fyn is reduced
and therefore, in addition to phosphorylation by c-Abl
[25], Fyn might also target Tyr394 in tau Indeed, Fyn
has previously been reported to phosphorylate both
Tyr18 and Tyr394 [19], although it is clear that Tyr394
is phosphorylated predominantly by c-Abl However,
impaired phosphorylation of Tyr394 did not appear
to influence the binding of tau to Fyn-SH2, as there
was no significant difference in the amount of Y394F
that cosedimented with GST–Fyn-SH2 when compared with wild-type tau This may be possible because, although Fyn-SH2 binds directly to phosphotyro-sines, the amino acid sequence context of the phosp-hotyrosine site is also important in SH2 domain recognition, a property that allows SH2 domains to display binding preferences for specific sites on target proteins [28]
Tyrosine phosphorylation does not modulate tau binding to Fyn-SH3
We and others have previously demonstrated that Fyn binds to tau predominantly through its SH3 domain, and this interaction is regulated by serine⁄ threonine phosphorylation of tau [20,21] To determine whether the tyrosine phosphorylation status of tau also affects the binding of tau to Fyn-SH3, CHO cells were cotransfected with Fyn together with either wild-type
or the YF mutant forms of tau Cell lysates containing equal amounts of tau were subjected to pulldown assays with GST–Fyn-SH3, and GST-bound proteins were then assessed by immunoblotting
Western blotting of lysates from pervanadate-treated cells with an antibody against total tau revealed decreased electrophoretic mobility of tau, with the appearance of an 68-kDa tau species in wild-type and all of the mutant forms of tau except for YallF and Y394F tau (Fig 3) In contrast to the results obtained with Fyn-SH2, wild-type tau and all of the YF mutant tau proteins were detected following pulldown with GST–Fyn-SH3 beads There were no significant differ-ences in the proportion of wild-type or mutant YF tau associated with Fyn-SH3 Furthermore, treatment of
Fig 2 The phosphorylation status of Tyr18 on tau is important for
Fyn-SH2–tau interaction CHO cells were cotransfected with Fyn
and V5-tagged wild-type (WT) or mutant YallF, Y18F, Y29F, Y197F,
Y310F or Y394F tau Cells were treated with 100 l M pervanadate
(P) or catalase (C) (A) CHO cell lysates and proteins pulled down
by GST–Fyn-SH2 beads on western blots labelled with antibodies
against tau (V5) or GST Numbers on the left indicate molecular
masses (kDa) (B) Bar chart showing the proportion of total tau
bound to Fyn-SH2 in CHO cells expressing wild-type or mutant tau.
Mean values ± SEM are shown relative to the amount of wild-type
tau bound to Fyn-SH2 N = 3 **P < 0.01, ***P < 0.005.
Fig 3 Tau interactions with Fyn-SH3 are not influenced by tyro-sine phosphorylation CHO cells were transiently cotransfected with Fyn and V5-tagged wild-type (WT) or mutant YallF, Y18F, Y29F, Y197F, Y310F or Y394F tau Cells were treated with pervana-date (P) or catalase (C) Cell lysates and proteins pulled down by GST–Fyn-SH3 beads were assessed on western blots labelled with antibodies against V5 or GST Numbers on the left indicate molecu-lar masses (kDa) N = 3.
