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Cited by 204Alzheimer's disease research and treatmentsProtein Kinase Regulation and GTPase SignalingDementia and Cognitive Impairment Research

Glycogen synthase kinase-3β phosphorylates tau protein at multiple sites in intact cells

Open access
Sperbera, Brian R., Leight, Susan, Goedert, Michel, Lee, V.M.-Y.·1995·Neuroscience Letters
DOI 10.1016/0304-3940(95)11902-9
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Abstract

Hyperphosphorylated tau protein is the major constituent of the paired helical filament (PHF), the major fibrous component of the neurofibrillary lesions of Alzheimer's disease (AD). Hyperphosphorylation of tau is believed to be the critical event that leads to filament assembly. Identification of the responsible protein kinases is therefore a key step towards an understanding of the pathogenesis of AD. Mitogen-activated protein kinase, glycogen synthase kinase-3 (GSK3) and neuronal cdc2-like kinase have been shown to phosphorylate tau protein in vitro at a number of sites that are phosphorylated in PHFs. In this study, we report that transient transfection of human GSK3 beta into Chinese hamster ovary cells stably transfected with individual human tau isoforms leads to hyperphosphorylation of tau at all the sites investigated with phosphorylation-dependent anti-tau antibodies. Thus, GSK3 beta is a protein kinase that phosphorylates tau protein in intact cells.

AI snapshot

Written from the paper’s full text.

TL;DR

Transient expression of a constitutively active human GSK3β mutant in CHO cells stably expressing human tau increased tau phosphorylation at multiple PHF-associated sites. This extends in vitro evidence that GSK3β can phosphorylate tau to an intact-cell system.

Key findings

  • Transfection of GSK3β(S9A) into CHO T40 and CHO T44 tau-expressing cells increased immunoreactivity with all phosphorylation-dependent anti-tau antibodies tested: PHF1 (Ser-396/Ser-404), T3P (Ser-396), AT270 (Thr-181), AT180 (Thr-231), and AT8 (Ser-202).
  • GSK3β expression also caused a decrease in electrophoretic mobility of tau consistent with phosphorylation, while total tau amount remained unchanged by immunoblot.
  • GSK3β was distributed throughout the cytoplasm and was not exclusively microtubule-associated; the c-myc-tagged protein ran at approximately 48 kDa in transfected cells.
  • Attempts to make cell lines stably expressing both GSK3β and tau were unsuccessful because CHO cells did not survive high sustained GSK3β activity.

Methods

  • Human GSK3β was mutated at Ser-9 to Ala to produce a constitutively active enzyme, tagged with c-myc, and subcloned into pSG5.
  • The construct was transiently transfected by DEAE/dextran into CHO cells stably expressing three-repeat tau (CHO T44) or two N-terminal insert four-repeat tau (CHO T40); >95% of these cells expressed tau, while about 2–10% expressed high GSK3β levels.
  • Tau phosphorylation was assessed 48 h after transfection by indirect immunofluorescence and immunoblotting with phosphorylation-dependent and phosphorylation-independent anti-tau antibodies.

Limitations

  • The paper states that the model system does not represent physiological conditions and that only a subset of tau transfectants expressed GSK3β, making increased tau phosphorylation modest and difficult to quantify across transient transfections.
  • The paper notes that GSK3β might have activated other kinases or inhibited phosphatases, although prior in vitro data support direct phosphorylation at many observed sites.
  • Evident from the text: the study uses overexpression of a constitutively active GSK3β mutant in CHO cells rather than endogenous GSK3β, and the antibody panel samples only a limited set of phosphorylation sites.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/0304-3940(95)11902-9

Cites

20 of 20 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

10 of 24 cited sources checked · 3 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    Prior studies of tau phosphorylation by these kinases were carried out in vitro without exception.

