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Tyrosine 394 Is Phosphorylated in Alzheimer's Paired Helical Filament Tau and in Fetal Tau with c-Abl as the Candidate Tyrosine Kinase

Derkinderen, Pascal, Scales, Timothy M. E., Hanger, Diane P., Leung, Kit-Yi, Byers, Helen L., Ward, Malcolm A., Lenz, Christof, Price, Caroline, Bird, Ian N., Perera, Timothy, Kellie, Stuart, Williamson, Ritchie, Noble, Wendy, Van Etten, Richard A., Leroy, Karelle, Brion, Jean-Pierre, Reynolds, C. Hugh, Anderton, Brian H.·2005·The Journal of Neuroscience
DOI 10.1523/jneurosci.1487-05.2005
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Abstract

Tau is a major microtubule-associated protein of axons and is also the principal component of the paired helical filaments (PHFs) that comprise the neurofibrillary tangles found in Alzheimer's disease and other tauopathies. Besides phosphorylation of tau on serine and threonine residues in both normal tau and tau from neurofibrillary tangles, Tyr-18 was reported to be a site of phosphorylation by the Src-family kinase Fyn. We examined whether tyrosine residues other than Tyr-18 are phosphorylated in tau and whether other tyrosine kinases might phosphorylate tau. Using mass spectrometry, we positively identified phosphorylated Tyr-394 in PHF-tau from an Alzheimer brain and in human fetal brain tau. When wild-type human tau was transfected into fibroblasts or neuroblastoma cells, treatment with pervanadate caused tau to become phosphorylated on tyrosine by endogenous kinases. By replacing each of the five tyrosines in tau with phenylalanine, we identified Tyr-394 as the major site of tyrosine phosphorylation in tau. Tyrosine phosphorylation of tau was inhibited by PP2 (4-amino-5-(4-chlorophenyl-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), which is known to inhibit Src-family kinases and c-Abl. Cotransfection of tau and kinases showed that Tyr-18 was the major site for Fyn phosphorylation, but Tyr-394 was the main residue for Abl.In vitro, Abl phosphorylated tau directly. Abl could be coprecipitated with tau and was present in pretangle neurons in brain sections from Alzheimer cases. These results show that phosphorylation of tau on Tyr-394 is a physiological event that is potentially part of a signal relay and suggest that Abl could have a pathogenic role in Alzheimer's disease.

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Figure 1 Mass spectrometric identification of phosphorylated tyrosine 394 in tau from PHF and in fetal tau by mass spectrometry. A , PHF tau was isolated from Alzheimer brain tissue and digested with trypsin for analysis by LC/MS/MS. The mass/charge ratio is plotted against intensity. Part of the EPI spectrum is shown of the doubly charged precursor peptide of m/z 1188.3 eluting at 31.9 min, TDHGAEIVYKpSPVVSGDTpSPR (tau residues 386 - 406). B , As in A but eluting at 32.7 min. The peak ions of m/z 1194 (b10) and 1211 (y12-98), which are not present in A , show that the precursor peptide is TDHGAEIVpYKSPVVSGDTpSPR [i.e., it is phosphorylated on residue 9 (Tyr-394) and not on residue 11 (Ser-396)]. The nomenclature is as described by Roepstorff and Fohlman (1984). C , Tau from human fetal brain tissue was isolated and purified by gel electrophoresis, digested with Asp-N, and analyzed by LC/MS/MS. The fragmentation spectrum is shown for the peptide DHGAEIV P YKSPVV P SG (tau residues 387- 401). The y ion series demonstrates that Tyr-394 is phosphorylated (ions of the m/z of y8 and above contain phosphate, whereas y6 and below do not), and that Ser-400 is dehydroalanine (mass 69 in all identified y ions) caused by loss of phosphate in the fragmentation reaction, whereas Ser-396 is intact nonphosphorylated serine.
