Vectors and small interfering RNAs (siRNAs)

Tau-V5 and Fyn-Myc expression constructs were generated using pENTR donor vectors that contained full-length human 2N4R tau and human Fyn [26]. pDONR223-PTK2B was a generous gift from William Hahn and David Root (Addgene plasmid # 23898). The human Pyk2 cDNA was derived from the pDONR223-PTK2B vector and subcloned into an mThy1.2 expression vector to generate Pyk2 transgenic mice. Pyk2-V5 and Pyk2-GFP vectors were generated by cloning the Pyk2 cDNA into pcDNA6.2/V5-DEST and pcDNA6.2/N-EmGFP-DEST vectors (Gateway cloning, Thermo Fisher Invitrogen). Fyn-GFP was constructed in a similar manner. Mutagenesis of Pyk2 to generate Pyk2Y402F, Tau to generate TauY18A (Y18 is a Fyn phosho-epitope) and Fyn to generate FynKD (K299A, kinase-dead), FynCA (Y531F, constitutively active), FynΔSH2 (SH2 domain (149-246 aa)-deleted) and FynΔSH3 (SH3 domain (82-143 aa)-deleted) constructs was performed following the manufacturer’s instructions (Q5 site-directed mutagenesis kit, New England Biolabs, E0554S). A DNA purification kit (NucleoBond Xtra Midi-EF, MACHEREY-NAGEL) was used for plasmid preparation. ON-TARGET plus human PTK2B siRNA (L-003165-00-0005) smart pool and non-targeting pool control siRNAs were purchased from Dharmocon. Co-transfection of the siRNA and the Tau-V5 plasmid was performed using the DharmaFECT 1 transfection reagent.

In vitro phosphorylation and dephosphorylation assays

Freshly thawed aliquots of recombinant proteins were briefly mixed and incubated in the appropriate kinase incubation buffer (20mM Tris-HCl, 10mM MgCl₂, 5mM dithiothreitol, 0.5mM EDTA, pH 7.5) depending on the assay. For instance, to test if phosphorylation was ATP-dependent, recombinant tau and Pyk2 were incubated with or without 2.5mM ATP. The phosphorylation reaction was carried out at 30°C for 1h, except for the time course analysis for which samples were taken at various time points throughout the reaction. For the in vitro dephosphorylation assay, a cell lysate from HEK cells co-transfected with Tau-V5 and Fyn-Myc and 50–100μg of a lysate taken from pR5 brains were included as positive controls to detect phospho-tau epitopes. Lambda phosphatase, alkaline phosphatase and Antarctic phosphatase (New England BioLabs) were used following standard protocols. Phosphorylation and dephosphorylation reactions were terminated by adding SDS sample buffer, and phosphorylation was determined by electrophoresis on 8% polyacrylamide gels followed by western blotting with phospho-tau specific antibodies.

Sequential extraction

Isolated hippocampal slices were homogenized with metal beads in RAB buffer (G-Bioscience, Cat# 786-91) using TissueLyser LT (Qiagen) in a 2ml tube for 4min at 4°C and maximum speed. The supernatant was removed and centrifuged at 21,000g for 90min at 4°C. The resultant supernatant (RAB-soluble proteins) was collected, and the pellet homogenized in the same volume of RIPA buffer (Cell Signaling Technology, #9806) and centrifuged at 21,000g for 90min at 4°C. The supernatant (RIPA-soluble proteins) was then collected. RAB/RIPA fractions were aliquoted and stored at – 80°C for further analysis.

Co-immunoprecipitation

Co-immunoprecipitation was performed following standard procedures. In brief, cells were washed in PBS and lysed in cold NP-40 buffer (50mM Tris-HCl, pH 7.4, 150mM NaCl, 2mM EDTA, 1% NP-40) supplemented with a protease/phosphatase inhibitor cocktail (Cell Signaling Technology). The lysate was briefly sonicated and centrifuged at 14,000rpm for 10min at 4°C. The supernatant was then collected and 1mg of total protein lysate was incubated with protein G-conjugated sepharose beads (GE Healthcare) with primary antibodies (Table 1) overnight at 4°C. For brain lysates, 500μg total protein lysate was precleared with protein A-conjugated sepharose beads (GE Healthcare) for 1h before incubation with the antibody-sepharose complex for 4h at 4°C. The beads were spun down, washed with lysis buffer or PBS buffer, and eluted with 2×SDS sample buffer, followed by western blotting.

