Phosphorylation of Tau Is Regulated by PKN*

Taizo TaniguchiDagger §, Toshio KawamataDagger , Hideyuki Mukai, Hiroshi HasegawaDagger , Takayuki Isagawa, Minoru YasudaDagger , Takeshi HashimotoDagger , Akira TerashimaDagger , Masamichi NakaiDagger , Yoshitaka Ono, and Chikako TanakaDagger

From the Dagger  Hyogo Institute for Aging Brain and Cognitive Disorders, Himeji 670-0981 and  Graduate School of Science, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan

Received for publication, August 15, 2000, and in revised form, November 26, 2000


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

For the phosphorylation state of microtubule-associated protein, tau plays a pivotal role in regulating microtubule networks in neurons. Tau promotes the assembly and stabilization of microtubules. The potential for tau to bind to microtubules is down-regulated after local phosphorylation. When we investigated the effects of PKN activation on tau phosphorylation, we found that PKN triggers disruption of the microtubule array both in vitro and in vivo and predominantly phosphorylates tau in microtubule binding domains (MBDs). PKN has a catalytic domain highly homologous to protein kinase C (PKC), a kinase that phosphorylates Ser-313 (= Ser-324, the number used in this study) in MBDs. Thus, we identified the phosphorylation sites of PKN and PKC subtypes (PKC-alpha , -beta I, -beta II, -gamma , -delta , -epsilon , -zeta , and -lambda ) in MBDs. PKN phosphorylates Ser-258, Ser-320, and Ser-352, although all PKC subtypes phosphorylate Ser-258, Ser-293, Ser-324, and Ser-352. There is a PKN-specific phosphorylation site, Ser-320, in MBDs. HIA3, a novel phosphorylation-dependent antibody recognizing phosphorylated tau at Ser-320, showed immunoreactivity in Chinese hamster ovary cells expressing tau and the active form of PKN, but not in Chinese hamster ovary cells expressing tau and the inactive form of PKN. The immunoreactivity for phosphorylated tau at Ser-320 increased in the presence of a phosphatase inhibitor, FK506 treatment, which means that calcineurin (protein phosphatase 2B) may be involved in dephosphorylating tau at Ser-320 site. We also noted that PKN reduces the phosphorylation recognized by the phosphorylation-dependent antibodies AT8, AT180, and AT270 in vivo. Thus PKN serves as a regulator of microtubules by specific phosphorylation of tau, which leads to disruption of tubulin assembly.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Dynamics and rearrangement of microtubule networks are modulated by microtubule-associated proteins (MAPs)1 (1, 2). Isolated MAPs show varying degrees of phosphorylation (3), and the state of phosphorylation, balanced by protein kinases and phosphatases, plays a pivotal role in modulating microtubule networks (4). In neurons, tau, a neuronal MAP, modulates microtubule organization during morphogenesis and process outgrowth, and the dynamic nature of tau-microtubule interactions has been observed even in the axons of mature neurons (5).

Although it has been shown that tau can be phosphorylated by more than 10 serine/threonine kinases in vitro (6), it has remained to be determined which kinases modulate tau function through phosphorylation in vivo. PKN is a fatty acid-activated serine/threonine kinase (7), has a catalytic domain highly homologous to the protein kinase C (PKC) family (8, 9), and is one of the direct targets of the small GTP-binding protein Rho (10-12). We reported that PKN phosphorylates tau both in vitro and in vivo and colocalizes with neurofibrillary tangles (NFTs), composed of aberrantly hyperphosphorylated tau in the brains of Alzheimer's disease patients (13). In the present work, we attempted to identify the phosphorylation sites on tau by PKN and PKC, and we examined the role of PKN in tau phosphorylation and microtubule assembly.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Recombinant Proteins-- A cDNA (clone T9) encoding the human brain tau (383 residues) was kindly provided by Prof. H. Mori (Osaka City University). All tau constructs used in this study (Fig. 1) were prepared by polymerase chain reaction amplification using clone T9 as the template. Substitution of serine with alanine was done using a Quick-Change Site-directed Mutagenesis Kit (Stratagene). After confirmation of the sequences, they were subcloned into the pGEX-4T vector (Amersham Pharmacia Biotech). Expression and purification of GST-tagged proteins were done according to the instructions of the manufacturer (Amersham Pharmacia Biotech). A pET23-NHis vector was constructed from the pET-23d vector (Novagen) to add 8 amino acids (MHHHHHHM) to the N terminus of the protein as a His tag. M and H indicate methionine and histidine, respectively. A cDNA (clone T9) was subcloned into pET23-NHis vector. Expression and purification of His-tagged tau protein was done according to the instructions of the manufacturer (Novagen). The numbering of amino acid residues was always based on the longest isoform of human tau (441 residues). Recombinant PKN enzyme was obtained as described (14). The cDNA encoding the open reading frames of rat PKC-alpha (15), -beta I (16), -beta II (16), -gamma (15), -delta (17), -epsilon (17), -zeta (18), and mouse PKC-lambda (generously provided by Dr. W. Ogawa, Kobe University) were inserted into the baculovirus transfer vector, and pBlueBacHis/GST and recombinant enzymes were extracted as described (14).


