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Okadaic acid enhances abnormal phosphorylation on tau proteins
Neuroscience Letters, 156 (1993) 67-69 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93l$ 06.00
67
NSL 09598
Okadaic acid enhances abnormal phosphorylation on tau proteins Y o s h i k o F u r i y a a, N a r u h i k o S a h a r a ~ a n d H i r o s h i M o r i ~'b aDepartment of Molecular Biology, Tokyo Institute of Psychiatry, Tokyo (Japan) and bDepartment of Neuropathology, Institute for Brain Research, University of Tokyo School of Medicine, Tokyo (Japan) (Received 1 February 1993; Revised version received 17 March 1993; Accepted 17 March 1993) Key words: Microtubule; Tau; Abnormal phosphorylation; Okadaic acid; Alzheimer's disease; Paired helical filament Tau proteins are one of the microtubule-associatedproteins (MAPs) and show promoting activity on microtubule assembly.Tau proves to be the major constituent of Alzheimer's paired helical filaments, in which tau is found to be different from normal tau in that it is abnormally phosphorylated. To examine the effect of the abnormal phosphorylation on microtubule assembly,we obtained abnormally phosphorylated tau that was made in vitro by hyperphosphorylation with ATP or with ATP and okadaic acid, a drug inhibiting phosphatase, mainly 1 and 2A. We confirmed the biochemical properties of abnormally phosphorylated tau based on its retarded gel mobility and immunoreactivityto anti-PHF. We found that abnormally phosphorylated tau was able to promote the polymerization of microtubules but showed less activity as compared with normally phosphorylated tau. This effect of ATP on abnormal phosphorylation of tau was enhanced when okadaic acid was added in the phosphorylation reaction mixture during microtubule assembly. It is of significance that phosphatase activity as well as kinase activity are involved in the formation of abnormal tau. The present evidence suggests the simultaneous occurrence of microtubule disassembly and the pathogenesis of paired helical filaments following the abnormal phosphorylation of tau.
Microtubules are one of the main cytoskeletons in all eukaryotic cells and found to be abundant in neuronal tissues. It has been established that there are several kinds of proteins (referred to as microtubule-associated proteins (MAPs)) which are co-purified with tubulin in microtubules during assembly and disassembly [6, 16, 18]. MAPs could regulate the cellular function through the dynamics of microtubule architecture. Tau proteins proved to be one of the microtubule-binding proteins that are different in molecular size from other high molecular-sized MAPs [20]. Tau has become more attractive after the finding that it is a component of Alzheimer's paired helical filaments (PHFs) [1,4, 11, 12, 17, 21, 22]. Tau in Alzheimer's P H F s suffers from abnormal phosphorylation [3, 7, 22] during the progression of the disease. Abnormally phosphorylated tau was released from microtubules and deposited intracellularly as the major component of neurofibrillary tangles. The switching mechanism of tau is interesting; what kind of trigger signal introduces it to be sorted from microtubules to neurofibrillary tangles. Tau occurs as a phosphoprotein in normal brains and can be
Correspondence: H. Mori, Department of Molecular Biology, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagayaku, Tokyo 156, Japan. Fax: (81) (3) 3329-8035.
