Spectroscopic studies of GSK3{beta} phosphorylation of the neuronal tau protein and its interaction with the N-terminal domain of apolipoprotein E

Abstract

Alzheimer disease neurons are characterized by extraneuronal plaques formed by aggregated amyloid-β peptide and by intraneuronal tangles composed of fibrillar aggregates of the microtubule-associated Tau protein. Tau is mostly found in a hyperphosphorylated form in these tangles. Glycogen synthase kinase 3β (GSK3β) is a proline-directed kinase generally considered as one of the major players that (hyper)phosphorylates Tau. The kinase phosphorylates mainly (Ser/Thr)-Pro motifs and is believed to require a priming activity by another kinase. Here, we use an in vitro phosphorylation assay and NMR spectroscopy to characterize in a qualitative and quantitative manner the phosphorylation of Tau by GSK3β. We find that three residues can be phosphorylated (Ser-396, Ser-400, and Ser-404) by GSK3β alone, without priming. Ser-404 is essential in this process, as its mutation to Ala prevents all activity of GSK3β. However, priming enhances the catalytic efficacy of the kinase, as initial phosphorylation of Ser-214 by the cAMP-dependent protein kinase (PKA) leads to the rapid modification by GSK3β of four regularly spaced additional sites. Because the regular incorporation of negative charges by GSK3β leads to a potential parallel between phospho-Tau and heparin, we investigated its interaction with the heparin/low density lipoprotein receptor binding domain of human apolipoprotein E. We indeed observed an interaction between the GSK3β-promoted regular phospho-pattern on Tau and the apolipoprotein E fragment but none in the absence of phosphorylation or the presence of an irregular phosphorylation pattern by the prolonged activity of PKA. Apolipoprotein E is therefore able to discriminate and interact with specific phosphorylation patterns of Tau.

FIGURE 1.

¹H-¹⁵N HSQC spectrum of PKA-GSK3β-phosphorylated ¹⁵N,¹³C-labeled Tau. See text for the phosphorylation conditions and Table 1 for the assignment of the identified peaks.

FIGURE 2.

A, SDS-PAGE (12%) kinetic study of the phosphorylation of Ser(P)-214-Tau incubated with a catalytic amount (1 μm) of recombinant GSK3β. Incubation times are indicated above the gel. B, 12% SDS-PAGE of wild type Tau primed by PKA at Ser-214 and submitted to the action of GSK3β (1st lane). 2nd lane, mutant Tau S404A not primed by PKA and submitted to the action of GSK3β. 3rd lane, mutant Tau S404A primed by PKA at Ser-214 and submitted to the action of GSK3β.

FIGURE 3.

NMR kinetic study of the phosphorylation of Ser(P)-214-Tau by GSK3β. A, ¹H-¹⁵N HSQC of ¹⁵N-labeled Tau before the action of PKA and GSK3β (A),¹H-¹⁵N HSQC spectra of ¹⁵N-labeled Ser(P)-214-Tau incubated for 3 (B), 4.5 (C), and 6 h (D) with 1 μm GSK3β at 30 °C. Peak identity was inferred from the HSQC assignments obtained on the PKA/GSK3β phosphorylated doubly labeled ¹⁵N-¹³C-Tau sample (Fig. 1 and Table 1).

FIGURE 4.

¹H-¹⁵N HSQC spectra of Tau and phosphorylated Tau at a final concentration of 100 μm without (red) or with (black) the 22-kDa N-terminal domain of human apolipoprotein E3:1–191 (apoE22K) at a final concentration of 200 μm to probe their interaction. Left panel, Tau is not phosphorylated; middle panel, Tau is phosphorylated 5 h by PKA (Tau-PKA), and right panel, Tau is phosphorylated at Ser-214 by PKA prior to phosphorylation by GSK3β during 6 h (Ser(P)-214-Tau-GSK3).

FIGURE 5.

Diagram of major phosphorylation sites identified in this study and resulting from the phosphorylation of prephosphorylated Tau at Ser-214 by GSK3β. The location of the priming site at Ser-214 (phosphorylated by PKA) is shown above the bar. The sites phosphorylated by GSK3β are shown below the bar. The diagram is a schematic representation of human Tau longest isoform present in the central nervous system (441 residues). P1 and P2 indicate the two proline-rich domains of Tau, and R indicates the four microtubule binding repeats.