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Fig. 1.   Schematic representation of various tau fragments. A schematic drawing of the entire tau protein (clone T9) is shown at the top of the figures, and His-tagged tau (His-tau) and GST-tagged deletion mutants and GST-tagged FR and MBD mutants are aligned below. 1, 2, 3, and 4 indicate 1MBD, 2MBD, 3MBD, and 4MBD, respectively. The numbers preceding and following each line denote the positions of the terminal aa residue of each fragment. A in FR and MBD mutants indicates the substitution of serine for alanine. In all cases, the numbering of the amino acid residues refers to the longest isoform of human tau (441 residues).

In Vitro Assay of Microtubule Assembly-- Tau (50 µg) was pretreated with either a catalytically active PKN (10 µg) or catalytically inactive PKN (10 µg) or without PKN in 100 µl of reaction mixture containing 50 mM Tris/HCl at pH 7.5, 5 mM MgCl2, 100 µM ATP, 1 mM dithiothreitol, 1 mM EDTA for 1 h at 30 °C and then added to 400 µl of assembly buffer (100 mM PIPES at pH 6.8, 1.25 mM EGTA, 1.25 mM GTP) containing 100 µg of tubulin (cytoskeleton) at 37 °C. Polymerization of microtubules was monitored by measuring absorbance at 350 nm (19, 20).

In Vitro Phosphorylation Assay-- The phosphorylation of recombinant tau proteins by recombinant enzymes was carried out at 30 °C in 25 µl of reaction mixture containing 20 mM Tris/HCl at pH 7.5, 4 mM MgCl2, 40 µM ATP, 50 ng of recombinant enzymes, and 61.6 kBq of [gamma -32P]ATP. After incubation for various times, the reaction was terminated by adding an equal volume of Laemmli's sample buffer. The samples were separated by 12% SDS-PAGE, and the phosphorylation was visualized and quantified using an instant imager (Packard Instrument Co.). The gels were also stained by Coomassie Brilliant Blue.

Mammalian Expression Constructs-- Mammalian expression construct of tau (Tau) was generated by insertion of cDNA encoding the open reading frames of tau (383 residues) into mammalian expression vector pSG5 (Stratagene). TauS320A was generated using a Quick-Change Site-directed Mutagenesis Kit (Stratagene) from Tau by mutating the serine residue (amino acid 320) to alanine. pTB701/PKN/AF3 (a catalytically active form of PKN; aPKN) was constructed by insertion of the cDNA fragment encoding amino acids 561-942 of PKN into mammalian expression vector pTB701 (21). pTB701/PKN/AF3(K644E) (a catalytically inactive mutant of PKN; iPKN) was generated by Quick-Change Site-directed Mutagenesis Kit (Stratagene) from aPKN by mutating the lysine residue (amino acid 644) at the ATP-binding site of the protein kinase domain to glutamic acid as described (22).