aberrantly phosphorylated in vitro during polymerization by microtubule-associated kinases [10]. Recently, phosphorylation of MAP- 1, MAP-2 and tau was found to deteriorate the assembly of microtubules [5, 9, 15, 18]. However, there are not enough data to understand the mechanism of abnormal phosphorylation of MAPs and, in particular, tau. We have examined the promotion activities of normal and abnormal tau on microtubule assembly. We have succeeded in making an abnormally phosphorylated tau with ATP and okadaic acid, a drug inhibiting phosphatases, mainly 1 and 2A, by shifting an equilibrium of phosphorylation reaction to hyperphosphorylation. The abnormal tau showed retarded mobility on gel electrophoresis and became reactive to anti-PHF antibody. We discuss the significance of abnormal phosphorylation on tau in terms of microtubule assembly and Alzheimer's pathogenesis. Porcine microtubules were prepared according to the method by Weingarten et al. [20]. Tau was prepared from two cycle-assembled microtubules. Microtubules were dissolved in 0.1 M glycine-HCl, p H 2.5, 0.75 M NaC1 and heated in a boiling bath for 10 min. Tau was obtained from the resulting supernatant. Abnormally phosphorylated tau was obtained by adding of 1 0 / I M ATP with or without okadaic acid (0.1/IM) in addition to 1 0 / I M G T P during the polymerization reaction. Tubulin was purified from pig brains according to the
68 method by Weingarten et al. [20]. Briefly, microtubules were obtained in a temperature-dependently assembled manner and purified by phosphocellulose chromatography. Tubulin and tau were estimated by measuring their absorbancy at 229 nm and 280 nm and by the BCA protein assay kit (Pierce Co.). We studied the biochemical behavior of normal and abnormally phosphorylated tau for their mobilities in gel electrophoresis and their immunoreactivities with antihuman tau antibody (Fig. la-c) and abnormal phosphorylation-specific anti-PHF antibody (Fig. l d-f). As expected, abnormally phosphorylated tau showed retarded mobility and PHF-related immunoreactivity with antiPHF antibody. We examined the biological activities of tau with or without abnormal phosphorylation. Fig. 2 shows the typical result of abnormal phosphorylation on tau. As repeatedly documented [13, 16, 20], normal tau was confirmed to promote the polymerization reaction of microtubules. As normal tau was in situ phosphorylated, dephosphorylated tau which was treated with alkaline phosphatase showed markedly increased activity as compared with normal tau (Fig. 2). This effect of phospho-
/ 0.08dephosphorylated
0.07 0.06 ~
F: C 0 "1-',q.
0
0.05 normal 0.040.030.020.01 0.00
o 200
S
10 Time(min)
ls
Fig. 2. Effect of normal tau and abnormally phosphorylated tau on microtubule assembly. Tau proteins and tubulin were prepared as described in the text. Microtubule polymerization reaction was monitored by the measurement of the absorbance at 410 nm. Polymerization reaction was performed in buffer A solution (100 mM Mes, pH 6.5, 1 mM EGTA, 1 mM MgC12, 15/IM GTE 10 j~g/ml leupeptine and 0.1/~g/ml pepstatine A). Tau (18/~g) was examined for their promoting activities of microtubule assembly; tubulin (59/.lg) with dephosphorylated tau, normally phosphorylated tau, abnormally phosphorylated tau with ATP (ATP-tau) and with ATP and okadaic acid (ATP/OA-tau). The inset shows Coomassiebrilliant blue R250 dye staining of electrophoretic profiles of dephospborylated, normally phosphorylated, abnormally phosphorylated (ATP and ATP/OA) tau (2.25/~g).
97.4 .... 68--
43-29 a
b
c
d
•
f
Fig. 1. Western blot analyses of normal tau and abnormally phosphorylated tau. Tau proteins were prepared from porcine brains as described in the text. SDS-polyacrylamide gel electrophoresis was performed and electrotransferred onto PVDF membrane (Millipore Co.). The membrane was treated with 3% gelatin in 500 mM NaCI, 20 mM Tris-HCI, pH 7.6, followed by anti-human tau antibody (pool 1) at a dilution of 1 : 10,000 (a-c) or affinity purified anti-PHF antibody at a dilution of 1 : 500 (d-f) [7]. The membrane was incubated with a secondary biotinyl anti-rabbit IgG antibody, avidin-biotin complex and developed with 4-chloro-l-naphthol. a,d: normal tau. b,e: ATP-mediated abnormally phosphorylated tau (ATP-tau). c,f: ATP and okadaic acid-mediated abnormally phosphorylated tau (ATP/OA-tau).