Figure 1 Mass spectrometric identification of phosphorylated tyrosine 394 in tau from PHF and in fetal tau by mass spectrometry. A , PHF tau was isolated from Alzheimer brain tissue and digested with trypsin for analysis by LC/MS/MS. The mass/charge ratio is plotted against intensity. Part of the EPI spectrum is shown of the doubly charged precursor peptide of m/z 1188.3 eluting at 31.9 min, TDHGAEIVYKpSPVVSGDTpSPR (tau residues 386 - 406). B , As in A but eluting at 32.7 min. The peak ions of m/z 1194 (b10) and 1211 (y12-98), which are not present in A , show that the precursor peptide is TDHGAEIVpYKSPVVSGDTpSPR [i.e., it is phosphorylated on residue 9 (Tyr-394) and not on residue 11 (Ser-396)]. The nomenclature is as described by Roepstorff and Fohlman (1984). C , Tau from human fetal brain tissue was isolated and purified by gel electrophoresis, digested with Asp-N, and analyzed by LC/MS/MS. The fragmentation spectrum is shown for the peptide DHGAEIV P YKSPVV P SG (tau residues 387- 401). The y ion series demonstrates that Tyr-394 is phosphorylated (ions of the m/z of y8 and above contain phosphate, whereas y6 and below do not), and that Ser-400 is dehydroalanine (mass 69 in all identified y ions) caused by loss of phosphate in the fragmentation reaction, whereas Ser-396 is intact nonphosphorylated serine.CC-BY-NC-SA · From the PDF
Figure 2 Tau is phosphorylated on tyrosine in pervanadate-treated COS-7 cells. COS-7 cells were transiently transfected with either empty vector (mock; right lane) or V5-tagged tau (WT; center lane) and treated for 20 min with pervanadate (100 𝛍 Msodiumorthovanadateand4mM H 2 O 2 and catalase). As a control, cells transfected with V5-tagged tau (2N4R; left lane) were treated with hydrogen peroxide and catalase but without sodium orthovanadate. Immunoblots were performed on duplicate membranes using 4G10 antiphosphotyrosine antibody (IP V5, Blot P-Tyr) or anti-tau TP70 antibody (IP V5, Blot Tau).
Figure 2 Tau is phosphorylated on tyrosine in pervanadate-treated COS-7 cells. COS-7 cells were transiently transfected with either empty vector (mock; right lane) or V5-tagged tau (WT; center lane) and treated for 20 min with pervanadate (100 𝛍 Msodiumorthovanadateand4mM H 2 O 2 and catalase). As a control, cells transfected with V5-tagged tau (2N4R; left lane) were treated with hydrogen peroxide and catalase but without sodium orthovanadate. Immunoblots were performed on duplicate membranes using 4G10 antiphosphotyrosine antibody (IP V5, Blot P-Tyr) or anti-tau TP70 antibody (IP V5, Blot Tau).CC-BY-NC-SA · From the PDF
Image 3
CC-BY-NC-SA · From the PDF
Figure 4 PP2, an Src family and c-Abl kinase inhibitor, prevents the pervanadate-induced tyrosine phosphorylation of tau. COS-7 cells were transiently transfected with V5-tagged tau andpretreatedfor1hwithPP2orPP3andthentreatedwithpervanadatefor20min,harvested, immunoprecipitated, and analyzed by Western blot analysis. Quantification was performed, and results show the mean values ± SEM from four separate experiments.