Transfection and Immunoanalysis-- CHO cells seeded at 50% confluency in a 60-mm dish were transfected with 5 µg of Tau and 2 µg of pTB701 vector (CHO-tau-Mock), 5 µg of Tau, and 2 µg of aPKN (CHO-tau-aPKN) and 5 µg of Tau, 2 µg of iPKN (CHO-tau-iPKN), and 5 µg of TauS320A and 2 µg of aPKN (CHO-tauS320A-aPKN) using 15 µl of LipofectAMINE (Life Technologies, Inc.). Twenty one hours after transfection, cells were treated with or without phosphatase inhibitors FK506 (600 nM), vanadate (100 µM), and okadaic acid (50 nM) for 3 h. Cell lysates were prepared, as described (23), and subjected to Western blot analysis using appropriate antibodies. Immunoprecipitation of human neuroblastoma SK-N-MC cells with tau1 and HIA3 were performed as described (13), and immunoprecipitates were subjected to Western blot analysis. Human neuroblastoma SH-SY5Y and SK-N-MC cells seeded at 70% confluency on coverslips in a 60-mm dish were transfected with 5 µg of aPKN or iPKN using 10 µl of a SuperFect Transfection Reagent (Qiagen). Forty to 44 h after transfection, cells were fixed with 4% paraformaldehyde, and the microtubule organization was analyzed by immunofluorescence staining using anti-PKN antibody (alpha C6) and anti-alpha tubulin antibody (YL1/2; Sera-Lab) as described (13).

Antibodies-- A polyclonal antibody, alpha C6, was raised in rabbit against a recombinant C-terminal protein corresponding to residues 863-946 of rat PKN (9). AT8, AT180, and AT270 (Innogenetics) are phosphorylation-dependent mouse monoclonal antibodies against tau, which recognize with proline-direct phosphorylation sites Ser-202/Thr-205, Thr-231, and Thr-181, respectively. tau1 (Roche Molecular Biochemicals) is a mouse monoclonal antibody against tau. A monoclonal antibody, HIHT1, was raised in mouse against a recombinant N-terminal protein corresponding to residues 1-190 without residues 45-102 of human tau. A polyclonal antibody, HIA3, was raised in rabbit against a synthetic phosphopeptide, CLSKVTS(P)KCGSL, where S(P) represents phosphoserine. An anti-alpha -tubulin antibody (YL1/2; Sera-Lab) is a rat antibody against alpha -tubulin.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Microtubule Assembly Is Inhibited by PKN Activation-- To examine effects of PKN activation on microtubule assembly, we carried out the in vitro assay of microtubule assembly. There was a marked increase in the rate of the turbidity when tau was added, whereas it was minimal without tau (Fig. 2A). The tau-induced increase in polymerization of tubulin was almost abolished by adding active PKN, which indicated that PKN impaired the ability of tau to promote microtubule assembly. On the other hand, microtubule assembly was uninhibited when inactive PKN was added to the reaction, indicating that enzymatic activity is required for inhibition of microtubule assembly (Fig. 2A).


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Fig. 2.   PKN disrupts microtubule assembly. A, tau phosphorylated by PKN has a reduced potential to promote microtubule assembly in vitro. Tau not treated with PKN (+tau only; red line), tau pretreated with a catalytically active PKN (+tau/aPKN; green line), or tau pretreated with a catalytically inactive PKN (+tau/iPKN; blue line) was added to the assembly buffer containing tubulin. Black line shows tubulin only. Polymerization of microtubules was monitored by measuring the absorbance at 350 nm. B, overexpression of PKN breaks down the microtubule array in SK-N-MC and SH-SY5Y cells. SK-N-MC (upper panels) cells and SH-SY5Y (lower panels) were transfected with either aPKN (right panels) or iPKN (left panels). Forty four hours after transfection, cells were fixed with 4% paraformaldehyde, and the microtubule organization was analyzed by double immunofluorescence staining for PKN (red) and alpha -tubulin (green). Note that PKN localizes in vesicular structures but is not colocalized with microtubules (arrow in right upper panel), which were dramatically lost compared with those in the cells overexpressing iPKN. Destruction of PKN-positive microtubules is observed in aPKN-overexpressing cells (arrowheads in right lower panel). Upper panels are at the same magnification and bar indicates 50 µm. Bars in lower panels indicate 10 µm.

