Recently, calpain, a calcium-dependent protease, was found to digest both p35 and p39 to their truncated products, p25 and p29, respectively (
- Patrick G.N.
- Zukerberg L.
- Nikolic M., De La
- Monte S.
- Dikkes P.
- Tsai L.H.
- Kusakawa G.
- Saito T.
- Onuki R.
- Ishiguro K.
- Kishimoto T.
- Hisanaga S.
). Furthermore, truncation of p35 to p25 and the subsequent formation of the p25-CDK5 complex result in neuronal cell death and hyperphosphorylation of tau (
- Lee M.S.
- Kwon Y.T., LI, M.
- Peng J.
- Friedlander R.M.
- Tsai L.H.
). From these findings, the following hypothesis emerged. Initially, various stress signals activate calpain by recruiting intracellular Ca2+
, and then the activated calpain digests p35 to p25. Finally, the p25-CDK5 complex hyperphosphorylates tau proteins, causing disruption of microtubule integrity and inevitable cell death (
- Smith D.S.
- Greer P.L.
- Tsai L.H.
Lack of knowledge about the regulation of CDK5 by its activators, p35 and p25, severely limits our understanding of the possible pathogenesis induced by CDK5. We have examined the kinetic characteristics ofin vitro phosphorylation of the longest isoform of human tau by CDK5 using recombinant proteins. This study clarified not only the kinetics of overall phosphorylation, but also the details of site-specific phosphorylation of human tau by CDK5 complexes.
This study determined the kinetic characteristics of in vitro phosphorylation of tau by CDK5 complexes using recombinant proteins. The p25-CDK5 complex was found to phosphorylate tau much faster than the p35-CDK5 complex. On the assumption that the time course of total phosphorylation can be approximated by a single exponential reaction, the time constant of the decline in phosphorylatable sites was determined, and the results suggest that p25 can accelerate the catalytic activity of CDK5 by ∼2.4-fold compared with p35.
We determined the stoichiometry (moles of Pi/mol of tau) of tau phosphorylation by CDK5 and found that the p25-CDK5 complex can phosphorylate tau faster. Furthermore, it can incorporate more phosphate into tau compared with the p35-CDK5 complex.
The stoichiometry of tau phosphorylation by neuronal cdc2-like protein kinase (brain-derived p25-CDK5 complex) was reported to be 3.8 mol of phosphate/mol of tau (
- Paudel H.K.
- Lew J.
- Ali Z.
- Wang J.H.
). Thus, the recombinant p25-CDK5 complex used in the present study is as potent as the naturally occurring CDK5 complex. Sironi et al.
- Sironi J.J.
- Yen S.H.
- Gondal J.A., Wu, Q.
- Grundke-Iqbal I.
- Iqbal K.
) studied in vitro
phosphorylation of tau at Ser262
by three different kinases (calcium/calmodulin-dependent protein kinase II, protein kinase A, and phosphorylase kinase) and found that total Pi
incorporation into tau by any of the three enzymes saturated after 100 min of reaction. Upon saturation of overall phosphorylation, calcium/calmodulin-dependent protein kinase II, protein kinase A, and phosphorylase kinase incorporated 0.9–1.2 mol of phosphate/mol of tau. Purified phosphorylase kinase from rabbit skeletal muscle reportedly phosphorylated tau to a stoichiometry of 2.1 mol of phosphate/mol of tau (
). Compared with these results, the stoichiometry of overall phosphorylation of tau by the p25-CDK5 complex is characteristically high. To evaluate the phosphorylation of tau by CDK5 quantitatively, kinetic parameters were determined using the Michaelis-Menten equation. The apparent K m
values for tau phosphorylation by the p35-CDK5 complex were 33 μm
and 2.6 min−1
, respectively. The K m
value for the p25-CDK5 complex was 27 μm
and was unchanged from that for the p35-CDK5 complex (p
< 0.05, n
= 4). Thek cat
value for the p25-CDK5 complex was 13 min−1
and was significantly larger than that for the p35-CDK5 (p
< 0.05, n
= 4). Thek cat
value for the p25-CDK5 complex was 6 times larger than that for the p35-CDK5 complex.
The kinetic parameters of CDK5 complexes were also determined using histone H1 as a substrate. The observed K m
value for the p35-CDK5 complex was in good agreement with previous results (
- Sharma P.
- Steinbach P.J.
- Sharma M.
- Amin N.D.
- Barchi Jr., J.J.
- Pant H.C.
). The difference in the K m
values for the p25-CDK5 and p35-CDK5 complexes was not significant, whereas thek cat
value for the p25-CDK5 complex was significantly larger than that for the p25-CDK5 complex. Thus, the p25-CDK5 complex is a much more potent kinase, and the p25 regulatory unit accelerates the reactivity of CDK5 without changing its affinity for tau.
For tau phosphorylation by phosphorylase kinase, theK m
and k cat
values were 6.9 μm
and 47.4 min−1
, respectively, indicating that phosphorylase kinase has a better affinity for tau and more efficient turnover of catalytic activity compared with CDK5 complexes (
). But the stoichiometry of total phosphorylation of tau by the p25-CDK5 complex is larger than that for the other tau kinases, including the p35-CDK5 complex. As a tau kinase, p25-CDK5 could contribute to the high phosphorylation stoichiometry of PHF-tau.