rylation was much enhanced in abnormally phosphorylated tau prepared in the presence of ATE We observed the same effect of okadaic acid alone instead of exogeneous ATP on tau phosphorylation (Furiya and Mori, unpublished). Such a deteriorative effect of abnormal phosphorylation was found to be most prominent on tau prepared with ATP and okadaic acid. We examined five independent prepared samples for porcine microtubules and two independent prepared samples for rat microtubules and obtained very similar results among them. The present finding was highly constant and indicated that tau could dually regulate microtubule polym-
69 e r i z a t i o n in a p h o s p h o r y l a t i o n - d e p e n d e n t m a n n e r as well as in a c a l c i u m - d e p e n d e n t m a n n e r [18]. Thus, p h o s p h o r y l a t i o n p l a y e d a key role in the d y n a m i c m e t a b o l i s m o f c y t o s k e l e t a l architecture as I n a g a k i et al. [8] first c l a i m e d for i n t e r m e d i a t e filaments. I f the d e c o n s t r u c t i o n o f the m i c r o t u b u l e n e t w o r k o c c u r r e d s i m u l t a n e o u s l y with o r p r i o r to the f o r m a t i o n o f A68 following release o f t a u f r o m microtubules, the present o k a d a i c acid-affected phosp h o r y l a t i o n m a y have a key function. K i n a s e activities u s u a l l y c o m p e t e with p h o s p h a t a s e activities resulting in a b a l a n c e o f p h o s p h o r y l a t i o n reactions in n o r m a l brains. It is, therefore, likely t h a t a b n o r m a l l y p h o s p h o r y l a t e d tau p o t e n t l y occurs as the m i n o r p o p u l a t i o n while n o r m a l l y p h o s p h o r y l a t e d t a u occurs as the m a j o r p o p u l a t i o n in n o r m a l brains. T h o u g h a b n o r m a l t a u is h a r d to detect in n o r m a l b r a i n s because o f its small a m o u n t , we c o u l d detect it in h o m o g e n a t e s o f norm a l b r a i n s when the s a m p l e was heavily l o a d e d for western b l o t t i n g ( E n d o h a n d M o r i , u n p u b l i s h e d ) . Hence, we think t h a t the p h o s p h o r y l a t i o n b a l a n c e m a y shift to hyp e r p h o s p h o r y l a t i o n in A D b r a i n s as d o c u m e n t e d by Sait o h et al. [19]. Since o k a d a i c acid c o u l d shift the p h o s p h o r y l a t i o n b a l a n c e to the h y p e r p h o s p h o r y l a t e d state in vitro, as p r e s e n t e d here, we w o u l d i m a g i n e t h a t o k a d a i c acid is a useful d r u g to m i m i c A D b r a i n c o n d i t i o n s a n d is a p p l i c a b l e in c u l t u r e d cells. P r e v i o u s r e p o r t s on the a b n o r m a l p h o s p h o r y l a t i o n o f t a u were focused o n the identification o f kinases a n d little a t t e n t i o n was p a i d to the possible i n v o l v e m e n t o f a p h o s p h a t a s e ( s ) on the pathogenesis o f n e u r o f i b r i l l a r y tangles in A l z h e i m e r ' s disease. We c a n n o t specify the m e c h a n i s m b y which such a shift was p r o d u c e d in p a t h o l o g i c a l brains. It m a y origin a t e f r o m the increased expression o f kinase activities like M A P kinase [2, 5], T P K I/II [10], M A P 2 kinase or cdc2 kinase [14] in A D brains. Alternatively, one m a y speculate on the possibility o f decreased expression o f p h o s p h a t a s e s o r on an increased a m o u n t o f p h o s p h a t a s e i n h i b i t o r s in A l z h e i m e r ' s disease brains. We t h a n k Y a s u o I h a r a for his k i n d gift o f a n t i - P H F a n t i b o d y a n d R i u k o E n d o h for her helpful technical assistance. 1 Brion, J.P., van den Bosch de Aguilar, P. and Flament-Durand, J., In: Traber and Gispen (Eds.), Advances in Applied Neurological Science: Senile Dementia of the Alzheimer Type, Springer, Berlin, 1985, pp. 164--174. 2 Drewes, G., Lichtenberg-Kraag, B., Doring, F., Mandelkow, E.-M., Biernat, J., Goris, J., Doree, M. and Mandelkow, E., Mitogen activated protein (MAP) kinase transforms tan protein into an Alzheimer-like state, EMBO J., 11 (1992) 2131-2138. 3 Grundke-Iqbal, I., Iqbal, K., Quinlan, M., Tung, Y.-C., Zaidi, M.S. and Wisniewski, H.M., Microtubule-associated protein tan, J. Biol. Chem., 261 (1986) 6084-6089. 4 Grundke-lqbal, I., lqbal, K., Tung, Y.-C., Quinlan, M.,