In human neuroblastoma SH-SY5Y and SK-N-MC cells transfected with aPKN or iPKN, microtubule organization was analyzed by double-labeling immunofluorescence histochemistry using an anti-PKN (alpha C6) and an anti-alpha -tubulin antibody (YL1/2). Many neurons overexpressing aPKN appeared atrophic compared with the cells nontransfected, mock-transfected, or overexpressing iPKN, whereas a subset of aPKN-overexpressing neurons remained normal in both size and shape and exhibited a dramatic loss and poorly organized networks of microtubules (Fig. 2B). On the contrary, the microtubule network had normal appearance in cells transfected with iPKN. Although PKN was well colocalized with perinuclear microtubules in iPKN-overexpressing cells, distinct localization of PKN from microtubules was often seen in the cells transfected with aPKN (arrow). Microtubules were disrupted to small fragments in these neurons when doubly immunolabeled (arrowheads). These data indicate that the cotransfection of tau and PKN resulted in a disruption of microtubule networks in neuronal cells.

PKN Phosphorylates Tau in Vitro-- We determined that PKN phosphorylated recombinant His-tagged tau (His-tau) as well as GST-tagged tau (GST-tau) (13) in vitro. The level of His-tau phosphorylation reached as high as 4 mol of Pi per monomer of tau protein (Fig. 3A). Predominant phosphorylation was seen in GST-tau-MBDs (241-385 aa), although PKN also phosphorylated GST-tau-FR (191-240 aa). On the contrary, PKN phosphorylated poorly GST-tau-NT (1-190 aa) and GST-tau-CT (386-441 aa) (Fig. 3B).


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Fig. 3.   Tau phosphorylation by PKN in vitro. A, time course of tau phosphorylation by PKN. Recombinant His-tau (12.5 pmol) was subjected to in vitro phosphorylation assay. The reaction was terminated at 0 (lane 1), 5 (lane 2), 10 (lane 3), 30 (lane 4), 60 (lane 5), 90 (lane 6), 120 (lane 7), 150 (lane 8), 180 (lane 9), 240 (lane 10), and 330 min (lane 11). Incorporated radioactivity was counted, and the stoichiometry of phosphorylation was calculated. Arrow indicates the time point when 30 ng of additional PKN was added to the assay mixture. B, PKN phosphorylates tau in FR and MBDs. GST-tagged deletion mutants were subjected to in vitro phosphorylation assay for 30 min. The assay was repeated at least three times. The representative data are shown. ARG, 32P incorporation visualized by an imaging analyzer after SDS-PAGE. CBB, Coomassie Brilliant Blue staining.

PKN Phosphorylates Ser-214 in Flanking Region (FR) of Tau-- We first determined PKN phosphorylation sites in the FR of tau. As PKN is a serine/threonine kinase, a candidate serine or threonine for phosphorylation site in FR of tau was substituted for alanine (Fig. 1), and an in vitro phosphorylation assay was performed. When Ser-214 was substituted by alanine, the phosphorylation of tau was significantly reduced (Fig. 4), indicating that Ser-214 is a PKN phosphorylation site in FR of tau.


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Fig. 4.   PKN phosphorylates Ser-214 in FR of tau in vitro. GST-tagged FR mutants were subjected to in vitro phosphorylation assay for 30 min. The assay was repeated at least three times, and representative data are shown. ARG, 32P incorporation was visualized using an imaging analyzer after SDS-PAGE. CBB, Coomassie Brilliant Blue staining.

PKN Phosphorylates Ser-258, Ser-320, and Ser-352 in MBDs of Tau-- PKN has a catalytic domain highly homologous to protein kinase C, which phosphorylates tau in MBDs (24). MBDs consist of four 31- or 32-amino acid repeats, 1MBD (244-274 aa), 2MBD (275-305 aa), 3MBD (306-336 aa), and 4MBD (337-378 aa). To identify the phosphorylation site(s) of PKN, every serine residue in each MBD was substituted by alanine (Fig. 1), and an in vitro phosphorylation assay was performed. PKN phosphorylated all the GST-1MBD tau fragments without S258A mutation, indicating that PKN phosphorylated Ser-258 in 1MBD (Fig. 5A). In the same manner, PKN phosphorylated Ser-320 in 3MBD and Ser-352 in 4MBD.