Western blotting

Equal amounts of protein were loaded (10–50μg depending on the assay) and resolved by SDS-PAGE in Tris-glycine-SDS buffer (Bio-Rad), followed by transfer onto low fluorescence polyvinylidene fluoride membranes (Bio-Rad). The membranes were blocked with Odyssey blocking buffer TBS (LICOR) for 1h, and then reacted with primary antibodies in Odyssey blocking buffer (LICOR) overnight at 4°C under gentle rocking. After washing, the corresponding IR Dye 680RD/800CW secondary antibodies (LICOR, 1:10,000 dilution) were added for 1h at room temperature under constant agitation. After washing, the membrane was scanned in the Odyssey Fc Imaging system (LICOR) for detection of an infrared signal.

Immunofluorescence

Cells grown on coverslips or microfluidic chamber devices were washed gently with PBS and fixed in 4% paraformaldehyde, followed by permeabilization with 0.3% Triton X-100. Paraffin-embedded brain sections were deparaffinized in xylene and then rehydrated prior to microwave-mediated antigen retrieval in citrate buffer. The cells or sections were then blocked for 1h in 5% goat serum with 1% bovine serum albumin (BSA), followed by incubation overnight with primary antibodies at 4°C. Free-floating slices were rinsed three times in PBS for 10min each, incubated in blocking solution (10% normal goat serum in PBS supplemented with 0.2% Triton X-100), and placed on an orbital shaker for 2h. Slices were incubated with primary antibodies at 4°C for 24h. Secondary antibodies were Alexa Fluor (405, 488, 555, 647)-labeled goat anti-mouse, goat anti-rabbit and goat anti-chicken IgG antibodies (Thermo Fisher, 1:500 dilution). DAPI was used to visualize the nuclei. Fluorescence images were taken with a 20×or 63×objective on a Zeiss LSM710 laser scanning confocal microscope or Diskovery spinning disk confocal microscopy at the Queensland Brain Institute’s Advanced Microscopy Facility.

Image analysis

For western blotting, band intensities from immunoblots were quantified using Image J software. They were first normalized to the intensity of an internal control protein and then normalized to the control group as indicated, except that phosphorylated proteins were generally normalized to the amount of total protein instead of the internal control. For the analysis of fluorescence intensities by Image J, all images were converted to 8-bit images and thresholded before the hippocampal area was selected as region of interest and the percentage area of positivity was determined. The sample size for each group is indicated in the figure legends.

Statistical analysis

The molecular and biochemical analysis was performed using a minimum of three biological replicates per condition. The statistical analyses were done using the Prism 7 software (GraphPad). The data distribution was assumed to be normal with similar variance between groups; however, datasets were first assessed with normality tests. Two groups were analyzed with the two-tailed unpaired Student’s t test when the datasets were normally distributed. Three or four groups were assessed with one-way ANOVA followed by a post hoc test (Dunnett’s test, or Sidak’s test) for multiple comparisons. Values are presented as the mean±standard error of mean (s.e.m). The sample size is indicated in the figure legends.

Pyk2 colocalizes and interacts with tau in neurons

The in vivo evidence of a role of Pyk2 in tau pathology led us to investigate the association of Pyk2 and tau in more detail, including under physiological conditions. We first observed immunoreactivity for both Pyk2 and its activated form in glial cells that were negative for the neuronal marker tau (Supplementary Figure 4A, B), reflecting the nature of Pyk2 as a prevalent focal adhesion kinase. In mature WT primary hippocampal neurons, Pyk2 was strongly present in cell bodies and tau-positive neurites, regardless of whether they were MAP2-positive or not (with MAP2 serving as dendritic marker) (Supplementary Figure 4C), indicating pronounced coexistence of Pyk2 and tau in dendrites and axons. To further confirm that Pyk2 is also localized to axons (where tau is enriched under physiological conditions), we used microfluidic chambers that allow for a better discrimination of dendrites and axons based on their length. Counterstaining for Pyk2, tau and MAP2 confirmed that Pyk2 expression extended into axons (Fig. 2A). Interestingly, Pyk2 and tau were detectable together in axons as early as neurons started to differentiate (Supplementary Figure 5A, B).