Wisniewski, H.M. and Binder, L.I., Abnormal phosphorylation of the microtubule-associated protein r (tau) in Alzheimer cytoskeletal pathology, Proc. Natl. Acad. Sci. USA, 83 (1986)4913-4917. Gustke, N., Steiner, B., Mandelkow, E.M., Meyer, H.E., Goedert, M. and Mandelkow, E., The Alzheimer-like phosphorylation of tau protein reduces microtubule binding and involves Ser-Pro and ThrPro motifs, FEBS Lett., 307 (1992), 199-205. Hirokawa, N., Quick-freeze, deep-etch visualization of the axonal cytoskeleton, Trends Neurosci., 9 (1986) 67-70. Ihara, Y., Nukina, N., Miura, R, and Ogawara, M,, Phosphorylated tau protein is integrated into paired helical filaments in Alzheimer's disease, J. Biochem., 99 (1986) 1807-1810. Inagaki, M., Nishi, Y., Nishizawa, K., Matsuyama, M. and Sato, C., Site-specific phosphorylation induces disassembly of vimentin filaments in vitro, Nature, 328 (1987) 649-652. Ishiguro, K., Omori, A., Takematsu, M., Sato, K., Arioka, M,, Uchida, T. and Imahori, K., Phosphorylation sites on tau by tan kinase I, a bovine derived kinase generating an epitope of paired helical filaments, Neurosci. Lett., 148 (1992) 202-206. 10 Ishiguro, K., Takamatsu, M., Tomizawa, K., Omori, A., Takahashi, M., Arioka, M., Uchida, T. and Imahori, K., Tau protein kinase I converts normal tau protein into A68-1ike component of paired helical filaments, J. Biol. Chem., 267 (1992) 10897-10901. 11 Kondo, J., Honda, T., Mori, H., Hamada, Y., Miura, R., Ogawara, M. and Ihara, Y., The carboxyl third of tau is tightly bound to paired helical filaments, Neuron, 1 (1988) 827-834. 12 Kosik, K.S., Joachim, C.L. and Selkoe, D.J., Microtubule-associated protein tau (r) is a major antigenic component of paired helical filaments in Alzheimer's disease, Proc. Natl. Acad. Sci. USA, 83 (1986) 4044 4948. 13 Raffaelli, N., Yamauchi, P.S. and Purich, D.U, Microtubule-associated protein autophosphorylation alters in vitro microtubule dynamic instability, FEBS Lett., 296 (1992) 21-24. 14 Ledesma, M.D., Correas, I., Avila, J. and Diaz-Nido, J., Implication of brain cdc2 and MAP2 kinases in the phosphorylation of tau protein in Alzheimer's disease, FEBS Lett., 308 (1992) 218-224. 15 Lindwall, G. and Cole, R.D., Phosphorylation affects the ability of tau protein to promote microtubule assembly, J. Biol. Chem., 259 0984) 5301 5305. 16 Murphy, D.B. and Borisy, G.G., Association of high-molecularweight proteins with microtubules and their role in microtubule assembly in vitro, Proc. Natl. Acad. Sci. USA, 72 (1975) 2696--2700. 17 Nukina, N. and Ihara, Y., One of the antigenic determinants of paired helical filaments is related to tan protein, J. Biochem., 99 (1986) 1541 1544. 18 Olmsted, J.B., Microtubule-associated proteins, Annu. Rev. Cell Biol., 2 (1986) 421-457. 19 Saitoh, T. and Dobkins, K.R., Increased in vitro phosphorylation of a Mr 60,000 protein in brain from patients with Alzheimer disease, Proc. Natl. Acad. Sci. USA, 83 (1986) 9764-9767. 20 Weingarten, M.D., Lockwood, A.H., Hwo, S.Y. and Kirschner, M.W., A protein factor essential for microtubule assembly, Proc. Natl. Acad. Sci. USA, 72 (1975) 1858-1862. 21 Wischik, C.M., Novak, M., Thogersen, H.C., Edwards, RC., Runswick, M.J., Jakes, R., Walker, J.E., Milstein, C., Roth, M. and Klug, A., Isolation of a fragment of tau derived from the core of the paired helical filamentr of Alzheimer disease, Proc. Natl. Acad. Sci. USA, 85 (1988) 4506-4510. 22 Wood, J.G., Mirra, S.S., Pollock, N.J. and Binder, L.I., Neurofibrillary tangles of Alzheimer disease share antigenic determinants with the axonal microtubule-associated protein tau (r), Proc. Natl. Acad. Sci. USA, 83 (1986) 4040-4043.