Figure 4 PP2, an Src family and c-Abl kinase inhibitor, prevents the pervanadate-induced tyrosine phosphorylation of tau. COS-7 cells were transiently transfected with V5-tagged tau andpretreatedfor1hwithPP2orPP3andthentreatedwithpervanadatefor20min,harvested, immunoprecipitated, and analyzed by Western blot analysis. Quantification was performed, and results show the mean values ± SEM from four separate experiments.CC-BY-NC-SA · From the PDF
Image 5
CC-BY-NC-SA · From the PDF
Figure 5 The Src family kinases, c-Src and Fyn, phosphorylate tau in transfected CHO cells. CHO cells were cotransfected with V5-tagged human tau and constructs expressing Src (Tau + Src), Fyn (Tau + Fyn), or empty vector (Tau + EV). Cells were harvested, and tau was isolated by immunoprecipitation (IP V5); blots were probed in A with4G10(IPV5,BlotP-Tyr),in B withTP70(IPV5,BlotTau),andin C withthepan-SrcantibodySRC-2demonstratingthatboth Src and Fyn were coimmunoprecipitated with tau (IP V5, Blot SRC-2). D shows Western blots of cell lysates before immunoprecipitation to demonstrate transfection of the kinases using SRC-2 antibody.
Figure 5 The Src family kinases, c-Src and Fyn, phosphorylate tau in transfected CHO cells. CHO cells were cotransfected with V5-tagged human tau and constructs expressing Src (Tau + Src), Fyn (Tau + Fyn), or empty vector (Tau + EV). Cells were harvested, and tau was isolated by immunoprecipitation (IP V5); blots were probed in A with4G10(IPV5,BlotP-Tyr),in B withTP70(IPV5,BlotTau),andin C withthepan-SrcantibodySRC-2demonstratingthatboth Src and Fyn were coimmunoprecipitated with tau (IP V5, Blot SRC-2). D shows Western blots of cell lysates before immunoprecipitation to demonstrate transfection of the kinases using SRC-2 antibody.CC-BY-NC-SA · From the PDF
Figure 6 c-Abl phosphorylates tau in both transfected CHO cells and by direct in vitro phosphorylation. A , CHO cells were cotransfected with V5-tagged wild-type human tau and c-Abl (Tau + c-Abl), c-Abl 𝚫 XB (Tau + c-Abl 𝚫 XB) expression vector constructs, or empty vector (Tau + EV). Both immunoprecipitated tau and total lysates were separated using an 8% gel. Western blot analysis was performed on all samples using 4G10 (Blot PTyr), TP70 (Blot Tau), or Abl (Blot Abl) antibodies. B , Recombinant tau was incubated in the absence or in the presence of recombinant c-Abl with ATP at 30°C for 30 min. The phosphorylation reactions were analyzed by Western blotting using 8% gels and 4G10 (Blot PTyr) and TP70 (Blot Tau). C , Cells were transfected, immunoprecipitated with anti-V5 antibody, and analyzed as in A using anti-Abl antibody for probing.
Figure 6 c-Abl phosphorylates tau in both transfected CHO cells and by direct in vitro phosphorylation. A , CHO cells were cotransfected with V5-tagged wild-type human tau and c-Abl (Tau + c-Abl), c-Abl 𝚫 XB (Tau + c-Abl 𝚫 XB) expression vector constructs, or empty vector (Tau + EV). Both immunoprecipitated tau and total lysates were separated using an 8% gel. Western blot analysis was performed on all samples using 4G10 (Blot PTyr), TP70 (Blot Tau), or Abl (Blot Abl) antibodies. B , Recombinant tau was incubated in the absence or in the presence of recombinant c-Abl with ATP at 30°C for 30 min. The phosphorylation reactions were analyzed by Western blotting using 8% gels and 4G10 (Blot PTyr) and TP70 (Blot Tau). C , Cells were transfected, immunoprecipitated with anti-V5 antibody, and analyzed as in A using anti-Abl antibody for probing.CC-BY-NC-SA · From the PDF
Figure7 Tyr-394 is the major site phosphorylated in tau by c-Abl, whereas Tyr-18 is the primary site phosphorylated by Fyn in transfected CHO cells. Cells were cotransfected with constructs expressing c-Abl 𝚫 XB ( A ), Fyn ( B ), or empty vector (EV) and with constructs for V5-tagged human tau (WT) or with V5-tagged mutants of tau (Y18F, Y29F, Y197F, Y310F, Y394F). Western blots were probed as indicated, with 4G10 (Blot PTyr), TP70 (Blot Tau), c-Abl s.c.-23 (Blot Abl), c-Abl [pY 412 ] (Blot Abl-PY), SRC-2 (Blot SRC-2), and Phospho-Src family, Tyr-416 (Blot Fyn-PY). The data presented are representative of three independent experiments.
