The development of cell processes induced by tau protein requires phosphorylation of serine 262 and 356 in the repeat domain and is inhibited by phosphorylation in the proline-rich domains

Abstract

The differentiation of neurons and the outgrowth of neurites depends on microtubule-associated proteins such as tau protein. To study this process, we have used the model of Sf9 cells, which allows efficient transfection with microtubule-associated proteins (via baculovirus vectors) and observation of the resulting neurite-like extensions. We compared the phosphorylation of tau23 (the embryonic form of human tau) with mutants in which critical phosphorylation sites were deleted by mutating Ser or Thr residues into Ala. One can broadly distinguish two types of sites, the KXGS motifs in the repeats (which regulate the affinity of tau to microtubules) and the SP or TP motifs in the domains flanking the repeats (which contain epitopes for antibodies diagnostic of Alzheimer's disease). Here we report that both types of sites can be phosphorylated by endogenous kinases of Sf9 cells, and that the phosphorylation pattern of the transfected tau is very similar to that of neurons, showing that Sf9 cells can be regarded as an approximate model for the neuronal balance between kinases and phosphatases. We show that mutations in the repeat domain and in the flanking domains have opposite effects. Mutations of KXGS motifs in the repeats (Ser262, 324, and 356) strongly inhibit the outgrowth of cell extensions induced by tau, even though this type of phosphorylation accounts for only a minor fraction of the total phosphate. This argues that the temporary detachment of tau from microtubules (by phosphorylation at KXGS motifs) is a necessary condition for establishing cell polarity at a critical point in space or time. Conversely, the phosphorylation at SP or TP motifs represents the majority of phosphate (>80%); mutations in these motifs cause an increase in cell extensions, indicating that this type of phosphorylation retards the differentiation of the cells.

Figure 1

Diagrams of tau isoforms or constructs and antibody epitopes. (A) Constructs: 1) htau40, the largest isoform of tau in the human CNS, containing four 31-residue repeats in the C-terminal half (numbered boxes) and two inserts near the N-terminus (411 residues). The regions flanking the repeats are labeled P1-P2 and R′ (hatched). 2) htau23, the smallest of the six isoforms generated by alternative splicing (352 residues). It lacks the N-terminal inserts and the second repeat. 3) Construct KXGA/R1, in which Ser262 in the KXGS motif of the first repeat is replaced by Ala. 4) Construct KXGA/R1/4, in which the serines in the KXGS motifs of repeats 1 and 4 are replaced by Ala (S262A and S356A). 5) Construct KXGA/R1/3/4, in which the serines in the KXGS motifs of repeats 1, 3, and 4 are replaced by Ala (S262A, S324A, and S356A). 6) Construct tau23/AP, in which all SP and TP motifs are replaced by AP. Construct tau23/AP/R1/4 is similar, but in addition Ser262 and Ser356 are changed into Ala. 7) Construct K19 represents the repeats only. (B) Antibody epitopes. Most phosphorylation-dependent antibodies react with SP and TP motifs in the flanking domains before or after the repeats.

Figure 2

Immunofluorescence of Sf9 cells transfected with different tau constructs. Cells were transfected with htau23 (A), KXGA/R1 (B), or KXGA/R1/3/4 (C) and immunostained 60 h after infection with antibodies DM1A (for tubulin, left) and K9JA (for tau, right). Transfection with htau23 (A) leads to many processes; when Ser262 is mutated to Ala (B), one still finds processes similar to tau23, but when all three KXGS motifs are changed into KXGA (C), the formation of processes is almost completely inhibited. Note that in the case of tau staining the cell bodies appear overexposed to image the more weakly stained processes. Bar, 50 μm.

Figure 3

Time course of process formation. (A) Quantitation from monolayer culture. Processes begin to appear at ∼30–40 h after infection; their frequency rises linearly thereafter. For tau23 (filled circles), the extrapolated onset is at t₀ = 34 h; the increase of process-bearing cells is 1.4% cells/h. Construct tau23/AP (open squares) is more efficient; processes appear earlier and increase faster (t₀ = 32 h; slope, 1.9%/h), showing that when the SP and TP sites remain unphosphorylated, the formation of processes is enhanced. Construct KXGA/R1/3/4 (filled triangles) is much less efficient (t₀ = 38 h; slope, 0.3%/h), showing that the phosphorylation of KXGS motifs enhances process formation. Construct KXGA/R1 behaves similar to tau23, whereas KXGA/R1/4 (open circles) behaves similar to KXGA/R1/3/4, indicating that two or more KXGS motifs must cooperate to change the response of the cells. Similarly, process formation is also inhibited with construct tau23/AP/R1/4 (diamonds), showing that the dominant effect of phosphorylation is in the repeats, not in the flanking regions. (B) Quantitation of process formation from suspension culture. This overemphasizes cells with stable processes, because labile ones are lost more easily by shear forces. Thus processes are less numerous, but qualitatively the trends are the same as in A; i.e., construct tau23/AP is most efficient, whereas KXGA/R1/3/4 induces almost no processes. Note the fourfold difference between tau23 and tau23/AP, indicating the greater stability of the processes, because the SP and TP motifs cannot be phosphorylated. Bars, standard deviations.

