Tau Protein Is Hyperphosphorylated in a Site-Specific Manner in Apoptotic Neuronal PC12 Cells

Authors


  • Lippincott Williams & Wilkins, Inc., Philadelphia

  • Abbreviations used: cdc2, cell division control protein kinase;cdk5, cyclin-dependent kinase 5; CK1α, casein kinase 1α; ERK,extracellular regulated kinase; GSK3β, glycogen synthase kinase 3β;HMW, high molecular weight; IEF, isoelectric focusing; MALDI-TOF,matrix-assisted laser desorption ionization-time of flight; NGF, nerve growthfactor; PHF, paired helical filament; RP, reverse-phase; SDS, sodium dodecylsulfate.

Address correspondence and reprint requests to Dr. G. V. W. Johnson atDepartment of Psychiatry and Behavioral Neurobiology, University of Alabama atBirmingham, Birmingham, AL 35294-0017, U.S.A. E-mail:gvwj@uab.edu

Abstract

Abstract: Alterations in the status of microtubules contribute tothe cytoskeletal rearrangements that occur during apoptosis. Themicrotubule-associated protein tau regulates microtubule dynamics and thus islikely to play an important role in the cytoskeletal changes that occur inapoptotic cells. Previously, we demonstrated that the phosphorylation of tauat the Tau-1 epitope was increased during neuronal PC12 cell apoptosis, andfurther that the microtubule binding of tau from apoptotic cells wassignificantly impaired because of altered phosphorylation. The fact that themicrotubule-binding capacity of tau from apoptotic cells was reduced to∼30% of control values indicated that sites in addition to those withinthe Tau-1 epitope were hyperphosphorylated during apoptosis. In this studyusing a combination of immunological and biochemical approaches, numeroussites were found to be hyperphosphorylated on tau isolated from apoptoticcells. Further, during apoptosis, the activities of cell division controlprotein kinase (cdc2) and cyclin-dependent kinase 5 (cdk5) were selectivelyand significantly increased. The association of these two protein kinases withtau was also increased during apoptosis. These findings are intriguing becausemany of the sites found to be hyperphosphorylated on tau during apoptosis arealso hyperphosphorylated on tau from Alzheimer's disease brain. Likewise,there are data indicating that in Alzheimer's disease the activities of cdc2and cdk5 are also increased.

Microtubules are dynamic structures that are essential for cell life and are likely to play an important role during cell death. Tau proteins are a family of differentially spliced isoforms (Lee et al., 1988; Goedert et al., 1989) that bind to and modulate microtubule dynamics (Drewes et al., 1998; Joshi, 1998), and thus are essential components of the neuronal cytoskeleton (Binder et al., 1985; Joshi, 1998). In addition to binding microtubules, tau interacts with the plasma membrane and thus may help coordinate membrane and microtubule interactions (Brandt et al., 1995). Tau's function as a microtubule binding protein is regulated by phosphorylation of specific Ser and Thr residues (Mandelkow and Mandelkow, 1998; Johnson and Hartigan, 1999). In general, increases in tau phosphorylation correlate inversely with its ability to bind and stabilize microtubules. However, the extent to which phosphorylation affects tau's microtubule-binding capacity is largely dependent on which sites are phosphorylated (Johnson and Jenkins, 1996; Mandelkow and Mandelkow, 1998; Johnson and Hartigan, 1999). Abnormal alterations in the phosphorylation state of tau that occur in certain neurodegenerative diseases, such as Alzheimer's disease, lead to impaired function and thus negatively affect microtubule stability and cytoskeletal dynamics (Johnson and Jenkins, 1996; Mandelkow and Mandelkow, 1998; Johnson and Hartigan, 1999).

Apoptosis is a dynamic, energy-dependent cell death process that is important for many physiological events (Vaux and Korsmeyer, 1999), and has also been implicated in pathological processes, such as cancer and neurodegenerative disease (Levine, 1997; Song and Steller, 1999; Stadelmann et al., 1999). During apoptosis, significant cytoskeletal rearrangements occur (Levee et al., 1996; Huot et al., 1998; Mills et al., 1999), and it is likely that tau and other cytoskeletal-regulating proteins play important roles during the process (Davis and Johnson, 1999b; Mills et al., 1999). Because the phosphorylation state of tau regulates its function (or dysfunction), it is important to determine how the site-specific phosphorylation state of tau is modulated during apoptosis.

PC12 cells can be differentiated into a neuronal phenotype by treatment with nerve growth factor (NGF) (Greene and Tischler, 1976) and undergo apoptosis in a reproducible manner upon withdrawal of both serum and NGF (Greene, 1978; Pittman et al., 1993). PC12 cells express both tau and high molecular weight (HMW) tau that contains exon 4a and/or exon 6, which are not present in tau (Couchie et al., 1992; Goedert et al., 1992). Previously, this laboratory has shown that both tau and HMW tau are hyperphosphorylated within Tau-1 epitope during apoptosis of neuronal PC12 cells (Davis and Johnson, 1999a). Furthermore, the microtubule-binding capability of tau and HMW tau isolated from apoptotic PC12 cells was significantly impaired, but restored on dephosphorylation (Davis and Johnson, 1999b). Given the extent to which the microtubule binding of tau from apoptotic cells was impaired, other sites, besides those within the Tau-1 epitope, are likely to be abnormally phosphorylated. Therefore, the present study focused on identifying specific sites on tau and HMW tau that are hyperphosphorylated during apoptosis and the protein kinases that are potentially involved in this process. Using a combination of immunoblotting with specific phospho-dependent tau antibodies and biochemical approaches, sites that are hyperphosphorylated on tau and HMW tau during apoptosis were identified. Further, data presented here indicate that an increase in the activities of cell division control protein kinase (cdc2) and cyclin-dependent kinase 5 (cdk5; also known as tau protein kinase II) may contribute in part to the hyperphosphorylation of tau and HMW tau during apoptosis.