    “However, without exception, these studies have been carried out in vitro.”
    Quote in the paperSubstantial claim3 of 5 sources checked
    • cites [2] Drewes, 1992, Mitogen activated protein (MAP) kinase transform tau protein into an Alzheimer-like state, EMBO J. source

      The indexed copy’s Full text section lists PDF page images rather than readable passages from the paper’s body. Searches for in vitro and in vivo tau phosphorylation found no passage stating that all prior studies of phosphorylation by these kinases were conducted in vitro; the reference list cannot establish the 'without exception' claim. (LLM, conf=0.75)

      Experimental — accuracy not yet measured
    • cites [4] Goedert, 1992, p42 MAP kinase phosphorylation sites in microtubule-associated protein tau are dephosphorylated by protein phosphatase 2A1, FEBS Lett. source

      The Introduction describes prior work in which a kinase purified from pig brain phosphorylated recombinant tau, and Methods §2.2 describes this paper’s phosphorylation assay using isolated tau and kinase. These passages support an in-vitro characterization, but the paper does not establish that every prior study of tau phosphorylation by the kinases was conducted in vitro. The indexed copy provides section labels but no page numbers for these passages. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [7] Hanger, 1992, Glycogen synthase kinase-3 induces Alzheimer's disease-like phosphorylation of tau: generation of paired helical filament epitopes and neuronal localization of the kinase, Neurosci. Lett. source
    • cites [12] Mandelkow, 1992, Glycogen synthase kinase 3 and the Alzheimer's disease-like state of microtubule-associated protein tau, FEBS Lett. source

      The discussion (p. 56) says tau phosphorylation by several common kinases has been studied in vitro and that whether those modifications occur in vivo remains uncertain. It does not establish the stronger claim that every study of tau phosphorylation by the kinases in question was carried out in vitro. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [14] Paudel, 1993, Brain prolinedirected protein kinase phosphorylates tau on sites that are abnormally phosphorylated in tau associated with Alzheimer's paired helical filaments, J. Biol. Chem. source
  • Premisecitation

    MAP kinase, GSK3, and neuronal cdc2-like kinase (cdk5) have been shown to phosphorylate tau in vitro at sites that are phosphorylated in PHFs.

    “Mitogen-activated protein kinase, glycogen synthase kinase-3 (GSK3) and neuronal cdc2-1ike kinase have been shown to phosphorylate tau protein in vitro at a number of sites that are phosphorylated in PHFs.”
    • Also stated in: § Abstract · plain
      “These include mitogen-activated protein (MAP) kinase, glycogen synthase kinase-3 (GSK3) and neuronal cdc2-1ike kinase (or cdk5) [2,4,7,12,14].”
    Quote in the paperSubstantial claim3 of 5 sources checked
    • cites [2] Drewes, 1992, Mitogen activated protein (MAP) kinase transform tau protein into an Alzheimer-like state, EMBO J. source

      The passage labeled Abstract in our indexed copy reports that MAP kinase phosphorylates tau and induces an Alzheimer-like state. It does not establish the claim for GSK3 or neuronal cdc2-like kinase (cdk5), and the passages retrieved for those terms provide no such result. The copy instead says that p34(cdc2) with cyclin A or B has only minor effects on tau phosphorylation; it does not identify p34(cdc2) as cdk5. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [4] Goedert, 1992, p42 MAP kinase phosphorylation sites in microtubule-associated protein tau are dephosphorylated by protein phosphatase 2A1, FEBS Lett. source

      The Discussion reports that p42 MAP kinase phosphorylates tau at sites abnormally phosphorylated in PHF tau. The Introduction also mentions other tau-phosphorylating kinases, but our copy does not identify them as GSK3 or neuronal cdc2-like kinase or establish the claimed result for those two enzymes. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [7] Hanger, 1992, Glycogen synthase kinase-3 induces Alzheimer's disease-like phosphorylation of tau: generation of paired helical filament epitopes and neuronal localization of the kinase, Neurosci. Lett. source
    • cites [12] Mandelkow, 1992, Glycogen synthase kinase 3 and the Alzheimer's disease-like state of microtubule-associated protein tau, FEBS Lett. source

      The Introduction and section 3.1 support in-vitro phosphorylation of tau by MAP kinase and GSK3 and describe Alzheimer-like phosphorylation. They do not establish the claim for neuronal cdc2-like kinase (cdk5): the Introduction instead discusses cell-cycle cdc2, reporting only slight phosphorylation and no Alzheimer-like immune response. Page numbers are unavailable in our copy. (LLM, conf=0.89)

      Experimental — accuracy not yet measured
    • cites [14] Paudel, 1993, Brain prolinedirected protein kinase phosphorylates tau on sites that are abnormally phosphorylated in tau associated with Alzheimer's paired helical filaments, J. Biol. Chem. source
  • Findingown data

    PHF1 immunoreactivity was significantly increased in cells expressing the GSK3β construct.