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Fig. 5.   Ser-320 in MBDs is phosphorylated by PKN but not by PKCs. A, PKN phosphorylates Ser-258, Ser-320, and Ser-352 in MBDs in vitro. GST-tagged MBD mutants were subjected to in vitro phosphorylation assay for 30 min. The assay was repeated at least three times, and representative data are shown. ARG, 32P incorporation was visualized using an imaging analyzer after SDS-PAGE. CBB, Coomassie Brilliant Blue staining. B, PKC subtypes phosphorylate Ser-258, Ser-293, Ser-324, and Ser-352 in MBDs in vitro. GST-tagged MBD mutants were subjected to in vitro phosphorylation assay for 30 min. The assay was repeated at least three times and representative data are shown. ARG, 32P incorporation was visualized using an imaging analyzer after SDS-PAGE. CBB, Coomassie Brilliant Blue staining.

PKC Subtypes Phosphorylate Ser-258, Ser-293, Ser-324, and Ser-352 in MBDs of Tau-- To compare the phosphorylation sites of PKN with those of PKC subtypes, an in vitro phosphorylation assay by PKC subtypes was done. PKC-alpha phosphorylated Ser-258, Ser-293, Ser-324, and Ser-352 in MBDs (Fig. 5B). We also determined the phosphorylation sites of 7 other subtypes, PKC-beta I, -beta II, -gamma , -delta , -epsilon , -zeta , and -lambda . Of all the subtypes examined- Ser-258, Ser-293, Ser-324- and Ser-352 were phosphorylated in MBDs. As summarized in Table I, we found that PKN does not phosphorylate the Ser-293 in 2MBD, which can be phosphorylated by PKCs, and that PKN and PKCs phosphorylate a different site, Ser-320 and Ser-324 in 3MBD, respectively.

                              
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Table I
Phosphorylation sites in each MBD

PKN Phosphorylates Tau in Vivo-- To examine the phosphorylation of tau by PKN in vivo, we developed a rabbit polyclonal antibody (HIA3) directed against the phosphorylated tau at Ser-320, a PKN-specific site for the phosphorylation of tau. Western blot analysis revealed that HIA3 did not react with nonphosphorylated recombinant His-tagged tau (Fig. 6A, lane 1) but did react with PKN-prephosphorylated His-tagged tau (Fig. 6A, lane 2). Moreover, HIA3 did not react with PKC-phosphorylated His-tagged tau (Fig. 6A, lanes 3 and 4). Thus HIA3 is a PKN-specific phosphorylation-dependent tau antibody. HIA3 did not react with CHO-tau-Mock (Fig. 6B, lanes 1 and 2) and CHO-tau-iPKN (Fig. 6B, lane 7) but did react with CHO-tau-aPKN (Fig. 6B, lanes 3 and 4), indicating that PKN phosphorylation of tau occurred in vivo. HIA3 did not react with CHO-tauS320A-aPKN (Fig. 6, lane 5 and 6), indicating that HIA3 recognizes only the phosphorylated Ser-320 of tau.


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Fig. 6.   Immunoblot analysis with a novel antibody, HIA3. A, HIA3 is a specific antibody for PKN phosphorylation. Recombinant His-tagged tau (1 ng) without (lane 1) or with prephosphorylation by PKN (lane 2), PKC-epsilon (lane 3), and PKC-zeta (lane 4) were subjected to SDS-PAGE followed by Western blotting analysis with HIA3 antibody (diluted 1:50,000). Arrowhead indicates the position of tau. B, HIA3 is a phosphorylation- dependent antibody for Ser-320. Five µg of total cell lysates from CHO-tau-Mock (lanes 1 and 2), CHO-tau-aPKN (lanes 3 and 4), CHO-tauS320A-aPKN (lanes 5 and 6), and CHO-tau-iPKN (lane 7) were subjected to SDS-PAGE followed by Western blot analysis with HIA3 antibody (diluted 1:50,000). Arrowhead indicates the position of tau. C, HIA3 recognizes Ser-320 phospho-tau in SK-N-MC neuroblastoma cells. Two hundred µg of total cell lysates from SK-N-MC neuroblastoma cells is immunoprecipitated with control mouse IgG (lane 1), tau1 antibody (lanes 2-4), control rabbit IgG (lane 5), or HIA3 antibody (lanes 6-8). Immunoprecipitates were subjected to SDS-PAGE followed by Western blot analysis with HIA3 antibody (lanes 1-4) or tau1 antibody (lanes 5-8). * and ** indicate the position of mouse and rabbit IgG, respectively. D, protease inhibitor treatments. CHO-tau-Mock (lanes 1, 4, 7, and 10), CHO-tau-aPKN (lanes 2, 5, 8, and 11), and CHO-tau-iPKN (lanes 3, 6, 9, and 12) were treated without (lanes 1-3) or with FK506 (600 nM) (lanes 4-6), vanadate (100 µM) (lanes 7-9), and okadaic acid (OA) (50 nM) (lanes 10-12), respectively, and were subjected to SDS-PAGE followed by Western blotting analysis with HIA3 (diluted 1:10,000). Arrowhead indicates the position of tau.