MATERIALS AND METHODS

Cell culture

PC12 cells were grown on rat tail collagen-coated Corning dishes in RPMI 1640 medium containing 10% horse serum, 5% FetalClone II (HyClone, Logan, UT, U.S.A.), 20 mM glutamine, and 100 units/ml streptomycin as described (Greene and Tischler, 1976). To induce differentiation to a neuronal phenotype, PC12 cells were maintained in medium containing 5% serum and 100 ng/ml NGF (Accurate Chemical Corp.) for 9-10 days. To induce apoptosis, serum and NGF were removed by replating the differentiated cells onto new collagen-coated dishes as described previously (Batistatou and Greene, 1991), and cells were maintained in serum-free medium with or without 100 ng/ml NGF for 48 h. Based on our previous studies (Davis et al., 1997; Davis and Johnson, 1999a,b), the majority of cells were in the apoptotic process, and the increase in tau phosphorylation was maximal, 48 h after serum and NGF removal. Therefore, this time point was chosen for the present studies.

Purification of tau and HMW tau

Tau and HMW tau were purified from control and apoptotic cells using a procedure based on the heat-stable characteristics of tau (Weingarten et al., 1975; Johnson et al., 1989), its elution profile from a C8 reverse-phase column (Hanger et al., 1998), and ammonium sulfate precipitation properties of tau obtained from small-scale tests. For each preparation, 50 100-mm plates of apoptotic PC12 cells (48 h of serum and NGF withdrawal) or 15 100-mm plates of control cells (plus NGF) were used as starting material. Apoptotic or control cells were harvested and sonicated in a high-salt lysis buffer containing 20 mM MES, pH 6.9, 1 mM EDTA, 1 mM EGTA, 1 mM MgSO4, 0.75 M NaCl, 0.5 mM phenylmethylsulfonyl fluoride, and 10 μl/ml protease inhibitor cocktail (Sigma). Cell lysates were spun at 16,000 g for 45 min at 4°C. The supernatant was collected, dithiothreitol was added to a final concentration of 2 mM, and the mixture was incubated in a boiling water bath for 10 min. The extract was cooled on ice, and heat-denatured proteins were removed by centrifugation at 16,000 g for 45 min at 4°C. Ammonium sulfate was added to the supernatant that contained the heat-stable proteins to 25% of saturation (P25) and kept on ice for 30 min. The precipitates were removed by spinning at 16,000 g for 30 min. The P25 supernatant was collected, ammonium sulfate was added to 50% saturation (P50), and the mixture was maintained at 4°C for 4 h. The P50 precipitate was collected by centrifugation at 16,000 g for 30 min. The tau-enriched P50 fraction was resuspended in 0.2 M NaCl in 5% CH3CN, 0.1% trifluoroacetic acid and applied onto an Aquapore RP-300 column (7 μm, 30 × 2.1 mm, C8 matrix; Perkin-Elmer). The tau protein was eluted with a linear gradient of CH3CN (5-50%) using reverse-phase (RP) HPLC (Dionex) as described previously (Hanger et al., 1998). The tau-containing fractions of RP-HPLC were identified by immunoblotting with the phosphate-independent tau antibody Tau5 antibody, and the appropriate tau-containing fractions were pooled. Using this protocol, tau and HMW tau eluted in the same fractions. RP-HPLC tau and HMW tau were used for immunoblotting, isoelectric focusing (IEF), and mass spectrometric analysis. Protein concentrations were determined using the bicinchoninic acid method (Pierce), and samples were stored at -80°C until use.

Immunoblot analysis with phosphate-dependent tau antibodies

Immunoblot analysis was carried out as described previously (Davis et al., 1997). In brief, purified proteins (0.25 μg) were separated by electrophoresis on 8% sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred to nitrocellulose, and probed with the indicated tau antibodies. After incubation with horseradish peroxidase-conjugated secondary antibodies, immunoblots were developed using enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech). Immunoblots were quantified using a Bio-Rad GS670 imaging densitometer. To examine the phosphorylation of tau, the following antibodies were used: PHF1 (a gift from Dr. P. Davies), Tau-1 (a gift from Dr. L. Binder), AT180 (Polymedco), AT270 (Polymedco), and 12E8 (a gift from Dr. P. Seubert). The epitopes recognized by each antibody and the dilutions used in the experiments are given in Table 1 [numbering based on the longest human (441 amino acids) (Goedert et al., 1989) and rat (432 amino acids) (Kosik et al., 1989) tau isoforms]. To determine total tau levels, the phosphate-independent monoclonal antibody Tau5 (a gift from Dr. L. Binder) or a polyclonal tau antibody (Dako) was used. To determine quantitatively the alterations in tau phosphorylation at different epitopes, all data obtained with the phospho-dependent antibodies were normalized to total tau levels in the samples and then the difference in phosphorylation between apoptotic and control tau was determined. The data are presented as percent increase over control. All data were analyzed using Student's t test and considered significantly different when p < 0.05.

Table 1. Phosphoepitopes of tau antibodies
 Phosphoepitopes 
AntibodyHumanRatAntibody dilution
  1. Except for Tau-1, the Ser/Thr residues listed must be phosphorylated for antibody reactivity. In the case of Tau-1, all residues within the epitope must be unphosphorylated (-P). The dilutions of the antibodies that were used in this study are also listed.

Tau1 S198, S199, S202, T205 (-P) S189, S190, S193, T196 (-P)1:7,000
PHF1 S396, S404 S387, S3951:1,000
12E8 S262, S356 S253, S3471:2,000
AT180 T231, S235 T222, S2261:1,000
AT270 T181 T1721:1,000

TABLE 1.