Open in a separate window

Fig.2

Localization and interaction of Pyk2 and tau. A) Representative immunofluorescence staining of Pyk2 in primary hippocampal neurons (DIV (days in vitro) 10) cultured in microfluidic chambers. Cells were counterstained for tau (all compartments including axons) and MAP2 (all compartments excluding axons). DAPI labels the nuclei. Scale bar: 100μm. B) Co-immunoprecipitation of Pyk2 and tau in RAB fractions of brain lysates obtained from WT and pR5 mice reveals weak interaction of endogenous Pyk2 and tau in vivo. Note that cleaved forms of endogenous Pyk2 were detected in the input lysate. IP, immunoprecipitation. Three independent experiments were performed. C) Endogenous Pyk2 is present in the crude synaptosomal fraction, together with tau. WT brain tissue was processed to obtain fractions including total homogenate (Hm), supernatant S1, pellet P1 and pellet P2, followed by immunoblotting with the indicated antibodies. D) Pyk2-V5-transfected hippocampal neurons (DIV16) stained with Pyk2 and V5, revealing strong localization of Pyk2 in dendritic spines. Scale bar: 50μm. E) WT hippocampal neurons (DIV16) stained with PSD95 (postsynaptic marker) and MAP2. Colocalization of endogenous Pyk2 and PSD95 shown in spines of a WT neuron indicated by arrowheads. Scale bar: 50μm.

We next examined whether there was a physical interaction between endogenous Pyk2 and tau. Using co-immunoprecipitation, we found that Pyk2 was able to interact with tau in WT brain lysates, although the interaction appeared not to be very strong; a similar interaction (with respect to co-immunoprecipitated full-length Pyk2) was found in brain lysates from pR5 mice that overexpress tau (Fig. 2B). In addition, we observed cleaved forms of endogenous Pyk2 in the input lysate, in agreement with what we had found for recombinant Pyk2 protein (see below).

Pyk2 has also been shown to be localized to the PSD, a scaffolding structure within dendritic spines [24, 25], where tau is also present [28]. We therefore performed fractionations of mouse brain tissue and obtained a synaptosomal (P2) fraction in which Pyk2 was present together with tau, as well as other PSD components such as PSD95 and Fyn (Fig. 2C). Furthermore, when Pyk2 was fused with the green fluorescent protein (Pyk2-GFP) for visualization and then expressed in cultured hippocampal neurons, we observed a colocalization with PSD95 in dendritic spines (Supplementary Figure 5C). In support, by tagging Pyk2 with the much smaller V5-tag (in order to rule out potential leakage of GFP), the same localization pattern was obtained, irrespective of whether an anti-V5 or anti-Pyk2 antibody was used (Fig. 2D). Moreover, labelling for endogenous Pyk2 consistently demonstrated its presence in dendritic spines (Fig. 2E). Together, these data demonstrate that Pyk2 colocalizes and interacts with tau in neurons.

Pyk2 phosphorylates tau in HEK293T cells

We next determined whether Pyk2 is a tau kinase. We therefore transfected HEK293T cells with a tau expression vector either alone or together with a Pyk2-GFP expression vector. As expected, Pyk2 expression significantly increased the amount of tau phosphorylated at Y18 (>5-fold, after normalization to total tau levels) compared to the tau-only transfection (Fig. 3A, B). Surprisingly, the level of total tau was also increased in the co-transfected group (~ 2-fold). However, these increases of pY18-tau and total tau in the co-transfected group were completely abolished when a phosphorylation-deficient mutant form of Pyk2 (Pyk2Y402F-GFP) was used (Fig. 3A, B). Of note: Only a long exposure time reveals Y18 phosphorylation and endogenous Pyk2 expression (Fig. 3A). In agreement with the biochemical analysis, immunofluorescence staining supports Y18 phosphorylation of tau by Pyk2 unless an inactive kinase is expressed (Fig. 3C).