    “Using PHF1 and antibody 9El0 in double immunofluorescence studies, we observed a significant increase in PHF1 immunoreactivity in cells expressing the GSK3fl construct (Fig. 2E,F).”
    Quote in the paperSubstantial claim
  • Findingown data

    GSK3β was distributed throughout the cytoplasm of CHO cells expressing the construct.

    “By indirect immunofluorescence using the c-myc MAb, GSK3fl was distributed throughout the cytoplasm of CHO cells which expressed the construct (Fig. 2A).”
    Quote in the paperNot a substantial claim
  • Findingown data

    Transient transfection of GSK3β into CHO T40 and CHO T44 cells resulted in increased immunoreactivity with all phosphorylation-dependent anti-tau antibodies investigated.

    “In this study, we report that transient transfection of human GSK3fl into Chinese hamster ovary cells stably transfected with individual human tau isoforms leads to hyperphosphorylation of tau at all the sites investigated with phosphorylation-dependent anti-tau antibodies.”
    • Also stated in: § GSK3I]S9Amyc: · plain
      “However, transient transfection of CHO T40 and CHO T44 cells with GSK3fl resulted in increased immunoreactivity with all the phosphorylation-dependent anti-tau antibodies investigated.”
    Quote in the paperSubstantial claim
  • May combine two claims. 6
  • Classified as probably not a substantial claim. 6
  • The quote only partly states this claim. 2

  • May combine two claims.

    Interpretationown data

    The results extend in vitro findings to an in vivo model system and indicate that GSK3β may be involved in tau phosphorylation in vivo.

    “They extend results obtained in vitro [7,12,14] to an in vivo model system, and indicate that GSK3fl may be involved in the phosphorylation of tau in vivo.”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [7] Hanger, 1992, Glycogen synthase kinase-3 induces Alzheimer's disease-like phosphorylation of tau: generation of paired helical filament epitopes and neuronal localization of the kinase, Neurosci. Lett. source
    • cites [12] Mandelkow, 1992, Glycogen synthase kinase 3 and the Alzheimer's disease-like state of microtubule-associated protein tau, FEBS Lett. source
    • cites [14] Paudel, 1993, Brain prolinedirected protein kinase phosphorylates tau on sites that are abnormally phosphorylated in tau associated with Alzheimer's paired helical filaments, J. Biol. Chem. source
    • Rests on an assumption: Findings in GSK3β-transfected CHO cells are relevant to GSK3β-mediated tau phosphorylation in vivo.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Phosphorylation of GSK3β at Ser9 by MAP kinase-activated protein kinase-1 and p70 S6 kinase decreases its activity by 95%.

    “Thus, phosphorylation of GSK3fl by MAP kinaseactivated protein kinase-1 and p70 $6 kinase at Ser 9 decreases its activity by 95% [17].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [17] Sutherland, 1993, Inactivation of glycogen synthase kinase-3 beta by phosphorylation: new kinase connections in insulin and growth-factor signaling, Biochem. J. source

      The indexed copy gives the 90–95% inactivation, the two kinases, and Ser-9 only in its Abstract, which cannot serve as evidence under this rubric. Searches for the activity reduction and Ser-9 found no Results or other body-text passage; the apparent full-text section contains links rather than the article text. (LLM, conf=0.70)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    By immunoblotting, the c-myc antibody recognized a band of approximately 48 kDa only in transfected CHO cells, consistent with recombinant GSK3β with a c-myc tail.

    “By immunoblotting the c-myc MAb recognized a band with an apparent molecular mass of ap- proximately 48 kDa only in transfected CHO cells (Fig. 3A), consistent with the apparent molecular mass of recombinant GSK3fl [ 19] with an added c-myc tail.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [19] Woodgett, 1990, Molecular cloning and expression of glycogen synthase kinase-3/factor A, EMBO J. source

  • May combine two claims.

    Interpretationcitation

    GSK3β probably was directly responsible for the increased immunoreactivities and mobility shifts observed, because in vitro studies demonstrated that tau can be directly phosphorylated by GSK3β at many of the same sites.