67
NSL 09598
Okadaic acid enhances abnormal phosphorylation on tau proteins Y o s h i k o F u r i y a a, N a r u h i k o S a h a r a ~ a n d H i r o s h i M o r i ~'b aDepartment of Molecular Biology, Tokyo Institute of Psychiatry, Tokyo (Japan) and bDepartment of Neuropathology, Institute for Brain Research, University of Tokyo School of Medicine, Tokyo (Japan) (Received 1 February 1993; Revised version received 17 March 1993; Accepted 17 March 1993) Key words: Microtubule; Tau; Abnormal phosphorylation; Okadaic acid; Alzheimer's disease; Paired helical filament Tau proteins are one of the microtubule-associatedproteins (MAPs) and show promoting activity on microtubule assembly.Tau proves to be the major constituent of Alzheimer's paired helical filaments, in which tau is found to be different from normal tau in that it is abnormally phosphorylated. To examine the effect of the abnormal phosphorylation on microtubule assembly,we obtained abnormally phosphorylated tau that was made in vitro by hyperphosphorylation with ATP or with ATP and okadaic acid, a drug inhibiting phosphatase, mainly 1 and 2A. We confirmed the biochemical properties of abnormally phosphorylated tau based on its retarded gel mobility and immunoreactivityto anti-PHF. We found that abnormally phosphorylated tau was able to promote the polymerization of microtubules but showed less activity as compared with normally phosphorylated tau. This effect of ATP on abnormal phosphorylation of tau was enhanced when okadaic acid was added in the phosphorylation reaction mixture during microtubule assembly. It is of significance that phosphatase activity as well as kinase activity are involved in the formation of abnormal tau. The present evidence suggests the simultaneous occurrence of microtubule disassembly and the pathogenesis of paired helical filaments following the abnormal phosphorylation of tau.
Microtubules are one of the main cytoskeletons in all eukaryotic cells and found to be abundant in neuronal tissues. It has been established that there are several kinds of proteins (referred to as microtubule-associated proteins (MAPs)) which are co-purified with tubulin in microtubules during assembly and disassembly [6, 16, 18]. MAPs could regulate the cellular function through the dynamics of microtubule architecture. Tau proteins proved to be one of the microtubule-binding proteins that are different in molecular size from other high molecular-sized MAPs [20]. Tau has become more attractive after the finding that it is a component of Alzheimer's paired helical filaments (PHFs) [1,4, 11, 12, 17, 21, 22]. Tau in Alzheimer's P H F s suffers from abnormal phosphorylation [3, 7, 22] during the progression of the disease. Abnormally phosphorylated tau was released from microtubules and deposited intracellularly as the major component of neurofibrillary tangles. The switching mechanism of tau is interesting; what kind of trigger signal introduces it to be sorted from microtubules to neurofibrillary tangles. Tau occurs as a phosphoprotein in normal brains and can be
Correspondence: H. Mori, Department of Molecular Biology, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagayaku, Tokyo 156, Japan. Fax: (81) (3) 3329-8035.
