Combinatorial Tau pseudophosphorylation: markedly different regulatory effects on microtubule assembly and dynamic instability than the sum of the individual parts

Abstract

Tau is a multiply phosphorylated protein that is essential for the development and maintenance of the nervous system. Errors in Tau action are associated with Alzheimer disease and related dementias. A huge literature has led to the widely held notion that aberrant Tau hyperphosphorylation is central to these disorders. Unfortunately, our mechanistic understanding of the functional effects of combinatorial Tau phosphorylation remains minimal. Here, we generated four singly pseudophosphorylated Tau proteins (at Thr(231), Ser(262), Ser(396), and Ser(404)) and four doubly pseudophosphorylated Tau proteins using the same sites. Each Tau preparation was assayed for its abilities to promote microtubule assembly and to regulate microtubule dynamic instability in vitro. All four singly pseudophosphorylated Tau proteins exhibited loss-of-function effects. In marked contrast to the expectation that doubly pseudophosphorylated Tau would be less functional than either of its corresponding singly pseudophosphorylated forms, all of the doubly pseudophosphorylated Tau proteins possessed enhanced microtubule assembly activity and were more potent at regulating dynamic instability than their compromised singly pseudophosphorylated counterparts. Thus, the effects of multiple pseudophosphorylations were not simply the sum of the effects of the constituent single pseudophosphorylations; rather, they were generally opposite to the effects of singly pseudophosphorylated Tau. Further, despite being pseudophosphorylated at different sites, the four singly pseduophosphorylated Tau proteins often functioned similarly, as did the four doubly pseudophosphorylated proteins. These data lead us to reassess the conventional view of combinatorial phosphorylation in normal and pathological Tau action. They may also be relevant to the issue of combinatorial phosphorylation as a general regulatory mechanism.

FIGURE 1.

Construction of pseudophosphorylated 4-repeat Tau. Schematic of four-repeat Tau, with positions of pseudophosphorylated Tau marked with arrows. Boxes labeled R1, R2, R3, and R4 correspond to the 18-amino acid imperfect repeats. Boxes labeled IR correspond to the three interrepeats. Exons 2, 3, and 10 are regulated by alternative splicing. All three exons are present in the Tau constructs used in this study.

FIGURE 2.

Tau-microtubule co-sedimentation assay. 15 μm tubulin dimers were incubated in the presence or absence of varying amounts of four-repeat Tau or the nonspecific protein BSA at 35 °C until steady state was achieved. The reaction was then centrifuged through a sucrose cushion. Microtubules and microtubule-bound Tau pellet, whereas non-polymerized tubulin and non-microtubule-bound Tau remain in the supernatant. SDS-PAGE analysis of the supernatant and pellet defines the percentages of tubulin in each fraction. The percentage of polymerized tubulin values are presented as mean ± S.E. (error bars).

FIGURE 3.

Effects of single and double pseudophosphorylation upon the ability of Tau to promote microtubule assembly. A, 15 μm tubulin dimers were incubated in the presence or absence of varying amounts of four-repeat Tau at 35 °C until steady state was achieved. The reaction was then centrifuged through a sucrose cushion. Microtubules and microtubule-bound Tau pellet, whereas non-microtubule bound Tau and non-polymerized tubulin remain in the supernatant. SDS-PAGE analysis of the supernatant and pellet defines the percentages of tubulin in each fraction. Statistical significance of the data based on Student's t test is presented in Table 1. B, effects of Tau pseudophosphorylation on the tubulin/Tau “packing ratio” in assembled microtubules. Data for tubulin and Tau content in assembled microtubules are from Fig. 3A and Tables 1 and 2. The values are given as mean ± S.E. (error bars) from at least three independent experiments.

FIGURE 4.

Examples of microtubule life history plots. Changes in the length of the microtubules grown in the absence of Tau (top), in the presence of 0.293 μm 4R WT Tau (1:40 Tau/tubulin dimer ratio; middle), or in the presence of 0.293 μm 4R S262D pseudophosphorylated Tau (1:40 Tau/tubulin dimer ratio; bottom) were plotted as a function of time. From these and additional life history plots, individual growth, shortening, and attenuation events were identified, and rates and lengths of each event were determined, as described under “Experimental Procedures.”

FIGURE 5.

Effects of various single and double pseudophosphorylation upon microtubule shortening (A) and growth (B) events. The positions of the ends of individual microtubules were tracked in the presence or absence of WT or pseudophosphorylated Tau at a 1:40 molar ratio of Tau/tubulin dimers, and life history plots were generated. From these data, individual growth, shortening, and attenuation events were identified. A, the average growth and shortening rates were calculated as the total growth or shortening length change for a particular reaction condition divided by the total time spent for growth or shortening events in that condition. Average growth and shortening lengths represent the average of independent growth and shortening events. Values are given as mean ± S.E. (error bars). n values for each condition are presented in Table 3. *, value significant at 95% confidence level compared with 4R WT Tau control conditions. **, value significant at 99.9% confidence level compared with 4R WT Tau control conditions.

FIGURE 6.

Effects of various single and double pseudophosphorylation upon catastrophe (A) and rescue (B) frequencies. A catastrophe is defined as conversion of a growing or attenuation event to a shortening event. Catastrophe frequencies were calculated as the total number of catastrophes divided by the total time spent growing and attenuated. A rescue is defined as a transition from shortening to either growth or attenuation. Rescue frequencies were calculated as the total number of rescues divided by the total amount of time spent shortening. Based upon the life history plots described in Fig. 4, catastrophe and rescue frequencies were calculated. Values are given as mean ± S.E. (error bars). *, value significant at 95% confidence level compared with 4R WT Tau control conditions. **, values significant at 99.9% confidence level compared with 4R WT Tau control conditions.

FIGURE 7.

Effects of various single and double pseudophosphorylation upon total time spent for attenuation, growth, or shortening. Based upon the life history plots described in Fig. 4, the percentage of time spent in each phase was calculated.

FIGURE 8.

Effects of various single and double pseudophosphorylation upon overall microtubule dynamics at steady state in vitro. Dynamicity is a calculated measure of overall dynamic activity (i.e. total length grown plus total length shortened divided by total time observed). Based upon the life history plots described in Fig. 4, dynamicity was calculated for all conditions. Values are given as mean ± S.E. (error bars). *, value significant at 95% confidence level compared with 4R WT Tau control conditions. **, values significant at 99.9% confidence level compared with 4R WT Tau control conditions.