Truncation of CDK5 Activator p35 Induces Intensive Phosphorylation of Ser202/Thr205 of Human Tau

Abstract

Hyperphosphorylated tau is a major component of neurofibrillary tangles, one of the hallmarks of Alzheimer's disease. CDK5 is a kinase that phosphorylates the tau protein, and its endogenous activator, p35, regulates its activity. Recently, calpain was found to digest p35 to its truncated product, p25. Several lines of evidence suggest that p25-CDK5 has much more powerful kinase activity and that it may cause abnormal hyperphosphorylation of tau. In this study, we have examined the kinetic characteristics of in vitro phosphorylation of the longest isoform of human tau by CDK5 and its activators using recombinant proteins. Although the kinase activity of CDK5 in phosphorylating tau was significantly higher in the presence of p25, the affinity of CDK5 for tau was not different. Aberrant phosphorylation of the tau protein is considered to play a decisive role in the pathogenesis of neurodegenerative disorders collectively called tauopathies (1). Tau is a neuronspecific, microtubule-associated protein that plays major roles in the assembly and stabilization of microtubules. In the brains of patients with Alzheimer's disease, abnormally phosphorylated tau protein, paired helical filament (PHF) 1 tau, becomes dissociated from neuronal microtubules and accumulates in PHFs, the filamentous network generated by self-aggregation of hyperphosphorylated forms of tau. All six adult isoforms of tau are known to be hyperphosphorylated in PHFs (2).Several lines of evidence suggest that proline-directed serine/threonine kinases, together with protein phosphatases, play an important role in the hyperphosphorylation of tau (3). Candidate protein kinases include mitogen-activated protein kinase, glycogen synthase kinase-3␤, and cyclin-dependent kinase-5 (CDK5). None of these enzymes can generate PHF-tau alone (4).CDK5, initially known as brain proline-directed protein kinase or neuronal cdc2-like protein kinase, phosphorylates tau at a high stoichiometry (5, 6). Unlike other tau kinases, the activity of CDK5 is regulated by endogenous regulatory proteins, p35 and p39. In this respect, CDK5 is distinctly different from other tau kinases (7).Recently, calpain, a calcium-dependent protease, was found to digest both p35 and p39 to their truncated products, p25 and p29, respectively (8, 9). Furthermore, truncation of p35 to p25 and the subsequent formation of the p25-CDK5 complex result in neuronal cell death and hyperphosphorylation of tau (10). From these findings, the following hypothesis emerged. Initially, various stress signals activate calpain by recruiting intracellular Ca 2ϩ , 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 (11).Several criticisms of this hypothesis have appeared. Among them, the observed accumulation of p25 in the brains of patients with Alzheimer's disease may be accounted for by postmortem degradation of p35 (12). In fact, p35 is notoriously unstable. Most brain-derived p35...

No difference in kinetics of tau or histone phosphorylation by CDK5/p25 versus CDK5/p35 in vitro

“…In contrast to the results of Hashiguchi et al (26), we find that the kinetic parameters of both enzymes for the phosphorylation of these substrates are the same within experimental error. In addition, we have examined the phosphorylation of individual target sites in tau by two-dimensional NMR.…”

“…To rigorously demonstrate its hyperactivity, however, it is necessary to show that the catalytic parameters associated with steady-state phosphorylation of substrates are different between the two enzymes. Hashiguchi et al (26) previously conducted such experiments, and concluded that the intrinsic activity of CDK5/p25 was indeed significantly higher than that of CDK5/p35. Their data, however, consisted of few data points and was fraught with large errors.…”

p53, Apaf-1, caspase-3, and -9 are dispensable for Cdk5 activation during cell death

“…The association between Cdk5 activation and cell death is seen in pathology: rat lungs infected with Streptococcus pneumoniae, 25 mouse limbs infected with Leishmania major, 26 preterm lamb lungs after ventilation, 27 cyclophosphamide-treated mouse embryos, 28 and several neurodisorders and neuronal injuries. [29][30][31][32][33][34][35][36][37] The regulatory signals and mechanisms by which Cdk5 is activated remain undefined.…”

Development of an Assay to Screen for Inhibitors of Tau Phosphorylation by Cdk5

“…Analysis of these data yielded a Km of 4 µM for tau and 35 µM for ATP, which are similar to the reported values. 36 The recovery of this filter assay was determined to be 36% by using 125 Iprotein. Different concentrations of tau were then incubated with the kinase, and the reaction was terminated with EDTA.…”