Trang 5CHO cells with pervanadate did not affect the interaction
of wild-type or mutant tau with Fyn-SH3 (Fig 3) These
findings indicate that, unlike the case for Fyn-SH2,
tyro-sine phosphorylation does not play a role in mediating
interactions between tau and Fyn-SH3, and this further
suggests that different, and possibly independent,
mech-anisms are involved in these interactions of tau with the
same protein
Interactions with Fyn-SH3 are regulated by key
PXXP motifs in tau
Tau has been shown to bind Fyn-SH3 through
spe-cific PXXP motifs, seven of which are present in
tau Six of these occur as three pairs of partially
over-lapping tandem sequences (tau residues Pro176–
Pro182, Pro200–Pro206 and Pro213–Pro219), and the
seventh exists as a separate motif at Pro233–Pro236
However, there is some discrepancy over which of
these PXXP motifs is most important for tau binding
to Fyn-SH3 In neuroblastoma cells, truncated tau
mutants lacking Pro233–Pro236 were used to
demon-strate that this region of tau is critical for the binding
of tau to Fyn-SH3 [20] Conversely, using synthetic
peptides, we found that Fyn binds strongly to Pro213–
Pro219 of tau, but exhibits little interaction with
Pro233–Pro236 [21] To further investigate which
PXXP motifs in tau are responsible for Fyn-SH3
binding, we generated alanine-substituted tau mutant
constructs, P216A and P233A, for V5-tagged wild-type
human tau
CHO cells were cotransfected with Fyn together
with V5-tagged wild-type, P216A mutant or P233A
mutant tau Cell lysates were analysed on western blots
probed with an antibody against V5 to confirm the
equivalence of tau protein expression in CHO cells
(Fig 4A) GST–Fyn-SH3 beads were used to pull
down bound tau in cell lysates Detection of bound
proteins by immunoblotting with an antibody against
V5 revealed tau bands of 64 kDa in the
GST–Fyn-SH3-bound fraction from cells transfected with each of
the tau constructs Densitometric analysis of
Fyn-SH3-bound tau as a proportion of that in corresponding
cell lysates showed that significantly less P216A tau
was pulled down by GST–Fyn-SH3 than by either
wild-type or P233A tau (P < 0.05) There were no
sig-nificant differences between the amounts of wild-type
or P233A tau bound by the GST–Fyn-SH3 beads
(Fig 4B) These results indicate that tau Pro216 plays
an important role in mediating the binding of
full-length wild-type tau to Fyn-SH3, in agreement with
the findings of our previous study with synthetic tau
peptides [21]
Tyrosine phosphorylation influences tau content
in neuronal DRMs Interactions between tau and Fyn are important for the cellular distribution of these proteins For example,
we have shown that the trafficking of tau to DRMs [9] and plasma membranes [5] is Fyn-dependent As we found that the tyrosine phosphorylation of tau influ-ences its interaction with Fyn, we therefore set out to investigate the role of tyrosine phosphorylation in intracellular tau trafficking
Primary cortical neurons cultured from wild-type (Fig 5A) and Fyn-deficient (Fig 5B) mice, with matched genetic backgrounds, were treated with per-vanadate or catalase, as above Cell homogenates were collected, and DRMs were isolated and concentrated Western blotting with an antibody against the DRM marker flotillin-1 was used to demonstrate that DRMs were successfully isolated from wild-type and Fyn-defi-cient mice (Fig 5) Similarly, immunoblotting with an antibody against Fyn revealed its enrichment in DRMs prepared from wild-type neurons, but not from neu-rons lacking Fyn Increased protein tyrosine phosphor-ylation following pervanadate treatment of wild-type
Fig 4 Pro216 in tau is important for its interaction with Fyn-SH3 CHO cells were transiently cotransfected with Fyn and V5-tagged wild-type (WT) or mutant P216A or P233A tau (A) CHO cell lysates and proteins pulled down by GST–Fyn-SH3 beads were probed with antibodies against tau (V5) and GST Numbers on the left indi-cate molecular masses (kDa) (B) Bar chart showing the proportion
of total tau pulled down by GST–Fyn-SH3 beads Values shown are mean fold change from control ± SEM N = 6 *P < 0.05.
Trang 6and Fyn-deficient neuronal cultures was detected with
the phosphotyrosine antibody 4G10 (Fig 5) There
was no apparent decrease in 4G10 immunoreactivity in
homogenates from Fyn-deficient neurons, and this
probably reflects compensation for the loss of Fyn by
other Src family kinases, as has been previously
dem-onstrated [29]