    “However, since in vitro studies [7,12,14] have demonstrated that tau can be directly phosphorylated by GSK3fl at many of the sites we observed in transfected cells, GSK3fl probably was directly responsible for the increased immunoreactivities and mobility shifts observed here.”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [7] Hanger, 1992, Glycogen synthase kinase-3 induces Alzheimer's disease-like phosphorylation of tau: generation of paired helical filament epitopes and neuronal localization of the kinase, Neurosci. Lett. source
    • cites [12] Mandelkow, 1992, Glycogen synthase kinase 3 and the Alzheimer's disease-like state of microtubule-associated protein tau, FEBS Lett. source
    • cites [14] Paudel, 1993, Brain prolinedirected protein kinase phosphorylates tau on sites that are abnormally phosphorylated in tau associated with Alzheimer's paired helical filaments, J. Biol. Chem. source

  • May combine two claims.

    Findingown data

    Phosphorylation of tau at Ser396 and Ser404 caused increased PHF1 and T3P immunoreactivities and decreased electrophoretic mobility relative to mock-transfected cells and human fetal tau standards.

    “Phosphorylation of tau at this site caused an increase in PHF1 and T3P immunoreactivities, as well as a decrease in electrophoretic mobility relative to mock-transfected CHO T40 or CHO T44 cells and human fetal tau standards (Fig. 3C,D).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    GSK3β expression increased tau phosphorylation at Thr181, Ser202, and Thr231.

    “Similar results were obtained for tau phosphorylation at Thr-181, Ser-202 and Thr-231.”
    • Also stated in: § GSK3I]S9Amyc: · plain
      “Increased AT270 (Fig. 2G,H), AT180 (Fig. 2I,J) and AT8 (Fig. 2K,L) staining in transiently transfected CHO T40 was observed by indirect immunofluorescence.”
    • Also stated in: § GSK3I]S9Amyc: · plain
      “Furthermore, increased phosphorylation at these sites was also observed by immunoblotting of transfected cells (Fig. 3E,F and data not shown).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Findingown data

    Approximately 2-10% of transfected cells expressed high levels of GSK3β.

    “Approximately 2-10% of cells expressed high levels of GSK3fl.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Many phosphorylated residues in PHF-tau are serines or threonines followed by proline.

    “Prior studies have shown that many phosphorylated residues in PHF-tau are serines or threonines followed by a proline [1,6,8,11].”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [1] Bramblett, 1993, Abnormal tau phosphorylation at Ser396 in Alzheimer's disease recapitulates development and contributes to reduced microtubule binding, Neuron source
    • cites [6] Goedert, 1993, The abnormal phosphorylation of tau protein at serine202 in Alz heimer's disease recapitulates phosphorylation during development source
    • cites [8] Hasegawa, 1992, Protein sequence and mass spectrometric analyses of tau in the Alzheimer's disease brain, J. Biol. Chem. source
    • cites [11] Lee, 1991, A68: a major subunit of paired helical filaments and derivatized forms of normal tau, Science source

  • May combine two claims.

    Interpretationreasoning

    The net increase in tau phosphorylation detected by immunoblot was modest because only a subset of the tau proteins were phosphorylated by GSK3β.

    “Thus, the net increase in tau phosphorylation as detected by immunoblot is modest since only a subset of the tau proteins are phosphorylated by GSK3fl.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Hyperphosphorylated tau is the major constituent of the paired helical filament (PHF), which is the major fibrous component of neurofibrillary lesions in Alzheimer's disease.

    “Hyperphosphorylated tau protein is the major constituent of the paired helical filament (PHF), the major fibrous component of the neurofibrillary lesions of Alzheimer's disease (AD).”
    • Also stated in: § Abstract · boosted
      “The major fibrous component of the neurofibrillary lesions is the paired helical filament (PHF) which has been shown to be composed of highly phosphorylated forms of microtubuleassociated protein tau (PHF-tau).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [18] Trojanowski, 1993, PHFτ (A68): from pathological marker to potential mediator of neuron dysfunction and degen eration in Alzheimer's disease, Clin. Neurosci.

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S2 (checkable), S4 (based).

    Premisenone given

    In vitro studies are not necessarily representative of in vivo events.

    “They suffer from the inherent limitation that in vitro studies are not necessarily representative of in vivo events.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    The CHO T44 and CHO T40 cell lines stably expressing tau had >95% of cells expressing high levels of the tau proteins.

