Tsuneko Uchida

Glycogen synthase kinase 3β is identical to tau protein kinase I generating several epitopes of paired helical filaments

We previously reported that tau protein kinase I (TPKI) induced normal tau protein into a state of paired helical filaments (PHF); this is further confirmed here by immunoblot analysis using several antibodies. We also present the amino acid sequence of TPKI, which is identical to glycogen synthase kinase 3β (GSK3β). Moreover, we found that TPKI activity was inseparable from GSK3 activity throughout the purification procedure. These results indicate that TPKI is identical to GSK3β.

A cdc2-related kinase PSSALRE/cdk5 is homologous with the 30 kDa subunit of tau protein kinase II, a proline-directed protein kinase associated with microtubule

We previously reported that tau protein kinase II (TPKII) from bovine brain was composed of 30 kDa and 23 kDa subunits. The 30 kDa subunit of TPKII can be regarded as a catalytic subunit because of its ATP‐binding activity. Antibodies directed against TPKII‐phosphorylated tau also reacted with tau phosphorylated by cdc2 kinase obtained from starfish oocytes, indicating that TPKII and cdc2 kinase phosphorylate the same sites. We determined the amino acid sequence of the 30 kDa subunit and found it to be homologous with a cdc2‐related kinase, PSSALRE/cdk5. Moreover, an antibody against PSSALRE/cdk5 reacted with the 30 kDa subunit. These results indicate that the 30 kDa subunit of TPKII is bovine homologue of PSSALRE/cdk5. Expression of the 30 kDa subunit mRNA was enhanced in juvenile rat brain. This result supports our previous hypothesis that the kinase works actively in juvenile brain.

Preferential labeling of Alzheimer neurofibrillary tangles with antisera for tau protein kinase (TPK) I/glycogen synthase kinase-3β and cyclin-dependent kinase 5, a component of TPK II

Abstract Using immunohistochemistry, we examined the localization of four types of proline-directed kinases in the brains of control rats and in the brains of non-demented aged human subjects, subjects with Alzheimer’s disease and those with Down’s syndrome. The four kinases were: cyclin-dependent kinase (cdk) 5, a component of tau protein kinase (TPK) II; TPK I/glycogen synthase kinase (GSK)-3β; GSK-3α; and mitogen-activated protein kinase (MAPK/ERK2). Each of these kinases has been reported to promote the hyperphosphorylation of tau protein in vitro. The kinases were located essentially in neurons, although the intensity and distribution of labeling varied. Antiserum for cdk5 showed the most preferential and consistent labeling of intraneuronal neurofibrillary tangles (NFT). Antiserum for TPK I/GSK-3β also labeled intraneuronal NFT. Double immunolabeling for TPK I/GSK-3β and tau1 showed that TPK I/GSK-3β was closely associated with NFT. Antiserum for GSK-3α labeled neurons weakly, and the intensity of labeling did not differ between neurons with and without NFT. Antiserum for MAPK labeled neurons in superficial cortical layers, but NFT appeared in both superficial and deep cortical layers. These findings suggest that cdk5 and TPK I/GSK-3β are the critically important kinases for the generation in vivo of hyperphosphorylated tau, the main component of the paired helical filaments in NFT.

Phosphorylation sites on tau by tau protein kinase I, a bovine derived kinase generating an epitope of paired helical filaments

Tau protein kinase I (TPKI) isolated from bovine brain has been determined to phosphorylate tau at four distinct sites by detecting modified Ser and Thr residues with protein sequencer. Ser199, Thr231, Ser396 and Ser413 were all found to have been phosphorylated by TPKI (numbering of amino acids was done in relation to the longest human tau [Neuron, 3 (1989) 519-526]). These phosphorylations generate an epitope of PHF (paired helical filaments) and eliminate the recognition of tau by the monoclonal antibody, tau-1. These results suggested that TPKI might be responsible for at least some of the phosphorylation of tau to induce PHF formation.

Identification of the 23 kDa subunit of tau protein kinase II as a putative activator of cdk5 in bovine brain

Tau protein kinase II (TPKII) was reported previously to be composed of a neuron‐rich cdc2‐related kinase (PSSALRE/cdk5) and 23 kDa subunit. Here we show that the 23 kDa subunit is a putative activator for the kinase activity. Amino acid sequence analysis revealed that the protein was novel and included a partial similarity of amino acids to a cyclin box important for the interaction with cdc2‐related kinase. These results suggest that the 23 kDa subunit, but not cyclin, activates cdk5 in neuronal cells, which no longer exhibit cell cycling but are terminally differentiated cells.