Open in a separate window

Fig.3

Pyk2 phosphorylates tau in HEK293T cells. A) Representative immunoblots of lysates from HEK293T cells transfected with V5-tagged tau and GFP-tagged Pyk2. Validation of the Y402F mutant form of Pyk2 by immunoblotting using a phospho-Y402-Pyk2 specific antibody. Sh, short exposure; lo, long exposure. Asterisk denotes the endogenous expression of Pyk2 in HEK cells using a longer exposure of the immunoblot in the absence of Pyk2-GFP overexpression. B) Quantification of immunoblots in (A) showing relative tau and pY18-tau levels. Mean±s.e.m, n=6 per group, one-way ANOVA, Dunnett’s multiple comparisons test, **p<0.01, ****p<0.0001. C) Representative immunofluorescence staining of HEK293T cells transfected with V5-tagged tau and GFP-tagged Pyk2 indicating a strong positive correlation of pY18-tau immunoreactivity and expression of the active form of Pyk2. Scale bar: 50μm. D) Immunoblots of lysates from HEK293T cells co-transfected with V5-tagged tau and PTK2B or control siRNA. E) Quantification of immunoblots in (D). Mean±s.e.m, n=3 per group, two-tailed t test, *p<0.05, ***p<0.001.

We next examined the loss-of-function effect of Pyk2 on tau phosphorylation. Since the pY18-tau antibody only detects human tau, we overexpressed a human tau construct in HEK cells that express endogenous Pyk2 protein (albeit at a relatively low level). Using siRNAs against human PTK2B gene, we successfully reduced endogenous Pyk2 levels by 66% (Fig. 3D, E; p<0.05). This significantly reduced pY18-tau levels by 90% compared to the control (p<0.001). Together, these data strongly suggest that Pyk2 is a tyrosine kinase of tau.

Pyk2 phosphorylates tau in vitro

To exclude the possibility of indirect phosphorylation, we attempted to validate the tyrosine kinase activity of Pyk2 for tau more directly. We therefore cloned His-tagged Pyk2 and tau cDNAs into bacterial expression vectors followed by affinity chromatography and size exclusion chromatography. Recombinant proteins were validated with Coomassie staining and immunoblotting using antibodies against the V5-tag, tau (HT7) and Pyk2 (Fig. 4A). As revealed with a C- and N-terminal anti-Pyk2 antibody, we observed more C- than N-terminally cleaved products, consistent with the pattern for V5, as the V5-tag was fused C-terminally. As expected, levels of pY18-tau were massively increased when tau was incubated with Pyk2 protein (Fig. 4B).

Open in a separate window

Fig.4

Pyk2 phosphorylates tau in vitro. A) Coomassie staining and immunoblots of purified recombinant His-tagged Tau-V5 and Pyk2-V5 proteins. The full-length forms of the recombinant proteins are indicated at the expected molecular weight. Carboxy-terminally cleaved Pyk2 was detected using both anti-V5 and anti-C-terminal Pyk2 antibodies. N-terminal and C-terminal Pyk2 antibodies were raised against an amino- and carboxy-terminal epitope of human Pyk2, respectively. B) Immunoblots of pY18-tau after incubation of recombinant tau with or without recombinant Pyk2 in the in vitro phosphorylation reaction. C) Immunoblots of pY18-tau after incubation of recombinant tau and Pyk2 with or without ATP in the in vitro phosphorylation reaction. D) Quantification of immunoblots in (C). Mean±s.e.m, n=3 per group, two-tailed t test, ***p<0.001. E) Representative immunoblots of a time course assay of Pyk2-catalyzed tau phosphorylation in vitro. Reactions were carried out at 30°C in the presence of 0.5μM recombinant Pyk2 and 1.9μM recombinant tau. F) Normalized pY18-tau levels in (E) were plotted as a function of incubation time, with fold-changes relative to time point ‘0 s’. The curve was generated by nonlinear regression. Three independent experiments were performed.

Of note, we consistently observed a basal level of pY18 phosphorylation for recombinant tau (Supplementary Figure 6A). In order to remove the basal phospho-groups from tyrosine residues, we incubated tau with multiple phosphatases but failed to achieve basal Y18 dephosphorylation, which may or may not be related to the conformation of tau, similar to what is known for its dephosphorylation by protein phosphatase 2A (Supplementary Figure B-D).