In-gel double digestion of tau and HMW tau and mass spectrometric analysis

RP-HPLC purified tau (with and without NGF) was electrophoresed on an 8% SDS-polyacrylamide gel. After electrophoresis, gels were stained with 0.5% Coomassie Brilliant Blue R250, 10% acetic acid, 20% methanol, and destained with 10% acetic acid, 20% methanol. The tau and HMW tau bands were excised from the gel and minced into small pieces. The gel pieces were rinsed extensively with 25 mM (NH4)2CO3, pH 8.0, 50% CH3CN, and dried on a Speedvac (Savant). For the first digestion, 10 μl of 0.1 μg/μl chymotrypsin (Boehringer Mannheim) in 25 mM (NH4)2CO3, pH 8.0, and 10 μl of 25 mM (NH4)2CO3, pH 8.0, were added to the dried gel pieces and incubated at 37°C for 14 h. To stop the first reaction, the gel pieces were dried down, 10 μl of 40 μM chymotrypsin inhibitor (Boehringer Mannheim) was added, and the mixture was incubated at room temperature for 30 min. The second digestion was started by adding 10 μl of 0.1 μg/μl trypsin (Boehringer Mannheim) in 25 mM (NH4)2CO3, pH 8.0, and 10 μl of buffer containing 25 mM (NH4)2CO3, pH 8.0, to the gel pieces, and incubated at 37°C for 20 h. The digested phosphopeptides in the gel were extracted with 5% trifluoroacetic acid, 50% CH3CN, and concentrated by Speedvac. As a control, a blank gel piece was also cut out and processed identically as the tau-containing gel pieces.

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis was performed by the UAB Mass Spectrometry Shared Facility. Masses of resulting peptides were compared with predicted masses for rat tau and HMW tau using the protein prospector program (http://donatello.ucsf.edu).

Two-dimensional IEF/SDS gel electrophoresis

The first dimension of IEF was carried out as described by O'Farrell et al. (1977) using a pH equilibrium system. In brief, 5% polyacrylamide disc gels that contained 8 M urea, 2% NP-40, 1.5% ampholine (pH 3.5-10) were prepared and prerun at 500 V for 1 h before the samples were loaded. Control and apoptotic RP-HPLC purified tau (0.5-1 μg) were dissolved in loading buffer containing 10 M urea, 2% NP-40, 1.5% ampholine (pH 3.5-10; Sigma), 5% β-mercaptoethanol, and 5 μg/μl soybean trypsin inhibitor as an internal standard (Sigma), and subsequently applied to each IEF gel and focused for 6 h at 1,500 V. The gels were removed from the tubes, incubated in 2× SDS stop solution for 30 min at 37°C, electrophoresed on an 8% SDS-polyacrylamide gel, and transferred to a nitrocellulose. To visualize the internal standard (trypsin inhibitor), nitrocellulose membranes were stained immediately with 0.5% Ponceau S solution in 5% acetic acid (Sigma), the position of trypsin inhibitor was marked, and then the blots were destained with distilled water, blocked, and probed with the appropriate antibodies.

Immunoprecipitation and kinase assays

Cells were lysed in lysis buffer containing 50 mM Tris-HCl, pH 7.4, 2.5 mM MgCl2, 0.14 M NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% NP-40, 0.5 mM phenylmethylsulfonyl fluoride, 10 μl/ml protease inhibitor cocktail (Sigma), 0.2 μM okadaic acid. Lysates were clarified by centrifugation at 20,000 g at 4°C for 45 min, and protein concentrations were determined. Extracts (500 μg) were incubated with either 35 μg of tau antiserum (Dako), 5 μg of anti-casein kinase 1α (CK1α) polyclonal antibodies (Santa Cruz), 4 μg of monoclonal antibodies against cdk5 (Santa Cruz), extracellular regulated kinase 1/2 (ERK1/2; also referred to as mitogen-activated protein kinase) (Upstate Biotechnology Inc.), or glycogen synthase kinase 3β (GSK3β) (Transduction Laboratories) at 4°C for 2 h. Fifty microliters of prewashed protein A beads (for polyclonal antibodies) or protein G beads (for the mouse monoclonal antibodies) (Amersham Pharmacia) were added to each reaction and then incubated at 4°C overnight. To precipitate cdc2, 50 μl of p13 Sepharose 6B (Upstate Biotechnology Inc.) was added directly to the extract and incubated at 4°C overnight (Zhang et al., 1994). After incubation, the agarose beads were washed extensively with lysis buffer and used for the kinase assays. To measure kinase activity in the immunoprecipitates, 25 μl of the beads was aliquoted and resuspended in 50 μl of kinase buffer containing 25 mM Tris-HCl, pH 7.4, 1 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and 3 μM human recombinant tau (PanVera). The reaction was started by adding 100 μM [γ-32P]ATP (350 dpm/pmol) and incubated at 30°C for 45 min. The reaction was stopped by adding an equal volume of 2× SDS stop solution. Proteins were resolved on a 10% SDS-polyacrylamide gel, and the phosphorylated proteins were analyzed either by phosphoimaging (Molecular Dynamics) or by autoradiography followed by densitometric scanning.

In vitro dephosphorylation assay

To determine if the changes in apoptotic tau and HMW tau were due to increases in phosphorylation, samples were dephosphorylated for some studies. RP-HPLC purified tau (1.5 μg) was incubated with 10 units of lambda protein phosphatase (BioLabs) in 50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 5 mM dithiothreitol, 0.01% Brij 35, 2 mM MnCl2 at 30°C for 2 h. The dephosphorylation reaction was stopped by adding either 2× SDS stop solution or IEF loading buffer, and the dephosphorylated tau was resolved either on an 8% SDS-polyacrylamide gel or by two-dimensional IEF/SDS electrophoresis. The proteins were transferred to a nitrocellulose, and the blots were probed with the indicated tau antibodies as described before.