A tau species of 50–55 kDa, corresponding to
endogenous mouse tau, was detected in lysates and
DRMs isolated from vehicle-treated wild-type and
Fyn-deficient neurons, confirming our previous
find-ings [9] In homogenate from wild-type, but not
Fyn-deficient, neurons, pervanadate treatment increased the
density of an 55-kDa tau band, suggesting increased
Fyn-dependent phosphorylation of tau in response to
pervanadate in wild-type neurons (Fig 5)
Interest-ingly, there was also an increase in the amount of
DRM-associated tau isolated from wild-type neurons
(Fig 5A) In contrast, pervanadate did not appear to
induce any change in the amount of tau in the DRM
fraction isolated from Fyn-deficient neurons (Fig 5B)
Quantitation of the amount of DRM-associated tau as
a proportion of the total tau in the corresponding cell lysates revealed a significant increase in the amount of tau present in DRMs from wild-type mice (P < 0.001), but not from Fyn-deficient mice, when compared with control (catalase-treated) neurons This finding suggests that increased tyrosine phosphoryla-tion of cellular proteins leads to enhanced trafficking
of tau to DRMs, and that this process is mediated by Fyn As we also found that the tyrosine phosphoryla-tion status of tau regulates its interacphosphoryla-tions with Fyn-SH2, it is possible that interactions between tau and Fyn-SH2 play an important role in regulating the intracellular trafficking of tau to membrane compartments
Discussion
Interactions between tau and Fyn play a critical role
in governing neuronal responses to elevated Ab levels
in models of AD [8], suggesting that disruption of the association of these proteins could represent a poten-tial therapeutic strategy for the treatment of AD Here,
we used GST fusion proteins of SH2 and Fyn-SH3 to further investigate the mechanisms by which tau and Fyn interact We determined that tau binds to both Fyn-SH2 and Fyn-SH3, and only the former of these associations is mediated by tyrosine phosphoryla-tion With the methods employed here, it was not pos-sible to accurately quantify differences in the relative proportions of tau capable of binding to Fyn-SH2 and Fyn-SH3, as there may be variations in the affinity of Fyn-SH2 and Fyn-SH3 for GST beads However, our data suggest that several-fold more tau binds to Fyn-SH3 than to Fyn-SH2
Tau binds predominantly to Fyn-SH3, which has a specific affinity for PXXP motifs in proteins [20,21] Tau binding to SH3 domains is regulated by the phos-phorylation of tau on specific serine⁄ threonine residues [21], and we show here that Fyn-SH3–tau interactions are not influenced by the tyrosine phosphorylation sta-tus of tau Using specific tau constructs in which either Pro216 or Pro233 was mutated to disrupt critical PXXP motifs implicated in Fyn binding, we have shown that Pro216 is especially important for the interaction of tau with Fyn-SH3, in line with our pre-vious work [21] These findings, however, contrast with those of a previous study in which a deletion mutant
of tau lacking residues 169–179 displayed a 90% reduction in its binding to Fyn-SH3 [20] The reason for this discrepancy is unclear; however, it is possible that the deletion mutants of tau used previously may have altered the tau folding⁄ conformation, thus mask-ing the interaction between P216A of tau and Fyn
Fig 5 Tyrosine phosphorylation influences tau content in neuronal
DRMs Western blots of homogenates and DRMs isolated from
neuronal cultures prepared from (A) wild-type (WT) and (B)
Fyn-defi-cient (Fyn) ⁄ )) mice Neurons were treated with pervanadate (P) or
catalase (C) Western blots were probed with antibodies against
total (phosphorylated and nonphosphorylated) endogenous tau
(Dako), the DRM marker flotillin-1, Fyn, or phosphotyrosine (4G10).
The arrow indicates an 55-kDa tau species that increases in
den-sity in response to pervanadate treatment of WT neurons
Num-bers on the left indicate molecular masses (kDa) The bar charts
below show the amount of tau in DRM fractions as a proportion of
total tau in cell lysates, expressed as the fold change from control.
Values shown are mean ± SEM N = 8 ***P < 0.005.
Trang 7In the work presented here, P216A and P233A
mutant tau proteins were correctly synthesized and
expressed in CHO cells, and both of these proteins
were able to interact, but to differing extents, with
Fyn-SH3 This suggests that the results shown here are
not artefactual, owing to incorrect folding of the tau