    “CHO cell lines stably expressing either three-repeat tau (CHO T44) or two N-terminal insert four-repeat tau (CHO T40) were prepared as described [1] and >95% of the CHO T44 and CHO T40 cells express high levels of these tau proteins.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [1] Bramblett, 1993, Abnormal tau phosphorylation at Ser396 in Alzheimer's disease recapitulates development and contributes to reduced microtubule binding, Neuron source

  • May combine two claims.

    Interpretationown data

    The activity of GSK3β in CHO cells was not isoform-specific, since both tau isoforms showed similar immunoreactivities.

    “The fact that both isoforms of tau showed similar immunoreactivities also suggests that the activity of GSK3fl in CHO cells was not isoformspecific.”
    Quote in the paperNot a substantial claim
13 of 13

  • The paper relies on this without support.

    Hyperphosphorylation of tau is the critical event leading to filament assembly.

    “Hyperphosphorylation of tau is believed to be the critical event that leads to filament assembly.”

  • The paper relies on this without support.

    Findings in GSK3β-transfected CHO cells are relevant to GSK3β-mediated tau phosphorylation in vivo.

    “The model system used here does not represent physiological conditions, but we used it to further understand how the phosphorylation of tau is regulated in the normal and AD brain.”
    “They extend results obtained in vitro [7,12,14] to an in vivo model system, and indicate that GSK3fl may be involved in the phosphorylation of tau in vivo.”
2 of 2

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • GSK3 is a proline-directed kinase that contributes to phosphorylation of tau at SP/TP sites.

    “GSK3 is a proline-directed kinase that contributes to phosphorylation of tau at serine or threonine followed by proline (i.e., SP/TP sites) [46–49]”

    From Stabilization of Microtubule-Unbound Tau via Tau Phosphorylation at Ser262/356 by Par-1/MARK Contributes to Augmentation of AD-Related Phosphorylation and Aβ42-Induced Tau Toxicity (2016) · cites this paper as [48]

    The Results report increased tau phosphorylation at several serine and threonine sites in cells expressing GSK3β. The introduction says GSK3α has been suggested to act as a proline-directed threonine kinase, but says GSK3β’s site specificity was not fully elucidated. The paper therefore supports GSK3β’s contribution to tau phosphorylation, but does not establish the claimed SP/TP specificity for GSK3β. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. Trojanowski, 1993, PHFτ (A68): from pathological marker to potential mediator of neuron dysfunction and degen eration in Alzheimer's disease, Clin. Neurosci., 1, 184

Cited by

Sorted by citations

Related

Authors
Sperbera, B. R., Leight, S., Goedert, M., & Lee, V. M.-Y.
Affiliations
  • University of Pennsylvania (US)
  • MRC Laboratory of Molecular Biology (GB)
Corresponding author
Lee, V.M.-Y.
ORCID
Goedert, Michel
Venue
Neuroscience Letters
Publisher
Elsevier BV
Published
1995-09
Volume
197
Issue
2
Pages
149-153
ISSN
0304-3940 (ISSN-L)
DOI
10.1016/0304-3940(95)11902-9
OpenAlex
W2078680897
Semantic Scholar
b65c92714a748a00c0908686620cc4d7ab64e748
Cited by
204
References
19
Citations by year
2026: 1 · 2025: 2 · 2024: 2 · 2023: 5 · 2022: 5 · 2021: 4 · 2020: 9 · 2019: 3 · 2018: 3 · 2017: 4 · 2016: 10 · 2015: 6 · 2014: 5 · 2013: 6 · 2012: 7
FWCI
4.22
Type
journal article
Language
en
Open access
Licence
Locations
  • Neuroscience Letters (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
National Institutes of Health
Topics
  • Health Sciences › Medicine › Physiology › Alzheimer's disease research and treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
  • Health Sciences › Medicine › Psychiatry and Mental health › Dementia and Cognitive Impairment Research
Keywords
glycogen synthase kinase-3β, tau protein, tau hyperphosphorylation, paired helical filaments, Alzheimer's disease, neurofibrillary tangles, protein kinases, tau isoforms, mitogen-activated protein kinase, phosphorylation sites
MeSH terms
Alanine, Amino Acid Sequence, Animals, Cricetinae, Humans, Molecular Sequence Data, Mutation, Phosphorylation, Serine, Transfection, Immunoblotting, CHO Cells, tau Proteins, Calcium-Calmodulin-Dependent Protein Kinases, Fluorescent Antibody Technique, Indirect, Glycogen Synthase Kinases, Glycogen Synthase Kinase 3

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