RESULTS

Tau and HMW tau are phosphorylated in a site-specific manner during apoptosis

Our laboratory demonstrated previously that tau and HMW tau are hyperphosphorylated at the Tau-1 site during differentiated PC12 cell apoptosis (Davis and Johnson, 1999a). Tau-1 antibody recognizes tau and HMW tau only when Ser195, Ser198, Ser199, Ser202, and Thr205 (numbering based on longest human brain tau isoforms) are not phosphorylated (Binder et al., 1985; Szendrei et al., 1993), and therefore decreases in immunoreactivity indicate an increase in phosphorylation at the Tau-1 epitope. To determine if other sites in both tau and HMW tau were also hyperphosphorylated during apoptosis, samples from control and apoptotic cells were probed with the antibodies listed in Table 1 that recognize different phosphoepitopes on tau. Samples were also probed with phosphate-independent antibodies (Tau5 or a polyclonal tau antibody) to determine the total levels of tau and HMW tau. Significant increases in phosphorylation at the epitopes of Tau-1, 12E8 (Seubert et al., 1995), PHF1 (Otvos et al., 1994), and AT180 (Illenberger et al., 1998) were observed in the apoptotic samples compared with the controls (Fig. 1). Further, differential phosphorylation between tau and HMW tau were detected (Fig. 1). For example, there was almost a fourfold increase in phosphorylation within the Tau-1 epitope for HMW tau and only a twofold increase in phosphorylation at the Tau-1 epitope for tau (Fig. 1). It should be noted that for quantification purposes the decrease in Tau-1 immuno-reactivity was expressed as an increase in phosphorylation (Fig. 1). Phosphorylation of the 12E8 epitope increased approximately twofold in HMW tau during apoptosis, but greater than sixfold in tau, and a similar trend was observed with PHF1. It is interesting that when the samples were probed with the AT180 antibody, only HMW tau showed a significant increase in phosphorylation in the apoptotic cells. There were no substantial differences in phosphorylation at the AT270 site between apoptotic tau and control tau (Fig. 1).

Figure 1.

Tau is hyperphosphorylated in a site-selective manner during apoptosis. A: Representative immunoblots probed with the indicated tau antibodies. RP-HPLC purified tau (0.25 μg) from apoptotic (-NGF) or control (+NGF) cells was loaded on each lane. B: Tau phosphorylation at the epitopes indicated was analyzed quantitatively. All values from the phospho-dependent antibodies were normalized to total tau levels in the sample (Tau5 immunoreactivity), and then the data for tau from the apoptotic (-NGF) cells were expressed as fold increase over control (+NGF) values (means ± SEM). During apoptosis, phosphorylation was increased significantly at the Tau-1, 12E8, and PHF1 epitopes for tau (solid columns) and HMW tau (open columns). No changes were observed at the AT270 epitope during apoptosis, and at the AT180 epitope an increase in phosphorylation occurred only in HMW tau. All data were from three independent experiments, and values were considered significantly different when *p < 0.05.

FIG. 1.

Tau and HMW tau from apoptotic cells are more acidic due to increased phosphorylation

To analyze further tau and HMW tau phosphorylation during apoptosis, two-dimensional IEF was carried out. The isoelectric point of individual proteins is dependent on many factors, such as amino acid composition and posttranslational modifications including phosphorylation and dephosphorylation (Steinberg et al., 1977). Phosphorylation tends to increase the negative charge of the protein and thus shift the protein's migration toward the anode (+), whereas dephosphorylation tends to decrease the negative charge of the protein and thus shifts the protein toward the cathode (-). Two-dimensional IEF analysis revealed that both tau and HMW tau from apoptotic cells exhibited greater migration toward the anode when compared with control tau and HMW tau (Fig. 2, left panels), indicating an increase in phosphorylation during apoptosis. To verify that the shift of apoptotic tau and HMW tau toward the anode resulted from an increase in phosphorylation, samples were dephosphorylated with lambda protein phosphatase prior to two-dimensional IEF analysis. Dephosphorylation of HMW tau from control and apoptotic cells with lambda phosphatase resulted in a pronounced shift toward the cathode (Fig. 2, right panels). Further, dephosphorylated HMW tau from control or apoptotic cells exhibited almost identical migration patterns. The predicted isoelectric point of rat tau is 8.26 as calculated by the protein prospector program (http://donatello.ucsf.edu), and thus it is a basic protein. Therefore, after dephosphorylation, tau did not migrate into the IEF gel during the first dimension, and hence was not detected on the blots (Fig. 2, right panels). In contrast, HMW tau is a more acidic protein with a predicted isoelectric point of 5.54 (http://donatello.ucsf.edu); therefore, dephosphorylated HMW tau migrated into the IEF gel in the first dimension and was detected on the immunoblots.

Figure 2.

Two-dimensional IEF/SDS gel electrophoresis analysis of tau and HMW tau from control (+NGF) and apoptotic (-NGF) cells. RP-HPLC purified tau (1 μg) from control and apoptotic cells was run on each gel with trypsin inhibitor (TI) as an internal standard (IP ≅ 4.6). The resulting blots were probed with the Tau5 antibody. Both HMW tau and tau from apoptotic cells (left lower panel) were shifted toward the anode (+) compared with HMW tau and tau from control cells (left upper panel), indicating that the proteins from the apoptotic cells were more acidic. However, when the samples were dephosphorylated (±NGF/DP) with lambda protein phosphatase, HMW tau shifted toward the cathode (-), and the migration patterns of HMW tau from both control and apoptotic cells were similar after dephosphorylation (right panels). These data demonstrate that the increased mobility of apoptotic tau and HMW tau toward the anode is due to increased phosphorylation. Because the isoelectric point of tau is very basic, after dephosphorylation it did not migrate in the IEF gel in the first dimension, and therefore only HMW tau was detected in the lambda phosphatase-treated samples.