mutants, at least not in the regions responsible for
SH3 binding Indeed, previous work by others showed
that the association between Fyn and tau was reduced,
but still maintained, when a tau mutant lacking an
entire PXXP motif was used in similar experiments
[20] We therefore conclude that Pro216 in tau plays
an important role in its binding to Fyn-SH3 However,
tau possesses seven PXXP motifs, five of which have
not been investigated, and we therefore cannot exclude
the possibility that other prolines in tau may be
impor-tant for its binding to Fyn-SH3
Here, we have demonstrated, for the first time, that
tau interacts with Fyn-SH2 Proteins harbouring SH2
domains bind to phosphorylated tyrosines on their
tar-get proteins, thereby coupling the activity of tyrosine
kinases with intracellular signalling pathways [28] In
our model system, in which exogenous tau and Fyn
were expressed in non-neuronal cells, we found that
only a small proportion of tau is associated with
Fyn-SH2, both under control conditions and following
per-vanadate treatment to induce tyrosine
phosphoryla-tion The magnitude of the increased tyrosine
phosphorylation that we observed following
pervana-date treatment is similar to that observed following
treatment of cells with physiological amounts of Ab
[30], and thus appears to have physiological relevance
The influence of this relatively small pool of
Fyn-SH2-bound tau on potential neuronal responses to
neuro-toxic insults such as Ab remains to be determined The
use of mutant tau constructs in which individual
tyro-sines were mutated to phenylalanine allowed us to
demonstrate that the tyrosine phosphorylation status
of tau significantly influences its ability to bind
Fyn-SH2 Indeed, we determined that Tyr18, the tau
resi-due apparently preferred by Fyn [14,17,18], plays a
dominant role in mediating the association of tau with
Fyn-SH2
Tau phosphorylated at Tyr18 has been detected in a
proportion of tangles in early AD brain [31], and in
paired helical filament tau extracted from AD brain
[17,26] Furthermore, Tyr18 phosphorylated tau has
been found in DRMs following treatment of neuronal
cells with Ab [14], and we have previously shown that
tau trafficking to DRMs in response to Ab treatment
of primary neurons is Fyn-dependent [9] Here, using
wild-type and Fyn-deficient neurons, we show that
Fyn mediates the tyrosine phosphorylation-induced
recruitment of tau to neuronal DRMs These mem-brane microdomains are widely recognized as hubs for intracellular signalling [32,33], and changes in the pro-tein composition of DRMs are associated with the induction of Ab-induced neurotoxicity [9] Ittner et al [11] have suggested that interactions between tau and Fyn in dendrites play a critical role in mediating Ab-induced neurotoxicity by influencing the stability of NMDA receptor–PSD-95 complexes Interestingly, both NMDA receptors and PSD-95 shuttle between DRMs and postsynaptic densities under certain condi-tions, including during learning [34], and PSD-95 plays
an important role in positioning NMDA receptors in DRMs [34] Thus, it is conceivable that tau and Fyn might exist in a complex with NMDA receptors and PSD-95 in neurons Activation of signalling pathways that lead to increased Fyn activity could therefore affect the tyrosine phosphorylation of tau, which could potentially modulate complex formation, and result in altered trafficking into neuronal membrane compartments
In summary, the results presented here suggest that tyrosine phosphorylation mediates the association of tau with Fyn-SH2, but not with Fyn-SH3 This dem-onstrates that different molecular mechanisms exist for these two distinct interactions of Fyn and tau, with probably disparate downstream consequences Further-more, these results support the view that non-micro-tubule associations of tau are important for normal physiological function in neurons, and reinforce the suggestion that tau is itself involved in intracellular sig-nalling pathways⁄ mechanisms As its interaction with Fyn is important for tau localization in neurons, regu-lation of the cellular signalling function of this micro-tubule-associated protein could also have significant implications during the progression of neurodegenera-tive diseases, such as AD, in which both tau and Fyn are implicated
Experimental procedures
Plasmids and cell transfection
