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First published online 4 January 2005
doi: 10.1242/jcs.01655

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Evidence that phosphorylation of the microtubule-associated protein Tau by SAPK4/p38 at Thr50 promotes microtubule assembly

¹ MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK
² MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

View larger version (19K): [in a new window]   Fig. 1. Phosphorylation of GST-hTau40 by different SAPK/p38 isoforms. (A) Human GST-Tau40 (hTau40) was labelled with Mg[-32P]ATP in the presence of p38, p38ß, SAPK3/p38 or SAPK4/p38, and subjected to SDS-PAGE. Phosphorylated hTau40 was excised from the gel, digested with trypsin and the peptides separated by chromatography. The column was developed with an acetonitrile gradient (broken line) and 32P-radioactivity is shown by the solid line. Phosphopeptides P1 to P13 are indicated; the asterisks indicate a phosphopeptide from the GST tag. (B) Identification of the sites in hTau40 phosphorylated by SAPK/p38s. The kinases used to phosphorylate Tau are indicated in brackets: , p38; ß, p38ß; , SAPK3/p38; , SAPK4/p38. P3 in Tau phosphorylated by p38 is a mixture of one to three di-phosphopeptides, whereas P5, for all p38 isoforms, is a mixture of two to four mono-phosphopeptides. P12 and P13 contained the same phosphopeptide, but the Met in P12 was oxidised. All residues were identified by a combination of techniques (MALDI-TOF, Q-TOF, MS/MS, solid phase sequencing and phospho-amino acid analysis) (Campbell and Morrice, 2002), with the exception of residue Thr69 for SAPK3/p38, which was identified only by HPLC. The phosphorylated residue(s) in each phosphopeptide is/are underlined and numbered according to the sequence of the longest Tau isoform in human brain. (C) The Tau isoform (hTau40) used in this study. The identified phosphorylation sites are shown. The C-terminal domain contains three or four repeats (R1-R4) that are flanked N-terminally by proline-rich regions (P1 and P2) which extend into the N-terminal or projection domain. The proline-rich regions contain most of the Ser/Thr-Pro motifs. The table indicates the in vitro phosphorylation sites on hTau40 phosphorylated by the different p38s.

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View larger version (27K): [in a new window]   Fig. 2. Characterisation of the antibody that recognises Tau phosphorylated at Thr50. GST-Tau was phosphorylated by 2 U/ml of SAPK3/p38. (A) 0.5 µg of GST-Tau non-phosphorylated (NK) or phosphorylated (SAPK3/p38) was electrophoresed and blotted with the Phos-Thr50 antibody raised against the peptide corresponding to residues 44-55 of Tau phosphorylated at Thr50 or with the Tau antibody that recognises both the unphosphorylated and phosphorylated proteins. (B) Different amounts of phosphorylated GST-Tau or GST-Tau(T50A) were spotted onto a nitrocellulose membrane. Membranes were incubated with 1 µg/ml of Tau antibody, antibody Phos-Thr50 or antibody Phos-Thr181. Immunoblots were carried out in the absence of competing peptide or protein (None), or in the presence of the unphosphorylated peptide immunogen (Thr50 peptide), the phosphopeptide immunogen (Phos-Thr50 peptide), phosphopeptide for another site (Phos-Thr153 peptide, Phos-Ser202 peptide or Phos-Thr69 peptide), a mixture of phosphopeptides (Phos-Thr153 peptide, Phos-Ser202 peptide and Phos-Ser235) or 0.2 mg/ml Phos-Tau(T50A) protein. Peptides were at 100 µg/ml. (C) Phosphorylation of Tau by p38 MAPKs in HEK293 cells. After transfection with plasmids encoding HA-Tau and HA-p38 or HA-p38ß, or myc-SAPK3/p38 or myc-SAPK4/p38, HEK293 cells were exposed for 15 minutes to 0.5 M sorbitol or 0.5 mM sodium arsenite, or for 30 minutes to UV-C (200 J/m2). Five micrograms of cell lysate was immunoblotted using antibody Phos-Thr50 and an antibody that recognises unphosphorylated and phosphorylated Tau equally. To examine activation of p38 MAPKs, 10 µg of cell lysate was used in the immunoblot. The p38 phospho-specific antibody also recognises phosphorylated p38ß, SAPK3/p38 and SAPK4/p38.