FIG. 2.

Mass spectrometry analysis

To more thoroughly assess alterations in tau phosphorylation during apoptosis, MALDI-TOF mass spectrometry analysis was performed. The masses of resulting peptides were compared with predicted masses for rat tau and HMW tau. Only the peptides that were found consistently in both control and apoptotic samples were analyzed further. A total of 21 peptides from tau in all control and apoptotic samples were detected (n = 3 separate preparations). Fourteen of these peptides contained either no amino acids that could be phosphorylated, or there were no differences in the phosphorylation states between control and apoptotic tau. However, the phosphorylation state of seven peptides from apoptotic tau increased when compared with the same peptides from control tau (Table 2). It is interesting that Ser315 (Ser324 in human tau) within the microtubule binding region was phosphorylated in apoptotic tau, but not in control tau (Table 2). Further, a peptide with the Tau-1 epitope and a peptide spanning the PHF1 epitope also showed increased phosphorylation in apoptotic tau compared with control tau (Table 2), thus supporting the immunoblot data. For HMW tau, a total of 19 peptides were found consistently in all samples. Seven of these 19 peptides from HMW tau were found to be hyperphosphorylated in the apoptotic samples when compared with control cells (Table 3). A peptide containing parts of the Tau-1 epitope was also found phosphorylated to a greater extent in apoptotic HMW tau compared with controls (Table 3). For both tau and HMW tau, some of the peptides were not detected during mass spectrometric analysis, and therefore all the sites found to be phosphorylated by immunoblot analysis could not be identified. Nevertheless, these findings clearly indicated that both tau and HMW tau are phosphorylated to a greater extent in apoptotic cells compared with controls during apoptosis.

Table 2. Mass spectrometric analysis of tau phosphopeptides from control and apoptotic cells
Peptide (AA)SequenceMasses of +NGF (predicted)Masses of -NGF (predicted)p
  1. Mass spectrometry data of tau phosphopeptides from control (+NGF) and apoptotic (-NGF) cells in which changes in phosphorylation were detected. Amino acid numbering is based on rat tau (431 amino acids). The actual mass is given for each peptide, and in parentheses the predicted masses are listed. The number of phosphates (P) on the peptide is also indicated. In the peptide sequences, potential phosphorylation sites are in bold, and peptides containing the Tau-1 and PHF1 epitopes are indicated. AA, amino acids; ND, not detected.

57-72STPTAEDVTAPLVEER 1,714.99 (1,714.85)1,715.04 (1,714.85)0
  ND1,873.92 (1,874.78)2
  1,955.91 (1,954.75)1,955.97 (1,954.75)3
  ND2,033.12 (2,034.71)4
142-154 IATPRGAATPGQK 1,267.67 (1,267.71)ND0
  ND1,427.88 (1,427.56)2
147-161 GAATPGQKGTSNATR 1,416.88 (1,416.72)ND0
  ND1,737.06 (1,736.58)4
189-200 (Tau-1)SSPGSPGTPGSR 1,086.64 (1,086.52)ND0
  ND1,326.63 (1,326.42)3
203-212TPSLPTPPTR 1,066.67 (1,066.59)1,066.57 (1,066.59)0
  ND1,146.71 (1,146.56)1
  ND1,385.91 (1,386.46)4
313-331 CGSLGNIHHKPGGGQVEVK 1,988.21 (1,988.03)1,988.04 (1,988.01)0
  ND2,068.95 (2,067.98)1
386-397 (PHF1) KSPVVSGDTSPR ND1,309.79 (1,309.62)1
  1,469.70 (1,469.55)ND3
  ND1,549.03 (1,549.51)4
Table 3. Mass spectrometric analysis of HMW tau phosphopeptides from control and apoptotic cells
Peptide (AA)SequenceMasses of +NGF (predicted)Masses of -NGF (predicted)p
  1. Mass spectrometry data of HMW tau phosphopeptides from control (+NGF) and apoptotic (-NGF) cells in which changes in phosphorylation were detected. Amino acid numbering is based on rat HMW tau (686 amino acids). The actual mass is given for each peptide and in parentheses the predicted masses are listed. The number of phosphates (P) on the peptide is also indicated. In the peptide sequences, potential phosphorylation sites are in bold. The peptide that contains the Tau-1 epitope is indicated. AA, amino acids; ND, not detected.

57-72STPTAEDVTAPLVEER 1,715.04 (1,714.85)1,714.96 (1,714.85)0
  ND1,954.95 (1,954.75)3
122-131SQSLLVEPGR 1,085.71 (1,085.60)1,085.65 (1,085.60)0
  ND1,165.66 (1,165.56)1
145-163THQQVPSMSGAPLPPQGLR 2,001.24 (2,001.03)2,001.16 (2,001.03)0
  ND2,239.20 (2,240.93)3
164-179 EATHQPLGTRPEDVER 1,835.11 (1,834.90)1,835.06 (1,834.90)0
  ND1,994.23 (1,994.84)2
426-439TPPGSGEPPKSGER 1,475.79 (1,475.65)1,475.80 (1,475.65)1
  ND1,635.94 (1,635.59)3
440-454 (Tau-1)SGYSSPGSPGTPGSR ND1,393.83 (1,393.65)0
  1,473.84 (1,473.60)ND1
  ND1,793.05 (1,793.47)5
457-469TPSLPTPPTREPK 1,420.91 (1,420.78)1,420.92 (1,420.78)0
  ND1,500.88 (1,500.75)1

TABLE 2.

TABLE 3.