A plasmid expressing either the longest isoform of human CNS tau, containing two N-terminal inserts (2N) and four microtubule-binding repeats (4R), 2N4R tau, was a generous gift from M Goedert (Medical Research Council Laboratory of Molecular Biology, Cambridge, UK) [35] Generation of 2N4R tau constructs, each with a single tyro-sine replaced by phenylalanine (Y18F, Y29F, Y197F, Y310F and Y394F), or all with five tyrosines replaced by phenylalanines (YallF), has been described previously [25]
To generate tau constructs with individual prolines replaced
Trang 8by alanines, a QuikChange XL site-directed mutagenesis
kit (Stratagene, Amsterdam, the Netherlands) was used
The primers used were as follows: P216A forward primer,
5¢-ACC CCG TCC CTT GCA ACC CCA CCC ACC-3¢;
P216A reverse primer, 5¢-GGT GGG TGG GGT TGC AA
G GGA CGG GGT-3¢; P233A forward primer, 5¢-GCA G
TG GTC CGG ACT CCA GCC AAG TCG CCG-3¢ (Primer
B); and P233A reverse primer, 5¢-CGG CGA CTT GGC
TGG AGT CCG GAC CAC TGC-3¢ P216A and P233A
were generated by use of appropriate primers with the
plas-mid expressing wild-type tau as template DNA The cDNA
sequence of the full insert was determined for each tau
con-struct For expression in mammalian cells, cDNA encoding
tau was subcloned into the pcDNA3.1⁄ V5-His-TOPOvector
(Invitrogen, Paisley, UK), yielding a construct with
C-termi-nal V5 and His tags, as described previously [25] Human
Fyn cDNA in a pSG5 vector was a gift from D Markby
(Sugen, San Francisco, CA, USA) Constructs for the
bacte-rial expression of GST have been described previously [21]
Plasmids expressing Fyn-SH3 and Fyn-SH2 were obtained
from S Anderson (University of Colorado Health Sciences
Center, Denver, CO, USA) SH2 and
GST–Fyn-SH3 were generated by subcloning Fyn-SH2 and Fyn-GST–Fyn-SH3
into the plasmid pGEX-5X-2
CHO cell culture and treatment
CHO cells were maintained in DMEM supplemented with
10% (v⁄ v) fetal bovine serum, 2 mM L-glutamine, and
100 UÆmL)1penicillin⁄ 100 lgÆmL)1streptomycin, and
incu-bated at 37C in a 5% CO2atmosphere Cells were plated
into six-well dishes and transfected 24 h later by using
Lipofectamine Plus Reagent (Invitrogen), following the
manufacturer’s instructions Pervandate and catalase were
prepared as described previously [25] Briefly, vanadate
stock solution was prepared as a 200 mM solution of
sodium orthovanadate (pH 10) Pervanadate was prepared
as a·100 stock by adding 50 lL of 200 mMsodium
ortho-vanadate and 1.6 lL of 30% (w⁄ w) hydrogen peroxide to
948.4 lL of water for 5 min at room temperature, giving
10 mM sodium orthovanadate and 16.3 mM hydrogen
per-oxide After 5 min at room temperature, the excess
hydro-gen peroxide was removed by addition of 200 lgÆmL)1
catalase (520 UÆmL)1) and incubation for an additional
5 min, as described by Huyer et al [27] Twenty-four hours
after transfection, cells were treated with either 100 lM
per-vanadate or 2 lgÆmL)1 catalase (control) for 20 min, prior
to harvesting for western blots, as described below
Preparation of GST fusion proteins and GST
pulldown
Generation of GST, GST–Fyn-SH2 and GST–Fyn-SH3
and subsequent coupling to glutathione beads have been
described previously [21] Following CHO cell transfection
and⁄ or cell treatments, cells were washed once with NaCl⁄ Pi and harvested into ice-cold lysis buffer (25 mM Tris⁄ HCl, pH 7.5, containing 10% (v ⁄ v) glycerol, 0.5% (w ⁄ v) Triton X-100) 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 150 mM sodium chloride, 10 mM sodium fluoride, and complete protease inhibitor cocktail (minus EDTA; Roche, Burgess Hill, UK), and allowed to solubi-lize for 15 min on ice prior to centrifugation at 15 000 g for
5 min at 4C to remove insoluble debris Following deter-mination of protein concentration by bicinchoninic acid protein assay (Thermo Scientific, Rockford, IL, USA), equal amounts of protein were incubated with GST, GST– Fyn-SH2 or GST–Fyn-SH3 beads for 1 h at 4C with rotation The beads were pelleted by centrifugation at 500 g for 1 min at 4C, the supernatant was discarded, and the beads washed three times with lysis buffer Laemmli sample buffer was added, and the samples were heated to 100C for 5 min to release bound proteins Proteins in CHO cell lysates were separated on 10–12% polyacrylamide gels, transferred to nitrocellulose, and probed with an antibody
to tau The amount of tau in each sample was quantified
by densitometry, and samples were adjusted by dilution in lysis buffer to ensure equal tau protein concentrations for subsequent determination of the relative amounts of GST-bound proteins
