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We have found that a 15 kDa protein (p15) produced by a fungus of Helicosporium genus induced characteristic morphological changes on neurites extending from rat cerebral cortex neurons in culture. In the presence of p15, neurite elongation from cortical neurons was markedly inhibited, and they extended short, slender, and less branched neurites. Laminin-induced promotion of neurite outgrowth was also dramatically suppressed. In contrast, p15 promoted nerve growth factor (NGF)-induced neurite outgrowth from PC12 cells, although treatment of cells with p15 alone had little effect. Partial amino acid sequence analysis of p15 revealed that it had limited homology to plant lectins. These results suggest that the mechanism of neurite outgrowth was considerably different between cortical neurons and PC12 cells and that p15 modulated neurite outgrowth in cell type-specific manners.

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.

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 3 beta (GSK3 beta). Moreover, we found that TPKI activity was inseparable from GSK3 activity throughout the purification procedure. These results indicate that TPKI is identical to GSK3 beta.

To study the phosphorylation state of tau in vivo, we have prepared antisera by immunizing rabbits with synthetic phosphopeptides containing phosphoamino acids at specific sites that are potential targets for tau protein kinase II. Immunoblot experiments using these antisera demonstrated that tau in microtubule-associated proteins is phosphorylated at Ser144 and at Ser315. Almost all tau 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 tau 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 tau by tau protein kinase I, a kinase responsible for abnormal phosphorylation of tau found in paired helical filaments of patients with Alzheimer's disease, was enhanced by prior phosphorylation of tau by tau protein kinase II. Thus, we suggest that tau protein kinase II is indirectly involved, at least in part, in the regulation of the phosphorylation state of tau in neuronal cells.

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.