Kayoko Tomizawa

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.

A Brain‐Specific Protein p25 Is Localized and Associated with Oligodendrocytes, Neuropil, and Fiber‐Like Structures of the CA3 Hippocampal Region in the Rat Brain

Abstract: Developmental expression and cellular localization of a novel brain‐specific 25‐kDa protein (p25), a substrate of tau protein kinase II, were investigated in the rat brain using polyclonal antibodies raised against peptides synthesized based on the p25 amino acid sequence. By western immunoblotting, p25 was found to be expressed only slightly in the embryonic period; the expression increased from 11 days up to 5 weeks of age, and continued to increase gradually until 1–2 years of age. Immunohistochemistry revealed distinct staining of glial cells in most regions of the central nervous system in the adult rat brain. These positively immunostained cells were especially abundant in the white matter, such as the corpus callosum, cingulum, external capsule, and internal capsule. The glial cells were identified as oligodendrocytes, and the nuclei of the cells remained unstained. Whereas the neuropil in most parts of the brain was immunostained less intensely than glias, the neuropil in the first and second layers of the cerebral cortex and the dentate gyrus was relatively strongly stained. Fiber‐like structures were also stained in the CA3 region of hippocampus.

A novel brain-specific 25 kDa protein (p25) is phosphorylated by a Ser/Thr-Pro kinase (TPK II) from tau protein kinase fractions

A novel brain‐specific 25 kDa protein (p25) was purified from a bovine brain extract. The protein was phosphorylated by Ser/Thr‐Pro kinase (TPK II) in tau protein kinase fractions at the Ser residues of Ser‐Pro sequences. Using immunoblot analysis, the protein was found only in brain extracts, and was most abundant in the brain regions such as cerebrum and hippocampus, but less abundant in cerebellum, medulla oblongata and olfactory bulb. The protein was detected in rat, bovine and human brain extracts, indicating that this protein specifically exists in mammalian brain tissues.

Tau-tubulin kinase phosphorylates tau at Ser-208 and Ser-210, sites found in paired helical filament-tau

Hyperphosphorylated tau protein is known to be a major component of the paired helical filaments (PHFs) that accumulate in the brain of Alzheimers patients. The kinase that phosphorylated Ser‐208 and Ser‐210 in PHF‐tau had remained unknown. We used anti‐pS208 and anti‐pS210 antibodies and Western blots to confirm that the tau‐tubulin kinase (TTK) phosphorylates tau at Ser‐208 and at Ser‐210. Using partial amino acid sequences of purified bovine brain TTK, a mouse cDNA of TTK was isolated and the sequence was determined. Its 963 bp coding region is composed of 320 amino acids and encodes a 36 kDa protein indistinguishable in size from authentic bovine brain TTK. Our immunoblot analysis demonstrated that TTK is ubiquitously distributed in the rat tissues, and that it is developmentally regulated in the rat brain.

Distribution of tau protein kinase I/glycogen synthase kinase-3β, phosphatases 2A and 2B, and phosphorylated tau in the developing rat brain

When trying to elucidate the role played by tau protein kinase I/glycogen synthase kinase-3beta (TPKI/GSK-3beta) in tau phosphorylation, it is important to consider the balance that exists between the various kinases and phosphatases that are involved in vivo. We studied developmental changes in the expressions of TPKI/GSK-3beta and phosphatases 2A and 2B in rat brains using immunoblot analysis. The expression of the kinase peaked postnatally at days 8-11 and returned then to low level after 5 weeks. Phosphatase 2A showed a similar pattern, increasing postnatally until day 14 and decreasing thereafter. On the other hand, phosphatase 2B was undetectable at the juvenile stage, but later its presence increased rapidly to peak at 5 weeks after birth, after which it was maintained at high levels throughout the adult stage. Immunohistochemical studies using the PAP method revealed details of the distribution of TPKI/GSK-3beta. At postnatal days 3-21 both gray and white matter were immunoreactive. Later, after 5 weeks, the immunoreactivity became more restricted to the gray matter. The staining of tau phosphorylated at Ser 199, Ser 396, and Ser 413 followed mostly the pattern of the kinase distribution throughout all stages of development. These data, therefore, confirm that TPKI/GSK-3beta is expressed primarily in neurons and especially in neurites until postnatal day 21, whereafter the distribution is concentrated mostly in the cell soma and the proximal neurite region.