Figure7 Tyr-394 is the major site phosphorylated in tau by c-Abl, whereas Tyr-18 is the primary site phosphorylated by Fyn in transfected CHO cells. Cells were cotransfected with constructs expressing c-Abl 𝚫 XB ( A ), Fyn ( B ), or empty vector (EV) and with constructs for V5-tagged human tau (WT) or with V5-tagged mutants of tau (Y18F, Y29F, Y197F, Y310F, Y394F). Western blots were probed as indicated, with 4G10 (Blot PTyr), TP70 (Blot Tau), c-Abl s.c.-23 (Blot Abl), c-Abl [pY 412 ] (Blot Abl-PY), SRC-2 (Blot SRC-2), and Phospho-Src family, Tyr-416 (Blot Fyn-PY). The data presented are representative of three independent experiments.CC-BY-NC-SA · From the PDF
Figure 8 Immunofluorescence labeling of Abl in a subpopulation of NFT-containing neurons. Hippocampal sections from an AD brain were labeled with anti-c-Abl antibody visualized with Alexa 488 green fluorophore ( A , D ) and with anti-phosphotau antibody AT8 visualized with Alexa 594 red fluorophore ( B , E ), followed by DAPI staining of nuclei. Merged images with DAPI staining are shown in C and F . A-C , An AT8-positive neuron (arrow) contains a tangle and also an Abl-positive granule ( A , arrowhead). An adjacent neuron (white circle) has a strong AT8-positive tangle but gives only weak and diffuse Abl staining. D-F , Three neurons containingstrongly AT8-positive NFT (asterisks), one of which contains an Abl-positive granule. At least 40 AT8-positive neurons were examined from each of three AD brains, and 6 ± 1% (SEM) of these also stained positively for Abl.
Figure 8 Immunofluorescence labeling of Abl in a subpopulation of NFT-containing neurons. Hippocampal sections from an AD brain were labeled with anti-c-Abl antibody visualized with Alexa 488 green fluorophore ( A , D ) and with anti-phosphotau antibody AT8 visualized with Alexa 594 red fluorophore ( B , E ), followed by DAPI staining of nuclei. Merged images with DAPI staining are shown in C and F . A-C , An AT8-positive neuron (arrow) contains a tangle and also an Abl-positive granule ( A , arrowhead). An adjacent neuron (white circle) has a strong AT8-positive tangle but gives only weak and diffuse Abl staining. D-F , Three neurons containingstrongly AT8-positive NFT (asterisks), one of which contains an Abl-positive granule. At least 40 AT8-positive neurons were examined from each of three AD brains, and 6 ± 1% (SEM) of these also stained positively for Abl.CC-BY-NC-SA · From the PDF

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Metadata

Authors
Derkinderen, P., Scales, T. M. E., Hanger, D. P., Leung, K.-Y., Byers, H. L., Ward, M. A., Lenz, C., Price, C., Bird, I. N., Perera, T., Kellie, S., Williamson, R., Noble, W., Van Etten, R. A., Leroy, K., Brion, J.-P., Reynolds, C. H., & Anderton, B. H.
Venue
The Journal of Neuroscience
Publisher
Society for Neuroscience
Published
2005-07-13
Volume
25
Issue
28
Pages
6584-6593
ISSN
  • 0270-6474
  • 1529-2401
DOI
10.1523/jneurosci.1487-05.2005
Cited by
169
References
2
Language
en
Licence
creativecommons.org/licenses/by-nc-sa/4.0/ (vor, 184 d)

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