HIA3 recognized its antigen among tau protein concentrated by immunoprecipitation with anti-tau antibody (tau1) from human neuroblastoma SK-N-MC cells. Furthermore, tau1 recognized its antigen among the protein concentrated by immunoprecipitation with HIA3 (Fig. 6C). These data suggest that Ser-320 phosphorylation occurs in SK-N-MC cells and would reflect some in vivo cellular processes.

Effects of phosphatase inhibitors on tau phosphorylation at Ser-320 by PKN were also examined to characterize the phosphorylation of tau by PKN in vivo. The immunoreactivity of phospho-tau at Ser-320 recognized by HIA3 increased by the treatment with phosphatase inhibitors FK506 (600 nM), orthovanadate (100 µM), and okadaic acid (50 nM) (Fig. 6D). FK506 induced a striking increase in the phosphorylation of tau at Ser-320 by PKN and in vanadate a moderate increase, but the effects of okadaic acid were weak (Fig. 6D). These data indicate that PKN phosphorylates tau at Ser-320 in vivo, and the phosphorylated Ser-320 is dephosphorylated by phosphatases, especially calcineurin.

PKN Activation Reduces Phosphorylation Level in Multiple Proline-directed Phosphorylation Sites in Vivo-- CHO cells transfected with tau and pTB701 vector (CHO-tau-mock) showed a hyperphosphorylated tau reacting with AT8, AT180, and AT270 which recognize with proline-direct phosphorylation sites Ser-202/Thr-205, Thr-231, and Thr-181, respectively (Fig. 7, lane 1). Cotransfection of CHO cells with tau and aPKN (CHO-tau-aPKN) reduced the phosphorylation state recognized by AT8 almost completely, that recognized by AT180 moderately, and that recognized by AT270 only slightly (Fig. 7, lane 2). The state of tau phosphorylation was unchanged when CHO cells were cotransfected with tau and iPKN (CHO-tau-iPKN) (Fig. 7, lane 3), indicating that inhibitory effects of PKN on tau phosphorylation depend on the kinase activity of PKN. The inhibitory effects of PKN on tau phosphorylation were not affected by phosphatase inhibitors FK506 (600 nM), orthovanadate (100 µM), and okadaic acid (50 nM) (Fig. 7, lanes 4-12), although these treatments did lead to recovery of the phospho-tau recognized by HIA3 (Fig. 6D).


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Fig. 7.   PKN suppresses some proline-directed phosphorylation states of tau in vivo. CHO-tau-Mock (lanes 1, 4, 7, and 10), CHO-tau-aPKN (lanes 2, 5, 8, and 11), and CHO-tau-iPKN (lanes 3, 6, 9, and 12) were treated without (lanes 1-3) or with FK506 (0.6 µM) (lanes 4-6), vanadate (100 µM) (lanes 7-9), and okadaic acid (OA) (50 nM) (lanes 10-12), respectively, and were subjected to SDS-PAGE followed by Western blotting analysis with HIHT1 (diluted 1:10,000), AT8 (diluted 1:10,000), AT180 (diluted 1:10,000), AT270 (diluted 1:10,000).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PKN Activation Disrupts Microtubule Organization-- Our data show that catalytically active PKN disrupts microtubule organization in neuroblastoma cells and that tau phosphorylated by PKN loses the potential to promote tubulin assembly in vitro. The stability of tubulin assembly is modulated by tau (2). PKN did not phosphorylate tubulin in vitro,2 thereby suggesting that PKN does not directly associate with tubulin. These findings suggest that the phosphorylation of tau by PKN resulted in disruption of microtubule organization. We reported (25) that PKN phosphorylates intermediate filament proteins such as neurofilaments, vimentin, and glial fibrillary acidic protein and that the phosphorylation by PKN inhibits polymerization. Furthermore, PKN interacts with alpha -actinin, an actin cytoskeletal-associated protein (26). These data imply that PKN may be involved in regulation of the cytoskeleton, such as formation of intermediate or actin filaments and the microtubule organization.