The levels of Ser/Thr-protein kinases are altered during apoptosis

The quantitative immunoblots with phospho-dependent tau antibodies, two-dimensional IEF, and mass spectrometric analysis clearly demonstrate that both tau and HMW tau are hyperphosphorylated in a site-specific manner during apoptosis. These data indicate that there is an increase in tau-directed protein kinase activity during apoptosis. Therefore, tau was immunoprecipitated from control and apoptotic cells, and the kinase activity that coimmunoprecipitated with tau was determined using recombinant human tau as the substrate. Because in apoptotic cells the levels of tau tend to decrease (Davis and Johnson, 1999a; see Fig. 3A), there was less tau in the precipitates from apoptotic cells (Fig. 3B). However, in Fig. 3A, the representative autoradiograph clearly demonstrates that there is an increase in the protein kinase activity associated with tau during apoptosis. Taudirected protein kinase activity was then normalized to the total tau levels in the coimmunoprecipitated samples. These data revealed that kinase activity associated with tau was significantly increased 75 ± 7.5% from apoptotic cells compared with control (n = 3 separate determinations).

Figure 3.

Tau-associated kinase activity is elevated during apoptosis. A: Immunoblot of cell lysates from control (+NGF) and apoptotic (-NGF) cells showing the amount of tau and HMW tau in the samples (40 μg of total protein/lane). B: Tau was immunoprecipitated from equivalent amounts of protein from control (+NGF) and apoptotic (-NGF) cells with a rabbit polyclonal tau antibody, and samples were probed with the mouse monoclonal antibody Tau5. Just tau is shown in this panel to demonstrate that there is less tau precipitated from the apoptotic cells compared with control cells. C: Tau-directed protein kinase activity that coimmunoprecipitated with tau was measured using [γ-32P]ATP, and recombinant human tau as the substrate. The representative autoradiograph shows the kinase activity that associated with tau from apoptotic and control cells, and demonstrates that the kinase activity associated with tau from apoptotic cells is increased significantly.

FIG. 3.

To identify the protein kinases that could potentially contribute to tau hyperphosphorylation during apoptosis, the expression levels of protein kinases that have been shown to phosphorylate tau in vitro or in situ were examined. It has been reported previously that cdc2 kinase levels are elevated during differentiated PC12 cell apoptosis (Davis et al., 1997), a finding confirmed in this study where a fourfold increase in cdc2 levels was observed in apoptotic cells compared with control cells (Fig. 4). The levels of cdk5, GSK3β, ERKs, and CK1 α were also examined in the present study. The level of cdk5 in apoptotic cells was significantly increased 65 ± 8% above the levels in control cells. In contrast, the levels of ERK1/2 and CK1α were the same in control and apoptotic cells, and the levels of GSK3β decreased 40 ± 4% in apoptotic cells compared with controls (Fig. 4). These data clearly demonstrate that the expression levels of protein kinases are selectively altered during apoptosis.

Figure 4.

Protein kinases are altered in a selective manner during apoptosis. Cell lysates were probed with the indicated antibodies. A: Representative immunoblots reveal that the levels of cdc2 and cdk5 were increased in apoptotic (-NGF) cell lysates compared with controls (+NGF), the levels of CK1α and ERK1/2 were unchanged, and the levels of GSK3β were decreased in apoptotic cells. B: Quantitative analysis of the alterations in the levels of kinases in apoptotic compared with control cells. Values are presented as percentage of control (means ± SEM, n = 3 separate experiments). Values were considered significantly different when *p < 0.05.

FIG. 4.

The activities of cdc2 and cdk5 are increased in apoptotic PC12 cells

To determine whether the alterations in the levels of the protein kinases during apoptosis resulted in similar changes in activity, each protein kinase was immunoprecipitated from control and apoptotic cells and the activity was measured. The data showed that the activity of cdc2 was elevated 410 ± 70% during apoptosis compared with controls (Fig. 5), which corresponds to the observed increase in levels (Fig. 4). Cdk5 activity in apoptotic cells increased 270 ± 30% compared with controls (Fig. 5). This increase in cdk5 activity in apoptotic cells was significantly greater than the observed increase in cdk5 levels (Fig. 4). As expected, there were no increases in the activity of the ERKs and CK1 α during PC12 cells apoptosis (Fig. 5). It is interesting that there was no change in GSK3β activity in apoptotic cells even though the levels of the protein were decreased.

Figure 5.

Protein kinase activities were selectively increased during apoptosis. Cell lysates from control and apoptotic cells were immunoprecipitated with the indicated antibody, and protein kinase activity was measured using recombinant human tau as the substrate. A: Representative autoradiographs showing the extent of tau phosphorylation with each immunoprecipitated protein kinase. B: Quantitative analysis of the alterations in the kinase activity in apoptotic compared with control cells. The kinase activity is presented as percent increase over control (means ± SEM, n = 3 separate experiments). The activities of cdc2 and cdk5 from apoptotic cells were increased significantly compared with that of controls. However, there were no significant differences in the activity of ERK, GSK3β, or CK1α during apoptosis. Values were considered significantly different when *p < 0.05.

FIG. 5.

Because the kinase activity associated with tau was increased during apoptosis and the activities of cdc2 and cdk5 significantly increased during apoptosis, the association of these protein kinases with tau was examined. In these studies, tau was immunoprecipitated from control and apoptotic PC12 cells and the precipitates were probed with antibodies against cdc2 or cdk5, and as a control precipitates were also probed for tau. These results demonstrate that the association of both kinases with tau increases during apoptosis (Fig. 6).

Figure 6.

Cdc2 and cdk5 associate with tau, and this association increases in apoptotic cells. Lysates from either apoptotic (-NGF) or control (+NGF) cells were immunoprecipitated with a tau polyclonal antibody (250 μg of protein was used to detect coimmunoprecipitation of cdc2 or cdk5, whereas for the immunoprecipitation of tau 40 μg of protein was used), and the precipitates were probed with the indicated monoclonal antibodies. Although less tau is precipitated from the apoptotic cells compared with control cells, there is more cdc2 and cdk5 present in the apoptotic tau precipitates, clearly demonstrating an increased association between these protein kinases and tau.