Figure 4

Phosphorylation of tau and tau constructs transfected in Sf9 cells. (A) 10% SDS-PAGE. Tau protein or mutants (tau23, KXGA/R1, KXGA/R1/3/4, and tau23/AP) were isolated from equal quantities of Sf9 cells transfected with the appropriate tau constructs and prepared in equal volumes of sample buffer. Equal volumes of protein solution representing the different harvest time points were loaded onto the SDS gel (10%). The amounts of loaded proteins correspond approximately to 0.4 μg (48 h), 2.5 μg (66 h), and 3 μg (73.5 h). As seen from the multiple and strongly shifted bands, tau23 is highly phosphorylated in a heterogeneous manner. The same applies to contructs KXGA/R1 and KXGA/R1/3/4 (which lack only one or three phosphorylation sites at KXGS motifs but retain all SP or TP sites). By contrast, construct tau23/AP shows very little phosphorylation and no shift, because it lacks most phosphorylation sites (note that the KXGS sites do not induce a shift; Drewes et al., 1995). By implication, the strong shift sites S409 and S416 (targets of PKA or CaMKII) are also not phosphorylated in Sf9 cells. (B) The same samples were immunoblotted with antibody 5E2, which reacts independently of phosphorylation and reflects the total amount of tau protein. Lane M, marker proteins.

Figure 5

Phosphorylation sites of baculovirus-expressed tau and tau constructs. Sf9 cell lysates (72 h after infection) were blotted and incubated with different phosphorylation-dependent and -independent mAbs. The tau constructs are, from left to right: 1) tau23/AP, 2) KXGA/R1, 3) htau23, 4) KXGA/R1/3/4, 5) untransfected Sf9 cell extract (control without tau), 6) Sf9 cells transfected with wild-type baculovirus (control without tau), 7) htau23 expressed in E. coli (unphosphorylated control), and 8) htau23 expressed in E. coli and phosphorylated with brain extract kinase activity (affecting largely the SP and TP sites and inducing a strong M_r shift; see Biernat et al., 1993). Antibodies, from top to bottom: 5E2 (a pan-tau antibody) recognizes all preparations that contain tau. Tau-1 shows only a moderate reaction with tau constructs expressed in Sf9 cells, because a single P site in the vicinity of residue 200 suffices to reduce the binding, but there is a strong reaction with unphosphorylated tau expressed in E. coli or tau23/AP. The antibodies AT-8 to SMI-34 are specific for different phosphorylated SP and TP motifs and therefore show no reaction with the tau23/AP mutant or with E. coli–expressed htau23. Antibody 12E8 recognizes only those constructs that contain Ser262 and/or Ser356 in the repeats. AT-100 recognizes tau phosphorylated at Thr212 and Ser214; this reaction is highly specific for Alzheimer tau but occurs in Sf9 cells as well, provided both sites are phosphorylatable (e.g., not in the tau23/AP mutant).

Figure 6

Two-dimensional phosphopeptide maps of tau constructs expressed in Sf9 cells and phosphorylated by endogenous kinases. (A) htau23 (higher magnification); (B) htau23/AP; (C) KXGA/R1/3/4; (D) construct K19 phosphorylated with PKA; (E) mixture of tau23 and K19 phosphorylated with PKA. In A the majority of spots are generated by phosphopeptides containing one or more of the 14 SP and TP motifs. The four phosphorylation sites in the repeats are highlighted and underlined (three KXGS motifs with S262, S324, and S356, plus S320). In B all SP or TP motifs are changed into AP so that the remaining major phosphorylation sites are Ser214 and those in the repeats, plus some unknown spots. In C the KXGS motifs in the repeats are changed into KXGA and are therefore absent from the map; besides the phosphorylated SP and SP motifs one observes S214 and S320. In D the repeat construct K19 was phosphorylated with PKA, showing only the sites S262, S324, S356, and S320. This sample was run together with tau23 in E to identify the sites. For details on analysis of phosphopeptides, see Illenberger et al. (1998).

Figure 7

Diagram illustrating the effects of tau phosphorylation on process formation. The left side shows the interaction between a microtubule and tau in different states of phosphorylation. The right side shows a cell body and a cell process whose length represents the efficiency of process formation. (A) Tau with unphosphorylated KXGS motifs in the repeats binds to microtubules (left), but if it becomes phosphorylated at KXGS motifs (filled circles), it detaches from microtubules, and these become dynamic. This is necessary, at least temporarily, for the nucleation of a cell process (independently of whether the SP or TP sites are phosphorylated; filled or empty diamonds in the flanking domains). (B) Tau phosphorylatable at the KXGS motifs in the repeats but not at the SP and TP motifs (Ala mutations in the flanking domains) allows the formation of long and stable processes once they are initiated. (C) If tau cannot be phosphorylated at KXGS motifs (Ala mutations in repeats) it remains tightly attached to microtubules. This blocks the initiation of cell processes and therefore inhibits their growth, independently of the phosphorylation at SP or TP motifs.