PKN Is a Tau Kinase; Identification of Phosphorylation Sites on Tau by PKN and PKC Subtypes-- We found that PKN phosphorylates recombinant His-tau as well as GST-tau in vitro. The level of phosphorylation reached as high as 4 mol of Pi per monomer of tau protein. In MBDs, PKN phosphorylated Ser-258 in 1MBD, Ser-320 in 3MBD, and Ser-352 in 4MBD. We also identified phosphorylation sites by PKC subtypes in MBDs. Therefore, there is no subtype specificity in phosphorylation of MBDs among the PKC subtypes examined. All subtypes of PKC examined in this study phosphorylated Ser-258 in 1MBD, Ser-293 in 2MBD, Ser-324 in 3MBD, and Ser-352 in 4MBD. Although PKN has a catalytic domain highly homologous to the PKC family, the phosphorylation patterns differ between PKN and PKC subtypes. These results indicate that each PKC-related protein kinase may have a specific function regarding tau phosphorylation. Moreover, it will be necessary to elucidate the meaning that PKN did not phosphorylate any of three serines in 2MBD, which is spliced out in the 3-repeat form of tau. PKN also phosphorylated Ser-214 in FR in vitro. The phosphorylation of Ser-214 has been noted with several kinases, including protein kinase A, and may play some roles for tau-microtubule interactions (27).

In Vivo Phosphorylation by PKN and Dephosphorylation by Calcineurin at Ser-320-- To determine whether PKN induces tau phosphorylation in vivo, we developed a phosphorylation-dependent anti-tau antibody (HIA3), which recognizes phospho-tau at Ser-320, a PKN-specific phosphorylation site. HIA3 reacted with CHO-tau-aPKN but not with CHO-tau-iPKN, thus indicating that PKN phosphorylated tau at Ser-320 in vivo. As the immunoreactivity of HIA3 in CHO-tau-aPKN increased by treatments with phosphatase inhibitors, there is functional involvement of phosphatase in dephosphorylation of phospho-tau at Ser-320 in vivo. FK506 binds with FKBP12, and the complex of FK506 and FKBP12 inhibits calcineurin (28). Vanadate is an inhibitor of protein tyrosine phosphatases and also inhibits serine/threonine phosphatase activities of calcineurin (29). Okadaic acid has been widely used as a protein phosphatase inhibitor in vitro as well as in cultured cells and animals. It inhibits protein phosphatase 2A strongly, protein phosphatase 1 moderately, and calcineurin weakly but does not inhibit protein phosphatase 2C (30). The dephosphorylation of Ser-320 was inhibited by these phosphatase inhibitors, which means that the key phosphatase in question is calcineurin. The findings of colocalization of PKN and calcineurin with tau in neurons (13)3 may support the hypothesis that tau phosphorylation at Ser-320 is regulated by cross-talk between PKN and calcineurin.