aberrantly phosphorylated in vitro during polymerization by microtubule-associated kinases [10]. Recently, phosphorylation of MAP- 1, MAP-2 and tau was found to deteriorate the assembly of microtubules [5, 9, 15, 18]. However, there are not enough data to understand the mechanism of abnormal phosphorylation of MAPs and, in particular, tau. We have examined the promotion activities of normal and abnormal tau on microtubule assembly. We have succeeded in making an abnormally phosphorylated tau with ATP and okadaic acid, a drug inhibiting phosphatases, mainly 1 and 2A, by shifting an equilibrium of phosphorylation reaction to hyperphosphorylation. The abnormal tau showed retarded mobility on gel electrophoresis and became reactive to anti-PHF antibody. We discuss the significance of abnormal phosphorylation on tau in terms of microtubule assembly and Alzheimer's pathogenesis. Porcine microtubules were prepared according to the method by Weingarten et al. [20]. Tau was prepared from two cycle-assembled microtubules. Microtubules were dissolved in 0.1 M glycine-HCl, p H 2.5, 0.75 M NaC1 and heated in a boiling bath for 10 min. Tau was obtained from the resulting supernatant. Abnormally phosphorylated tau was obtained by adding of 1 0 / I M ATP with or without okadaic acid (0.1/IM) in addition to 1 0 / I M G T P during the polymerization reaction. Tubulin was purified from pig brains according to the
68 method by Weingarten et al. [20]. Briefly, microtubules were obtained in a temperature-dependently assembled manner and purified by phosphocellulose chromatography. Tubulin and tau were estimated by measuring their absorbancy at 229 nm and 280 nm and by the BCA protein assay kit (Pierce Co.). We studied the biochemical behavior of normal and abnormally phosphorylated tau for their mobilities in gel electrophoresis and their immunoreactivities with antihuman tau antibody (Fig. la-c) and abnormal phosphorylation-specific anti-PHF antibody (Fig. l d-f). As expected, abnormally phosphorylated tau showed retarded mobility and PHF-related immunoreactivity with antiPHF antibody. We examined the biological activities of tau with or without abnormal phosphorylation. Fig. 2 shows the typical result of abnormal phosphorylation on tau. As repeatedly documented [13, 16, 20], normal tau was confirmed to promote the polymerization reaction of microtubules. As normal tau was in situ phosphorylated, dephosphorylated tau which was treated with alkaline phosphatase showed markedly increased activity as compared with normal tau (Fig. 2). This effect of phospho-
/ 0.08dephosphorylated
0.07 0.06 ~
F: C 0 "1-',q.
0
0.05 normal 0.040.030.020.01 0.00
o 200
S
10 Time(min)
ls
Fig. 2. Effect of normal tau and abnormally phosphorylated tau on microtubule assembly. Tau proteins and tubulin were prepared as described in the text. Microtubule polymerization reaction was monitored by the measurement of the absorbance at 410 nm. Polymerization reaction was performed in buffer A solution (100 mM Mes, pH 6.5, 1 mM EGTA, 1 mM MgC12, 15/IM GTE 10 j~g/ml leupeptine and 0.1/~g/ml pepstatine A). Tau (18/~g) was examined for their promoting activities of microtubule assembly; tubulin (59/.lg) with dephosphorylated tau, normally phosphorylated tau, abnormally phosphorylated tau with ATP (ATP-tau) and with ATP and okadaic acid (ATP/OA-tau). The inset shows Coomassiebrilliant blue R250 dye staining of electrophoretic profiles of dephospborylated, normally phosphorylated, abnormally phosphorylated (ATP and ATP/OA) tau (2.25/~g).
97.4 .... 68--
43-29 a
b
c
d
•
f
Fig. 1. Western blot analyses of normal tau and abnormally phosphorylated tau. Tau proteins were prepared from porcine brains as described in the text. SDS-polyacrylamide gel electrophoresis was performed and electrotransferred onto PVDF membrane (Millipore Co.). The membrane was treated with 3% gelatin in 500 mM NaCI, 20 mM Tris-HCI, pH 7.6, followed by anti-human tau antibody (pool 1) at a dilution of 1 : 10,000 (a-c) or affinity purified anti-PHF antibody at a dilution of 1 : 500 (d-f) [7]. The membrane was incubated with a secondary biotinyl anti-rabbit IgG antibody, avidin-biotin complex and developed with 4-chloro-l-naphthol. a,d: normal tau. b,e: ATP-mediated abnormally phosphorylated tau (ATP-tau). c,f: ATP and okadaic acid-mediated abnormally phosphorylated tau (ATP/OA-tau).