Involvement of τ Protein Kinase I in Paired Helical Filament‐Like Phosphorylation of the Juvenile τ in Rat Brain

Abstract: τ protein kinase I (TPKI) phosphorylates τ and forms paired helical filament epitopes in vitro. We studied temporal expression and histochemical distribution of τ phosphoserine epitopes at sites known to be phosphorylated by TPKI. Antibodies directed against phosphorylated Ser199 (anti‐PS 199) or phosphorylated Ser396 (C5 or anti‐PS 396) were used. TPKI is abundantly expressed in the young rat brain and the highly phosphorylated juvenile form of τ occurs in the same period. The activity peak of TPKI coincided with the high level of phosphorylation of Ser199 and Ser396 in juvenile τ at around postnatal day 8. By immunohistochemistry on the hippocampus and neocortex of 3–11‐day‐old rats, phosphorylated Ser396 was found in young axonal tracts and neuropil, where TPKI immunoreactivity was also detected. TPKI and phospho‐Ser199 immunoreactivities were also detected in the perikarya of pyramidal neurons. TPKI immunoreactivity had declined to a low level and phosphorylated serine immunoreactivities were undetectable in the sections of adult brain. These findings implicate TPKI in paired helical filament‐like phosphorylation of juvenile form of τ in the developing brain.

A novel tau-tubulin kinase from bovine brain

During purification of tau protein kinase I and II from the bovine brain extract, a new tau protein kinase was detected and purified with phosphocellulose, gel filtration, S‐Sepharose and AF‐Heparin column chromatography. The molecular mass of the enzyme was determined to be 32 kDa by gel filtration and activity staining on SDS‐PAGE. The enzyme is a Ser/Thr protein kinase phosphorylating tau, β‐tubulin, MAP2 and α‐casein. Employing many synthetic peptides, the recognition site of this enzyme appears to be ‐SR‐. The enzyme requires no second messenger and is inhibited with high concentration of heparin, but not by inhibitors of CKI. These results indicate that this enzyme, tau‐tubulin kinase is novel and distinct from TPKI, II and CKI, II.

Expression, purification and crystallization of a human tau-tubulin kinase 2 that phosphorylates tau protein

Tau-tubulin kinase 2 (TTBK2) is a Ser/Thr kinase that putatively phosphorylates residues Ser208 and Ser210 (numbered according to a 441-residue human tau isoform) in tau protein. Functional analyses revealed that a recombinant kinase domain (residues 1-331) of human TTBK2 expressed in insect cells with a baculovirus overexpression system retains kinase activity for tau protein. The kinase domain of TTBK2 was crystallized using the hanging-drop vapour-diffusion method. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 55.6, b = 113.7, c = 117.3 A, alpha = beta = gamma = 90.0 degrees. Diffraction data were collected to 2.9 A resolution using synchrotron radiation at BL24XU of SPring-8.

Identification and tissue-specific expression of a novel isoform of Semaphorin 3D

BACKGROUND Semaphorins are a family of secreted and membrane-associated proteins involved in axon guidance in the developing brain as well as morphogenesis in various organs. There has been no report on the expression of different transcripts of the genes encoding Class 3 Semaphorins with different protein structures. METHODS Molecular cloning of rat Semaphorin 3D gene and the expression analysis at gene and protein levels were performed. RESULTS We have isolated two cDNAs encoding rat Sema 3D, a Class 3 Semaphorin. One clone is predicted to encode a protein with a structure common to Class 3 Semaphorins. The other clone encodes a novel isoform of Sema 3D lacking half of the C2-type Ig domain and the entire basic region; this isoform is predicted to have a different structure from Class 3 Semaphorins. Analysis of protein expression using a cell culture system revealed that this splice variant isoform is not secreted into the media, whereas the classical Class 3 isoform is a secreted protein. The expression of each isoform shows tissue-specificity. GENERAL SIGNIFICANCE Our present findings suggest that gene regulation, via an alternative splicing mechanism, affects not only the tissue-specificity of Sema 3D expression, but also the distance over which it can act.