View larger version (22K): [in a new window]   Fig. 3. Phosphorylation of endogenous Tau in SH-SY5Y human neuroblastoma cells. (A) SH-SY5Y cells were incubated for 6 hours in medium with (1) or without (2) serum and exposed for 15 minutes to 0.5 M sorbitol or 0.5 mM sodium arsenite, or for 30 minutes to UV-C (200 J/m2). Endogenous Tau was immunoprecipitated with an anti-Tau antibody from 1.0 mg of cell lysate and immunoblotted using the Phos-Thr50 antibody or an antibody that recognises both unphosphorylated and phosphorylated Tau. To examine SAPK/p38 activation, 50 µg of cell lysate was used in the immunoblot with the p38 phospho-specific antibody, as shown in Fig. 2. (B) SH-SY5Y cells were preincubated for 1 hour with or without 10 µM SB203580 or 10 µM PD184352 prior to a 15 minutes exposure to 0.5 M sorbitol. Tau protein was immunoprecipitated and immunoblotted as indicated in A. Endogenous SAPK4/p38 was immunoprecipitated from 10 mg cell lysate with anti-SAPK4/p38 antibody, and immunoblotted using the p38 phospho-specific antibody, or with an antibody that recognises both unphosphorylated and phosphorylated SAPK4/p38. Activation of other p38s was examined as described in A. 60 µg of cell lysates were immunoblotted with an antibody that recognises MAPKAP-K2 phosphorylated at Thr334 or with an antibody that recognises both unphosphorylated and phosphorylated MAPKAP-K2.

View larger version (32K): [in a new window]   Fig. 4. Knock-down of SAPK3/p38 and SAPK4/p38 inhibits Tau phosphorylation at Thr50 after osmotic stress. (A) Comparison of substrate specificities of different SAPK/p38s. Each kinase (0.2 U/ml) was assayed under initial rate conditions, as described in Materials and Methods. The final concentrations of the Thr50-peptide (black bars) or MBP (grey bars) were 100 µM and 20 µM, respectively. Substrate phosphorylation is plotted as a percentage of the maximal phosphorylation. Results in A are shown as the mean±s.e.m. for triplicate determinations from a single experiment. (B) SH-SY5Y cells were transfected in the absence of siRNA (empty pSuper vector or control GAPDH siRNA), or in the presence of siRNA against SAPK4/p38 (pS1, pS2 and pS3 constructs) or siRNA against SAPK3/p38 (S3mix and pS3mix) as detailed in Materials and Methods. SAPK4/p38 from 10 mg lysate or SAPK3/p38 from 1 mg lysate was immunoprecipitated and immunoblotted as indicated above. (C) Endogenous Tau was immunoprecipitated from the cell lysates indicated in panel B, and immunoblotted as indicated in Fig. 3.

View larger version (29K): [in a new window]   Fig. 5. Effects of Tau mutations T50E and T50A on tubulin polymerisation and reassembly of microtubules. (A) Polymerisation of tubulin promoted by GST-Tau(WT), GST-Tau(T50A) and GST-Tau(T50E) was monitored over time by measuring turbidity. A typical experiment is shown; similar results were obtained using at least three separate preparations of each protein. (B) HEK293 cells transfected with Tau(WT), Tau (T50E) or Tau(T50A) were treated with nocodazole for 2 hours. The microtubule network in transfected cells is shown 20 minutes after nocodazole had been washed out. Immunofluorescence staining was performed using a monoclonal antibody that recognises ß-tubulin. The percentages of cells with re-polymerising microtubules were determined by counting five different optical fields containing at least 200 cells per field. ** indicates that the differences between the three groups were statistically significant (P<0.01). (C) HEK293 cells transfected with Tau(WT), Tau(T50A) or Tau(T50E) were exposed to 0.5 M sorbitol for 15 minutes. Five micrograms of cell lysate was immunoblotted using antibody AT270, which recognises phospho-Thr181 in Tau, and an antibody that recognises unphosphorylated and phosphorylated Tau.

View larger version (83K): [in a new window]   Fig. 6. Phosphorylation of Thr50 in PHF-Tau from Alzheimer's disease brains. Immunoblots of PHF-Tau with either Phos-Thr50 antibody (1 µg/ml) in the presence of 50 µg/ml of hTau46 (the 412 amino acid form), to further ensure the specificity of the antibody, or anti-Tau serum BR134 (1:1,000). Lanes 1 and 2, PHF-Tau prepared from two Alzheimer's disease brains; lane 3, mixture of recombinant human brain Tau isoforms.

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