rylation was much enhanced in abnormally phosphorylated tau prepared in the presence of ATE We observed the same effect of okadaic acid alone instead of exogeneous ATP on tau phosphorylation (Furiya and Mori, unpublished). Such a deteriorative effect of abnormal phosphorylation was found to be most prominent on tau prepared with ATP and okadaic acid. We examined five independent prepared samples for porcine microtubules and two independent prepared samples for rat microtubules and obtained very similar results among them. The present finding was highly constant and indicated that tau could dually regulate microtubule polym-
69 e r i z a t i o n in a p h o s p h o r y l a t i o n - d e p e n d e n t m a n n e r as well as in a c a l c i u m - d e p e n d e n t m a n n e r [18]. Thus, p h o s p h o r y l a t i o n p l a y e d a key role in the d y n a m i c m e t a b o l i s m o f c y t o s k e l e t a l architecture as I n a g a k i et al. [8] first c l a i m e d for i n t e r m e d i a t e filaments. I f the d e c o n s t r u c t i o n o f the m i c r o t u b u l e n e t w o r k o c c u r r e d s i m u l t a n e o u s l y with o r p r i o r to the f o r m a t i o n o f A68 following release o f t a u f r o m microtubules, the present o k a d a i c acid-affected phosp h o r y l a t i o n m a y have a key function. K i n a s e activities u s u a l l y c o m p e t e with p h o s p h a t a s e activities resulting in a b a l a n c e o f p h o s p h o r y l a t i o n reactions in n o r m a l brains. It is, therefore, likely t h a t a b n o r m a l l y p h o s p h o r y l a t e d tau p o t e n t l y occurs as the m i n o r p o p u l a t i o n while n o r m a l l y p h o s p h o r y l a t e d t a u occurs as the m a j o r p o p u l a t i o n in n o r m a l brains. T h o u g h a b n o r m a l t a u is h a r d to detect in n o r m a l b r a i n s because o f its small a m o u n t , we c o u l d detect it in h o m o g e n a t e s o f norm a l b r a i n s when the s a m p l e was heavily l o a d e d for western b l o t t i n g ( E n d o h a n d M o r i , u n p u b l i s h e d ) . Hence, we think t h a t the p h o s p h o r y l a t i o n b a l a n c e m a y shift to hyp e r p h o s p h o r y l a t i o n in A D b r a i n s as d o c u m e n t e d by Sait o h et al. [19]. Since o k a d a i c acid c o u l d shift the p h o s p h o r y l a t i o n b a l a n c e to the h y p e r p h o s p h o r y l a t e d state in vitro, as p r e s e n t e d here, we w o u l d i m a g i n e t h a t o k a d a i c acid is a useful d r u g to m i m i c A D b r a i n c o n d i t i o n s a n d is a p p l i c a b l e in c u l t u r e d cells. P r e v i o u s r e p o r t s on the a b n o r m a l p h o s p h o r y l a t i o n o f t a u were focused o n the identification o f kinases a n d little a t t e n t i o n was p a i d to the possible i n v o l v e m e n t o f a p h o s p h a t a s e ( s ) on the pathogenesis o f n e u r o f i b r i l l a r y tangles in A l z h e i m e r ' s disease. We c a n n o t specify the m e c h a n i s m b y which such a shift was p r o d u c e d in p a t h o l o g i c a l brains. It m a y origin a t e f r o m the increased expression o f kinase activities like M A P kinase [2, 5], T P K I/II [10], M A P 2 kinase or cdc2 kinase [14] in A D brains. Alternatively, one m a y speculate on the possibility o f decreased expression o f p h o s p h a t a s e s o r on an increased a m o u n t o f p h o s p h a t a s e i n h i b i t o r s in A l z h e i m e r ' s disease brains. We t h a n k Y a s u o I h a r a for his k i n d gift o f a n t i - P H F a n t i b o d y a n d R i u k o E n d o h for her helpful technical assistance. 1 Brion, J.P., van den Bosch de Aguilar, P. and Flament-Durand, J., In: Traber and Gispen (Eds.), Advances in Applied Neurological Science: Senile Dementia of the Alzheimer Type, Springer, Berlin, 1985, pp. 164--174. 2 Drewes, G., Lichtenberg-Kraag, B., Doring, F., Mandelkow, E.-M., Biernat, J., Goris, J., Doree, M. and Mandelkow, E., Mitogen activated protein (MAP) kinase transforms tan protein into an Alzheimer-like state, EMBO J., 11 (1992) 2131-2138. 3 Grundke-Iqbal, I., Iqbal, K., Quinlan, M., Tung, Y.-C., Zaidi, M.S. and Wisniewski, H.M., Microtubule-associated protein tan, J. Biol. Chem., 261 (1986) 6084-6089. 4 Grundke-lqbal, I., lqbal, K., Tung, Y.-C., Quinlan, M.,