Animals and culture of primary cortical neurons
Wild-type and Fyn-deficient (Fyn) ⁄ )) mice were obtained from The Jackson Laboratory (Bar Habor, Maine, USA) Fyn) ⁄ )mice were maintained on a mixed B6129F2⁄ J back-ground, and the same strain was used to provide wild-type controls All animal work was licensed under the Animals (Scientific Procedures) Act 1986, reviewed by the ethical review panel of King’s College London, Institute of Psychia-try and the Home Office inspectorate, and performed in accordance with the European Communities Council Direc-tive of 24 November 1986 (86⁄ 609 ⁄ EEC) Primary cortical neurons were prepared from embryonic day 16 wild-type and Fyn-deficient mouse embryos, as described previously [9] Cells were plated onto six-well dishes coated with poly(D -lysine) (5 lgÆmL)1) at a density of 1· 106
cells per well, and cultured in Neurobasal medium (Invitrogen) containing 2% (v⁄ v) B-27 serum-free supplement, 0.5 mM L-glutamine,
100 UÆmL)1penicillin, and 100 lgÆmL)1streptomycin Cells were incubated at 37C in a 5% CO2atmosphere for 7 days prior to use To obtain total cell lysates from CHO cells and primary cortical cultures, cells were washed in NaCl⁄ Tris and harvested in lysis buffer, as described above
Isolation of DRMs
DRMs were isolated as described previously [36] Briefly, primary cortical neurons were washed in NaCl⁄ Tris and lysed in isolation buffer containing 1% (w⁄ v) Chapso in
Trang 9MBS buffer (25 mMMes, 150 mMsodium chloride, pH 6.5)
containing 10 mMmagnesium chloride, 10 mMsodium
fluo-ride, 2 mMsodium orthovanadate, 1 mMEGTA, and 5 mM
dithiothreitol Lysates were homogenized by 20 strokes in a
Dounce homogenizer, and incubated on ice for 30 min
One millilitre of homogenate was mixed with 1 mL of
90% (w⁄ v) sucrose in MBS buffer, and placed in a 12-mL
ultracentrifuge tube A discontinuous 5%–35%–45%
sucrose gradient was formed by layering 6 mL of
35% (w⁄ v) sucrose in MBS buffer on top of the 2-mL
homogenate, followed by 4 mL of 5% (w⁄ v) sucrose in
MBS buffer Samples were centrifuged at 180 000 g for
18 h at 4C in a Beckman SW41 rotor (Beckman
Instru-ments, Fullerton, CA, USA) Twelve 1-mL fractions were
collected from the top of each gradient To concentrate
DRMs in fractions 4–5, 10 mL of MBS buffer was added
and the samples were centrifuged at 110 000 g for 1 h at
4C in a Beckman SW41 rotor The resulting pellet was
solubilized in 100 lL of 20 mMTris⁄ HCl (pH 7.4)
contain-ing 8M urea, 10 mMNaF, 2 mMNa3VO4and 5 mM
dith-iothreitol Samples were mixed with Laemlli buffer and
heated at 100C for 5 min prior to analysis by SDS ⁄
PAGE
SDS⁄ PAGE and western blotting
Denaturing PAGE and western blotting were performed as
described previously [37] Briefly, separated proteins were
blotted onto nitrocellulose membranes (Whatman,
Maid-stone, UK) and blocked in 5% (w⁄ v) nonfat
milk⁄ 0.05% (v ⁄ v) Tween-20 in NaCl ⁄ Pifor 1 h After
block-ing, membranes were incubated overnight at 4C in blocking
solution containing primary antibody The antibodies used
were directed against tau (total tau, rabbit polyclonal; Dako,
Glostrup, Denmark), V5 (mouse monoclonal; Invitrogen),
phosphotyrosine (4G10, mouse monoclonal; Millipore,
Bill-erica, MA, USA), Fyn (mouse monoclonal; Santa Cruz
Bio-technology, Santa Cruz, CA, USA) and GST (mouse
monoclonal; GE Healthcare, Little Chalfont, UK) After
three washes in NaCl⁄ Picontaining 0.05% (v⁄ v) Tween-20,
blots were incubated with the appropriate
fluorophore-conju-gated goat secondary antibody (IRDye800 goat anti-rabbit;
Rockland, Gilbertsville, PA, USA or Alexa Fluor 680 goat
anti-mouse; Invitrogen) for 1 h at ambient temperature
Pro-teins were visualized with the Odyssey imaging system
(Li-Cor Biosciences, Cambridge, UK) Scanned images were
analysed with the public domain IMAGEJ program (http://
www.rsb.info.nih.gov/ij/)
Statistics
Statistical analyses were performed by t-test or ANOVA
with GRAPHPAD PRISM5.0 (GraphPad Software Inc., La
Jolla, CA, USA) Data are presented as mean ± standard
error of the mean (SEM)
Acknowledgements
We thank the following for gifts of materials: M Goedert (MRC Laboratory of Molecular Biology, Cambridge, UK) for tau 2N4R cDNA; S Anderson (University of Colorado Health Sciences Center) for pGEX constructs; and D Markby (Sugen, San Fran-cisco, CA, USA) for human Fyn cDNA This work was supported by Alzheimer’s Research UK, the Alz-heimer’s Society, and the Medical Research Council
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