FIG. 6.

p35 is predominantly cleaved to p25 during apoptosis

p35 is the regulatory subunit of cdk5 (Tsai et al., 1994; Tang and Wang, 1996; Patrick et al., 1999); however, a truncated form of p35, p25, was shown recently to activate cdk5 inappropriately, and when overexpressed in cells, p25 induced apoptosis (Patrick et al., 1999). To determine whether the levels of p35 and p25 are altered during PC12 cell apoptosis, blots were probed with an antibody to the C-terminus of p35 (C-19, Santa Cruz) that recognizes both p35 and p25. A representative im- munoblot shown in Fig. 7 clearly demonstrates that during apoptosis the levels of p25 increase with a concomitant decrease in p35 levels. These data suggest that the increases in activity of cdk5 not only are the result of increased cdk5 levels, but also may be due to increases in the production of p25, and thus increased activation of cdk5.

Figure 7.

p35 is proteolyzed to p25 in apoptotic cells. Lysates from apoptotic (-NGF) and control (+NGF) cells were probed with a polyclonal antibody against the C-terminus of p35 that recognizes both p35 and p25. The positions of p35 and p25 are indicated. The blot shows that the p35 proteolysis occurs in apoptotic cells.

FIG. 7.

DISCUSSION

In a previous study, we demonstrated that during apoptosis tau is hyperphosphorylated within the Tau-1 epitope (Davis and Johnson, 1999a), and further that the microtubule-binding capacity of tau isolated from apoptotic cells is dramatically decreased (Davis and Johnson, 1999b). Because of the amplitude of the phosphorylation-dependent decrease in the microtubule-binding capacity of tau from apoptotic cells, it was likely that other sites were also hyperphosphorylated. In this study, we clearly demonstrate that during apoptosis tau is hyperphosphorylated at numerous sites. Further, we demonstrate that the activities of cdc2 and cdk5 are selectively and significantly elevated in apoptotic cells, and both protein kinases show an increased association with tau from apoptotic cells.

Apoptosis is a dynamic process that results in profound morphological changes (Mills et al., 1999). For these apoptotic morphological changes to occur, the cytoskeleton must undergo dynamic rearrangement. Indeed, there are increasing numbers of studies demonstrating alterations of cytoskeletal components during apoptosis (Guenal et al., 1997; van Engeland et al., 1997; Huot et al., 1998; Mills et al., 1999). Microtubules as a dynamic and essential component of the cytoskeleton likely play an integral role during apoptotic cell death (Mills et al., 1999). Tau acts as a regulator of microtubule dynamics through its ability to stabilize microtubules and promote microtubule assembly (Johnson and Jenkins, 1996), a function that is negatively regulated by its phosphorylation state (Lindwall and Cole, 1984; Gustke et al., 1992). In apoptotic cells, the microtubule-binding capacity of tau was reduced significantly (Davis and Johnson, 1999b), and in this study we found that the phosphorylation of several key sites known to regulate the microtubule-binding capacity of tau were increased during apoptosis. For example, the KXGS motif was phosphorylated in three of the four microtubule binding repeats in apoptotic tau. Previous studies have found that phosphorylation of these sites significantly decreases the ability of tau to bind and stabilize microtubules (Biernat et al., 1993; Trinczek et al., 1995). Further, both by immunoblotting with the PHF1 antibody and by mass spectrometric analysis, the phosphorylation of Ser396 (Ser387 in rat tau) was found to be increased in tau from apoptotic cells. Phosphorylation of Ser396 reduces the microtubule-binding capacity of tau (Bramblett et al., 1993), and therefore phosphorylation of this site is likely to contribute to the impaired microtubule binding of tau from apoptotic cells. Overall, the increases in the site-specific phosphorylation state of tau that occur during apoptosis likely contribute to the destabilization of the microtubule network (Mills et al., 1999), and hence the cytoskeletal changes that occur during apoptosis. It should be noted that, in agreement with our findings, Nuydens et al. (1997) also observed an increase in tau phosphorylation during apoptosis. In contrast, another study reported that tau was dephosphorylated during apoptosis based primarily on immunocytochemical findings (Mills et al., 1998). The reason for these apparent disparate findings is unknown; however, in this previous study a subclone of the PC12 cell line called PC6-3, which displays significant differences from the parental cell line, was used. Further the protocol to induce apoptosis in the PC6-3 cells also differed from the present study (Mills et al., 1998). Nonetheless, given the findings of this present study, and the previous results demonstrating that the microtubule-binding capacity of tau from apoptotic cells is impaired in a phosphorylation-dependent manner (Davis and Johnson, 1999b), it is clear that increased tau phosphorylation occurs during neuronal PC12 apoptosis induced by removal of trophic support.

