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.