Inhibition of Proline-directed Phosphorylations of Tau by PKN-- Hyperphosphorylated tau is the major constituent of the paired helical filament (PHF), the major fibrous component of neurofibrillary tangles (NFT) seen in Alzheimer brains. Many of the hyperphosphorylated sites in PHF are proline-directed. We have reported that PKN is closely associated with NFT at an early stage of PHF formation (13). The present study revealed that PKN phosphorylates tau in vitro and in vivo. We therefore examined the effect of PKN activation on proline-directed phosphorylation, and we found that PKN reduced the level of phosphorylation of multiple proline-direct phosphorylation sites recognized by AT8, AT180, and AT270 in vivo. There are at least three possible mechanisms to explain our observations. First, PKN may activate phosphatases that dephosphorylate the phosphate from Ser-202/Thr-205, Thr-231, and Thr-181 recognized by AT8, AT180, and AT270, respectively. FK506, vanadate, and okadaic acid, however, did not affect the tau phosphorylation recognized by AT8, AT180, and AT270, although they effectively recovered the phospho-tau recognized by HIA3. These data imply that PKN may not activate protein phosphatases. Second, PKN may inactivate other tau kinases that phosphorylate serine or threonine recognized by AT8, AT180, and AT270. One of the candidate kinases would be GSK3, which can phosphorylate Ser-202/Thr-205, Thr-231, and Thr-181 recognized by AT8, AT180, and AT270, respectively (31-33). Furthermore, GSK3 phosphorylated by mitogen-activated protein kinase-activated protein kinase-1 (also known as p90Rsk), p70 ribosomal S6 kinase (p70S6K), and protein kinase B (also known as AKT/RAC) would be consequently inactivated by phosphorylation (34). In the same manner, PKN may inhibit GSK3 by phosphorylation, and the level of phosphorylation recognized by AT8, AT180, and AT270 might decrease in vivo. We found that PKN and PKCs directly inhibit GSK3-beta activity by phosphorylating at least Ser-9 of GSK3-beta and suppress the GSK3-beta -induced AT8 and AT270 immunoreactivity of tau, although they did enhance the GSK3-beta -induced AT180 immunoreactivity in vitro (35). The discrepancy between in vitro and in vivo experiments implies that tau kinases other than GSK3-beta may associate with PKN-induced suppression of the tau phosphorylation at Thr-231 recognized by AT180. Third, PKN may induce a conformational change of tau by phosphorylation and consequently make tau inaccessible either by other tau kinases such as GSK3 or by phosphorylation-dependent tau antibodies such as AT8, AT180, and AT270. Indeed tau prephosphorylated by PKN was less phosphorylated by GSK3-beta in vitro (35).

The present study revealed that PKN phosphorylates tau at specific sites and consequently triggers disruption of microtubule organization. The phosphorylation at Ser-320 in 3MBD is regulated by cross-talk of PKN with calcineurin. Moreover, phosphorylations recognized by AT8, AT180, and AT270 and reduced by PKN in vivo are the most prominent phosphorylations observed in Alzheimer PHFs. On the other hand, free fatty acids such as arachidonic acid and linoleic acid, which can activate PKN in vitro (36), stimulate the polymerization of tau and amyloid beta  peptides in vitro (37). These data suggest that tau phosphorylation by PKN may play crucial roles in the pathology of Alzheimer's disease by regulating tau-microtubule interactions. Thus PKN may serve as a point for therapeutic intervention in neurodegenerative disease including Alzheimer's disease.

    ACKNOWLEDGEMENTS

We are grateful to Prof. H. Mori (Osaka City University) for tau cDNA (clone T9), Dr. W. Ogawa (Kobe University) for mouse PKC-lambda cDNA, and Dr. M. Hasegawa (Tokyo University) for technical advice on the in vitro assay of microtubule assembly. We thank M. Sumida, M. Obana, and A. Hori for their excellent technical assistance and Y. Kitamura, ASK Co. and D. Akagi (ASK Co.) for developing the HIHT1 and HIA3 antibody. We also thank M. Ohara for reading the manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Hyogo Institute for Aging Brain and Cognitive Disorders, 520 Saisho-ko, Himeji 670-0981, Japan. Tel.: 81-792-95-5511; Fax: 81-792-95-8199; E-mail: tanigu@hiabcd.go.jp.

Published, JBC Papers in Press, December 4, 2000, DOI 10.1074/jbc.M007427200

2 H. Mukai, unpublished data.

3 T. Kawamata unpublished data.

    ABBREVIATIONS

The abbreviations used are: MAP, microtubule-associated protein; MBD, microtubule binding domain; PKC, protein kinase C; NFT, neurofibrillary tangle; FR, flanking region; PHF, paired helical filament; CHO, Chinese hamster ovary; GST, glutathione S-transferase; PIPES, 1,4-piperazinediethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; aa, amino acids.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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