Phosphorylated tau can promote tubulin assembly

Abstract

Phosphorylation can affect the function of microtubule-associated protein tau. Here, the human brain tau with 441 amino acids was phosphorylated by cyclic-AMP-dependent protein kinase (PKA) or glycogen synthase kinase-3beta. PKA-phosphorylated tau (2.7 mol phosphates/mol) does not promote tubulin assembly as judged by spectrophotometric and atomic force microscopy measurements, unless trimethylamine N-oxide (TMAO), a natural occurring osmolyte, is included in these assays. TMAO is also found to promote tubulin assembly of glycogen synthase kinase-3beta-phosphorylated tau (1.6 mol phosphates/mol). TMAO does not act by causing a chemical dephosphorylation of phosphorylated tau, but it acts to overcome the functional deficit caused by phosphorylation. PKA-phosphorylated tau binds to tubulin in the presence of TMAO and lowers the critical concentration of tubulin needed for assembly. From these data, we conclude that PKA-phosphorylated tau retains the ability to bind tubulin and promote tubulin assembly. TMAO is required, however, to sensitize the reaction. Possible uses of TMAO in relation to studies of tubulin assembly in vitro, in intact cells, and in relation to Alzheimer's disease are presented in this report.

Figure 1

(a) Spectrophotometric measurement of tubulin assembly. Tubulin was incubated (30°C, 15 min) alone (solid circles) or with TMAO (solid triangles), and the assemblies were monitored by an increase in the absorbance at 350 nm. By using the same conditions, tubulin was assembled in the presence of tau (open circles) or tau and TMAO (open triangles). Finally, tubulin was assembled with PKA-phosphorylated tau in the presence (open squares) or absence (solid squares) of TMAO. The phosphorylation stoichometry of PKA-phosphorylated tau was 2.7 ± 0.1 phosphates/mol. (b) SDS/PAGE analysis of tau proteins. Nonphosphorylated tau (lane 1) and PKA-phosphorylated tau with 2.8 phosphates/mol (lane 2) were analyzed in a 10% SDS/PAGE gel. Proteins in the gel were visualized by a Coomassie brilliant blue R staining. (c) Radioautogram of ³²P-labeled tau in a SDS/PAGE analysis. Tau was phosphorylated with PKA in the presence of [γ-³²P]ATP. Any aggregates of phosphorylated tau–TMAO (lane 1) and phosphorylated tau—tubulin–TMAO (lane 2) were collected by centrifugation and analyzed by SDS/PAGE.

Figure 2

AFM images of tubulin assembly. Tubulin (a) was assembled by using standard reaction conditions (Fig. 1) and fixed. The mixture was then spotted onto a mica disc, dried, and scanned by AFM. Other reaction mixtures that included tau (b), TMAO (c), tau + TMAO (d), PKA-phosphorylated tau with 2.7 phosphates/mol (e), or PKA-phosphorylated tau + TMAO (f) were also scanned. The sample height is color coded, as indicated by the height scale bar. Note field sizes for a, c, and e (2 μm × 2 μm) are smaller than those for b, d, and f (25 μm × 25 μm) to highlight features. The heights of MTs shown in b, d, and f are all about 20 nm, in general.

Figure 3

The determination of tubulin-critical concentrations. Six assembly groups were analyzed: (i) tubulin alone (open circles); (ii) tubulin plus TMAO (close circles); (iii) tubulin plus tau (open triangles); (iv) tubulin, tau, plus TMAO (closed triangles); (v) tubulin plus phosphorylated tau (open squares); (vi) tubulin, phosphorylated tau, plus TMAO (closed squares). The detailed assembly conditions are described in Materials and Methods. The lines are the results after a linear curve fitting processing for each group.

Figure 4

Analysis of tubulin assembly with GSK-3β-phosphorylated tau. (a) Spectrophotometric measurement of tubulin assembly. Reaction mixtures contained tubulin alone (solid circles) or tubulin + TMAO (solid triangles). Other reactions contained tubulin in the presence of tau (open circles), tau + TMAO (open triangles), GSK-3β-phosphorylated tau (solid squares), or GSK-3β-phosphorylated tau + TMAO (open squares). The phosphorylation stoichometry of GSK-3β-phosphorylated tau is 1.6 phosphates/mol. (b) AFM image of the reaction mixture that contained tubulin, GSK-3β-phosphorylated tau, and TMAO.