Localization and Developmental Changes of τ Protein Kinase I/Glycogen Synthase Kinase‐3β in Rat Brain

Abstract: τ protein kinase I (TPKI) purified from bovine brain extract has been shown to phosphorylate τ and to form paired helical filament (PHF) epitopes and was found recently to be identical to glycogen synthase kinase‐3β (GSK‐3β). Before elucidating a role of TPKI/GSK‐3β in PHF formation, it is necessary to investigate the normal function of the enzyme. To study the distribution and developmental changes of the enzyme, specific polyclonal antibodies were prepared against TPKI and GSK‐3α. Immunoblot analysis demonstrated that TPKI was nearly specifically localized in the brain of adult rats. The level of TPKI in the rat brain was high at gestational day 18, peaked on postnatal day 8, and then decreased rapidly to a low level, which was sustained up to 2 years. Immunohistochemistry indicated primarily neuronal localization of TPKI. Growing axons were stained most intensely in the developing cerebellum, but the immunoreactivity became restricted to the gray matter in the mature tissue. Parallel fibers had a high level of TPKI and also stained intensely for τ. These findings indicate that τ is one of the physiological substrates of TPKI and suggest that the enzyme plays an important role in the growth of axons during development of the brain.

A serine/threonine proline kinase activity is included in the tau protein kinase fraction forming a paired helical filament epitope

Previously we partially purified a novel protein kinase which phosphorylated tau and formed a paired helical filament (PHF) epitope. In this paper we show that the kinase fraction contains a protein kinase activity recognizing serine/threonine proline sequence. The kinase phosphorylated tau at the tau-1 site previously reported as one of the phosphorylation sites on PHF by other groups. The kinase also phosphorylated extraordinarily insoluble portion located on C-terminal region of tau in PHF. It is worth considering that tau phosphorylated by this kinase activity is incorporated into PHF.

Analysis of phosphorylation of tau with antibodies specific for phosphorylation sites.

Previously, we determined sites of tau protein phosphorylation by tau protein kinase (TPK) I/glycogen synthase kinase 3 beta (GSK-3 beta) and TPKII/(cyclin-dependent kinase 5 (CDK5) + p23). We prepared antibodies specific for these sites of tau phosphorylated by TPKI and TPKII, using chemically synthesized phosphopeptides as antigens. Each antibody specifically reacts with each phosphorylation site. With these antibodies, it was confirmed that TPKI and TPKII are responsible for these phosphorylation sites, as reported previously, except that Ser404 is also weakly phosphorylated by TPKI alone. It was also observed that TPKII-phosphorylation enhances TPKI-phosphorylation. These results indicate that these antibodies are useful tools for investigation of the phosphorylation of tau by TPKI and TPKII.

τ Protein Kinase II Is Involved in the Regulation of the Normal Phosphorylation State of τ Protein

Abstract: To study the phosphorylation state of τ in vivo, we have prepared antisera by immunizing rabbits with synthetic phosphopeptides containing phosphoamino acids at specific sites that are potential targets for τ protein kinase II. Immunoblot experiments using these antisera demonstrated that τ in microtubule‐associated proteins is phosphorylated at Ser144 and at Ser315. Almost all τ variants separated on two‐dimensional gel electrophoresis were phosphorylated at Ser144 and nearly one‐half of them at Ser315. Phosphorylation at Ser144 and at Thr147 of τ isolated from heat‐stable brain extracts was shown to be developmentally regulated, with the highest level of phosphorylation found at postnatal week 1. In vitro phosphorylation of τ by τ protein kinase I, a kinase responsible for abnormal phosphorylation of τ found in paired helical filaments of patients with Alzheimers disease, was enhanced by prior phosphorylation of τ by τ protein kinase II. Thus, we suggest that τ protein kinase II is indirectly involved, at least in part, in the regulation of the phosphorylation state of τ in neuronal cells.

Precursor of cdk5 activator, the 23 kDa subunit of tau protein kinase II: Its sequence and developmental change in brain

Tau protein kinase II (TPKII) is shown by immunoprecipitation to be a complex composed of two subunits, a catalytic subunit, cdk5, and regulatory subunit, p23. By sequence analysis of p23 CDNA, p23 was found to occupy a region from the 99th amino acid residue to the C‐terminus of a novel protein with a molecular weight of 34,000 Da, suggesting that this 34 kDa protein is a precursor of p23 (pre‐p23). These findings suggest that p23 results from the processing of the precursor protein, pre‐p23. The precursor mRNA was expressed most abundantly in rat brain just before and after birth. Expression of pre‐p23, but not of cdk5, mRNA changed, coinciding with the developmental change of TPKII activity, suggesting that its expression controls the phosphorylation of tau by the TPKII/TPKI system in the neonatal brain. p23 appears to be a cdk5 activator in neuronal cells.