Wisniewski, H.M. and Binder, L.I., Abnormal phosphorylation of the microtubule-associated protein r (tau) in Alzheimer cytoskeletal pathology, Proc. Natl. Acad. Sci. USA, 83 (1986)4913-4917. Gustke, N., Steiner, B., Mandelkow, E.M., Meyer, H.E., Goedert, M. and Mandelkow, E., The Alzheimer-like phosphorylation of tau protein reduces microtubule binding and involves Ser-Pro and ThrPro motifs, FEBS Lett., 307 (1992), 199-205. Hirokawa, N., Quick-freeze, deep-etch visualization of the axonal cytoskeleton, Trends Neurosci., 9 (1986) 67-70. Ihara, Y., Nukina, N., Miura, R, and Ogawara, M,, Phosphorylated tau protein is integrated into paired helical filaments in Alzheimer's disease, J. Biochem., 99 (1986) 1807-1810. Inagaki, M., Nishi, Y., Nishizawa, K., Matsuyama, M. and Sato, C., Site-specific phosphorylation induces disassembly of vimentin filaments in vitro, Nature, 328 (1987) 649-652. Ishiguro, K., Omori, A., Takematsu, M., Sato, K., Arioka, M,, Uchida, T. and Imahori, K., Phosphorylation sites on tau by tan kinase I, a bovine derived kinase generating an epitope of paired helical filaments, Neurosci. Lett., 148 (1992) 202-206. 10 Ishiguro, K., Takamatsu, M., Tomizawa, K., Omori, A., Takahashi, M., Arioka, M., Uchida, T. and Imahori, K., Tau protein kinase I converts normal tau protein into A68-1ike component of paired helical filaments, J. Biol. Chem., 267 (1992) 10897-10901. 11 Kondo, J., Honda, T., Mori, H., Hamada, Y., Miura, R., Ogawara, M. and Ihara, Y., The carboxyl third of tau is tightly bound to paired helical filaments, Neuron, 1 (1988) 827-834. 12 Kosik, K.S., Joachim, C.L. and Selkoe, D.J., Microtubule-associated protein tau (r) is a major antigenic component of paired helical filaments in Alzheimer's disease, Proc. Natl. Acad. Sci. USA, 83 (1986) 4044 4948. 13 Raffaelli, N., Yamauchi, P.S. and Purich, D.U, Microtubule-associated protein autophosphorylation alters in vitro microtubule dynamic instability, FEBS Lett., 296 (1992) 21-24. 14 Ledesma, M.D., Correas, I., Avila, J. and Diaz-Nido, J., Implication of brain cdc2 and MAP2 kinases in the phosphorylation of tau protein in Alzheimer's disease, FEBS Lett., 308 (1992) 218-224. 15 Lindwall, G. and Cole, R.D., Phosphorylation affects the ability of tau protein to promote microtubule assembly, J. Biol. Chem., 259 0984) 5301 5305. 16 Murphy, D.B. and Borisy, G.G., Association of high-molecularweight proteins with microtubules and their role in microtubule assembly in vitro, Proc. Natl. Acad. Sci. USA, 72 (1975) 2696--2700. 17 Nukina, N. and Ihara, Y., One of the antigenic determinants of paired helical filaments is related to tan protein, J. Biochem., 99 (1986) 1541 1544. 18 Olmsted, J.B., Microtubule-associated proteins, Annu. Rev. Cell Biol., 2 (1986) 421-457. 19 Saitoh, T. and Dobkins, K.R., Increased in vitro phosphorylation of a Mr 60,000 protein in brain from patients with Alzheimer disease, Proc. Natl. Acad. Sci. USA, 83 (1986) 9764-9767. 20 Weingarten, M.D., Lockwood, A.H., Hwo, S.Y. and Kirschner, M.W., A protein factor essential for microtubule assembly, Proc. Natl. Acad. Sci. USA, 72 (1975) 1858-1862. 21 Wischik, C.M., Novak, M., Thogersen, H.C., Edwards, RC., Runswick, M.J., Jakes, R., Walker, J.E., Milstein, C., Roth, M. and Klug, A., Isolation of a fragment of tau derived from the core of the paired helical filamentr of Alzheimer disease, Proc. Natl. Acad. Sci. USA, 85 (1988) 4506-4510. 22 Wood, J.G., Mirra, S.S., Pollock, N.J. and Binder, L.I., Neurofibrillary tangles of Alzheimer disease share antigenic determinants with the axonal microtubule-associated protein tau (r), Proc. Natl. Acad. Sci. USA, 83 (1986) 4040-4043.