Our analysis of the total phosphorylation of tau revealed a difference in the catalytic activity of CDK5 complexes; site-specific phosphorylation must be clarified for a better understanding of CDK5. We have employed two independent approaches: phosphopeptide mapping of the tryptic digest of tau and Western blot analysis with phosphorylation-dependent anti-tau antibodies.
By comparing the phosphopeptide maps for the early and saturated stages of phosphorylation, we found that 32Piincorporation into spot 1 was very slow. In contrast,32Pi incorporation into the other four spots was almost complete within 120 min, suggesting that the time course of overall phosphorylation of tau depends on the phosphorylation kinetics of Ser202, Ser235, and Ser404. We further examined this unreported characteristic of site-specific phosphorylation of Ser202 and Thr205.
We have made the following observations. (a) The p35-CDK5 complex promoted 32Pi incorporation into spot 1 very slowly; and (b) the p25-CDK5 complex promoted32Pi incorporation into spot 1 more rapidly, but only after 2 h. These results raise the possibility of sequential phosphorylation of Thr205 after Ser202.
To investigate 32Pi incorporation, we conducted phosphopeptide mapping after having occluded possible phosphorylation sites with unlabeled ATP. Newly incorporated32Pi was visualized as an intense spot on the map. After occlusion with the p25-CDK5 complex, p25-CDK5 promoted32Pi incorporation into spot 1 only.32Pi incorporation into any of the major spots did not happen. These findings suggest that phosphorylation of Ser202 was already saturated and that only Thr205 was slowly phosphorylated over the extended time of incubation. In contrast, the p35-CDK5 complex failed to occlude both spots 1 and 2, as suggested by the intense signal on the two spots. The occlusion experiment also confirmed that slow phosphorylation by CDK5 complexes happens only at Ser202and Thr205.
To clarify further the time dependence of phosphorylation of Ser202
, Western blot analysis of tau was carried out. The three phosphorylation-dependent anti-tau antibodies have distinct epitopes. AT8 reacts with tau only when multiple sites around Ser202
, including Ser199
, and Thr205
, are phosphorylated. Single phosphorylation of any of the residues is not enough for AT8 reactivity (
- Goedert M.
- Jakes R.
- Vanmechelen E.
- Preuss U.
- Doring F.
- Illenberger S.
- Mandelkow E.M.
). Thus, AT8 is useful in detecting phosphorylation of Ser202
for proline-directed kinases, including CDK5 complexes. In contrast, Tau-1 requires an absolutely non-phosphorylated epitope around Ser202
- Szendrei G.I.
- Lee V.M.
- Otvos Jr., L.
). In the present study, Tau-1 reacted with recombinant tau quite well. However, phosphorylation of Ser202
caused loss of Tau-1 reactivity. The pS202 antibody is raised against phosphorylated Ser202
of human tau.
AT8 reactivity was latent, whereas the Tau-1 signal started to decline without delay, suggesting that Ser202 and Thr205 cannot be phosphorylated simultaneously. Either Ser202 or Thr205 must be phosphorylated first. The phosphorylated Ser202 signal (immunoreactivity of the pS202 antibody) started to develop as soon as the Tau-1 signal disappeared. Although phosphorylation of Thr205 as a first step of sequential phosphorylation cannot be ruled out, the initial phosphorylation of Ser202 and the subsequent phosphorylation of Thr205 seem to be the principal route to double phosphorylation.
Many of the pathologic epitopes on hyperphosphorylated tau in the brains of Alzheimer's disease patients were thought to be generated by sequential phosphorylation by different tau kinases. For example, phosphorylation of a specific amino acid residue by CDK5 is a prerequisite for the subsequent kinase action of glycogen synthase kinase-3β (
- Sengupta A., Wu, Q.
- Grundke-Iqbal K.
- Singh T.
- Alvarrez A.
- Toro R.
- Cacere A.
- Maccioni R.B.
). Phosphorylation of Thr212
by glycogen synthase kinase-3β is known to facilitate protein kinase A action on Ser214
- Zheng-Fischhofer Q.
- Biernat J.
- Mandelkow E.M.
- Illenberger S.
- Godemann R.
- Mandelkow E.
). Such hierarchy among tau kinases (
- Jicha G.A.
- O'Donnell A.
- Weaver C.
- Angeletti R.
- Davies P.
) makes it complicated to clarify the mechanism of abnormal phosphorylation of tau.
We have shown evidence for the sequential phosphorylation of Ser202 and Thr205 of tau by the p25-CDK5 complex. Phosphorylation of Thr205 occurred only after Ser202 was phosphorylated. As a result, phosphorylation of Thr205 was extremely slow compared with phosphorylation of other sites such as Ser235 and Ser404. In addition, the p35-CDK5 complex had weak or negligible kinase action on Thr205.
Our findings strongly suggest that cleavage of p35 to p25 regulates not only the overall kinase activity of CDK5, but also the sequential phosphorylation of Ser202 and Thr205. The association of CDK5 with a particular activator unit, p35 or p25, provides a novel mechanism of controlling kinase characteristics.