We next carried out a kinase assay by co-incubating Pyk2 and tau protein with or without ATP. Y18 phosphorylation by Pyk2 was increased>1.8-fold in the presence of ATP, indicating that this reaction is ATP-dependent (Fig. 4C, D). Furthermore, although basal pY18-tau levels were significant, we were able to establish the time course of the Pyk2-catalyzed tau phosphorylation reaction over a 60min timeframe, showing a time-dependent increase of the pY18-tau signal that was accompanied by a slight molecular weight shift (Fig. 4E, F). A typical enzyme-substrate reaction dynamics was indicated by fitting a curve generated by nonlinear regression to the data (Fig. 4F). Together, our in vitro findings suggest that Pyk2 is a direct tyrosine kinase of tau.

Pyk2 interacts with the tau kinase Fyn

Src family kinases are the most thoroughly studied kinases linked to tau tyrosine phosphorylation [38–40]. Of these, Fyn is one of the most significant kinases in phosphorylating tau under pathological conditions [26, 28, 30]. We therefore sought to determine whether Fyn is involved in Pyk2-mediated tau phosphorylation. We first confirmed the tyrosine kinase activity of Fyn in relation to tau by co-transfecting tau and Fyn expression constructs in HEK293T cells, revealing that pY18-tau levels were induced>6.6-fold by Fyn (Fig. 5A, B). Increased tau levels (2.5-fold) were also observed when Fyn was co-transfected.

Open in a separate window

Fig.5

Pyk2 interacts with the tau tyrosine kinase Fyn. A) Immunoblots of cell lysates from HEK293T transfected with V5-tagged tau and Myc-tagged Fyn, indicating that Fyn robustly phosphorylates tau at Tyr18. Sh, short exposure; lo, long exposure. B) Quantification of immunoblots in (A) indicating elevation of pY18-tau levels by Fyn overexpression. Mean±s.e.m, n=4 per group, two-tailed t test, ****p<0.0001. C) Representative fluorescence image of HEK293T cells overexpressing Fyn-RFP and Pyk2-GFP. DAPI labels the nuclei. Scale bar: 10μm. D) Immunoprecipitation of Pyk2 and Fyn expressed in HEK293T cells reveals their interaction. E) Immunoprecipitation of Pyk2 and truncated forms of Fyn (FynΔSH2-GFP, SH2 domain-deleted Fyn; FynΔSH3-GFP, and SH3 domain-deleted Fyn) reveals the role of SH2 domain in the interaction. F) Immunoprecipitation of Fyn together with Pyk2 or a phosphorylation-deficient (inactive) Y402F mutant form of Pyk2. G) Quantification of immunoblots (F) indicating a reduced interaction between Fyn and inactive Pyk2. Mean±s.e.m, n=4 per group, two-tailed t test, ***p<0.001.

We next addressed the relationship of Pyk2 and Fyn, considering that both enzymes can phosphorylate tau at residue Y18. Co-transfection of GFP-tagged Pyk2 and Fyn revealed significant colocalization (Fig. 5C), which was validated by immunoprecipitation (Fig. 5D). Fyn, like any Src family kinase member, engages its conserved SH2 and SH3 domains to mediate protein interactions [41]. We therefore used Fyn constructs that lacked either domain and established that the SH2 domain was responsible for the Fyn/Pyk2 interaction (Fig. 5E). Given that a motif containing phosphorylated tyrosine is a general binding substrate for SH2, we also investigated whether the phosphorylated Y402 epitope of Pyk2 was involved in this interaction. As expected, interaction between the Pyk2 mutant Y402F and Fyn was reduced by more than 72% relative to that of control Pyk2 (Fig. 5F, G; p<0.001). Together, this demonstrates that Pyk2 interacts with Fyn via the autophosphorylation epitope pY402 of Pyk2 and the SH2 domain of Fyn.

We thank Dr. Di Xia for assistance in making overexpression constructs, and Rowan Tweedale for critically reading the manuscript. This study was supported by the Estate of Dr. Clem Jones AO, the State Government of Queensland, the Federal Government of Australia (ACT900116), and by grants from the Australian Research Council [DP160103812, LE130100078] and the National Health and Medical Research Council of Australia [GNT1127999, GNT1037746] to JG.

Authors’ disclosures available online (https://www.j-alz.com/manuscript-disclosures/18-0054r1).