It is interesting that many of the sites that we found to be phosphorylated on apoptotic tau are also hyperphosphorylated on paired helical filament (PHF) tau from Alzheimer's disease brain (Morishima-Kawashima et al., 1995; Hanger et al., 1998). These include sites within the Tau-1 epitope (Szendrei et al., 1993), Thr212 and Ser214 (Thr203 and Ser205 in rat tau) (Illenberger et al., 1998), sites within the PHF1 epitope as well as Ser400 and Thr403 (Ser391 and Thr394 in rat tau) (Otvos et al., 1994), and sites within the 12E8 epitope (Seubert et al., 1995). Further, like tau from apoptotic cells (Davis and Johnson, 1999b), the microtubule-binding capacity of PHF tau is decreased significantly, but restored upon dephosphorylation (Yoshida and Ihara, 1993; Alonso et al., 1994; Lee, 1995). These findings are intriguing as there is increasing evidence that apoptosis or processes with apoptotic features play a role in the neuronal death that occurs in Alzheimer's disease brain. Activation of the protease caspase-3 is considered to be a central factor in apoptosis, and recently the presence of activated caspase-3 was detected in neurons with apoptotic morphology in Alzheimer's disease brain, but not in control brains (Stadelmann et al., 1999). Further, amyloid precursor protein is a substrate of caspase-3, and proteolysis of amyloid precursor protein by caspase-3 increases the rate of amyloid β-peptide formation in cells (Gervais et al., 1999). Based on studies in apoptotic neurons or neuronal-like cells, cell-cycle proteins are often increased during, and are important players in, the apoptotic cascade (Heintz, 1993). Further, staining of Alzheimer's disease brain with antibodies that recognize mitotic phosphoepitopes is increased significantly compared with that of control brains (Vincent et al., 1996), and there is also up-regulation of different cell-cycle proteins in Alzheimer's disease brain (Busser et al., 1998). In addition, the activity of cdc2 was increased in Alzheimer's disease brain and cdc2/cyclin B1 coprecipitated with PHF tau (Vincent et al., 1997). It is interesting that in neuronal PC12 cell apoptosis (Gao and Zelenka, 1995; Davis et al., 1997), and in other models of apoptosis, there is a significant increase in the levels and activity of cdc2 (Gao and Zelenka, 1995). Additionally, there is a significant increase in the association of cdc2 with tau in apoptotic cells. This is of interest because certain protein kinases that phosphorylate tau have been shown to be tightly associated with PHF tau, including casein kinase I (Kuret et al., 1997) and protein kinase A (Jicha et al., 1999). Although it is clear that cdc2 is elevated in Alzheimer's disease brain and in apoptotic cells, and in vitro cdc2 phosphorylates sites on tau that are phosphorylated on PHF tau and tau from apoptotic cells (Ledesma et al., 1992; Vulliet et al., 1992), it remains to be determined whether cdc2 is involved directly or indirectly in modulating tau phosphorylation in situ.

Cdk5 must be associated with its regulatory subunit p35 to be active (Tsai et al., 1994). Cdk5 can also be activated by p25 a truncated version of p35, and the cdk5/p25 may be more active than the cdk5/p35 complex (Patrick et al., 1999). Cdk5 copurifies with microtubules (Ishiguro et al., 1992) (and was originally called tau protein kinase II) and phosphorylates tau at numerous Ser/Thr-Pro sites (Imahori et al., 1998; Michel et al., 1998). In apoptotic cells, cdk5 activity and p25 levels were significantly elevated, concomitant with an increase in tau phosphorylation at specific epitopes. Based on immunohistochemical data, several earlier studies suggested that there was a correlation between apoptosis and increased cdk5 expression (Ahuja et al., 1997; Henchcliffe and Burke, 1997; Zhang et al., 1997). In a recent report, it was demonstrated that transfection of cells with cdk5/p25 resulted in tau hyperphosphorylation and apoptosis, which was not observed when cdk5/p35 was transfected into the cells (Patrick et al., 1999). In that study, the levels of p25 were also found to be increased in Alzheimer's disease brain, and it was suggested that there was deregulation of cdk5 due to increases in the p25 regulatory subunit (Patrick et al., 1999), and this could contribute to the increases in tau phosphorylation in Alzheimer's disease. Furthermore, cdk5 immunoreactivity is apparently increased in pretangle neurons with early stages of neurofibrillary tangles (Pei et al., 1998). Interestingly, we found that the increase in cdk5 activity in apoptotic cells was significantly greater than the increase in cdk5 levels that were observed. A plausible explanation for these findings is that the increase in p25 levels in apoptotic cells is responsible for the greater increase in cdk5 activity. Given the fact that the calcium-activated protease calpain is able to convert p35 to p25 (Kusakawa et al., 2000; Lee et al., 2000), it can be hypothesized that this protease may also be responsible for converting p35 to p25 in apoptotic neuronal PC12 cells. As was observed with cdc2, there was a significant increase in the association between cdk5 and tau in apoptotic cells. In contrast to these findings, a recent report suggested that phosphorylation of tau reduces the association between cdk5/p25 and tau (Sobue et al., 2000). However, in this previous report, the phosphorylation sites that regulated the association between tau and cdk5/p25 were not determined, and the majority of the studies were done in vitro (Sobue et al., 2000). Therefore, it is difficult to make a direct comparison between our present findings and this previous report (Sobue et al., 2000) until further studies are carried out. Indeed, Patrick et al. (1999) presented immunohistochemical data showing colocalization of hyperphosphorylated tau and p25 immunoreactivity in Alzheimer's brain suggesting that the two proteins interact.

In apoptotic neuronal PC12 cells, the levels of GSK3β decreased significantly, but there was no decrease in activity. GSK3β is regulated by both tyrosine and serine phosphorylation. Tyrosine phosphorylation on Tyr216 increases GSK3β activity (Hughes et al., 1993), whereas Ser9 phosphorylation decreases activity (Cross et al., 1995; Welsh et al., 1996). Ser9 on GSK3β is phosphorylated by Akt (Cross et al., 1995; Welsh et al., 1996). Further, Akt activation appears to be critical for neuronal survival (Dudek et al., 1997), and inhibiting Akt may facilitate the apoptotic process (Zhou et al., 1998). Therefore, it can be hypothesized that during apoptosis the Ser9 phosphorylation of GSK3β decreases, resulting in increased activity of the remaining GSK3β, and hence GSK3β activity remains unchanged even though GSK3β levels decreased.

HMW tau is produced from an 8-kb mRNA generated by alternative splicing from the same gene that encodes for tau (Goedert et al., 1989; Couchie et al., 1992). Although little is known about the specific function and regulation of HMW tau, the general functional properties are likely to be very similar to those of tau. In previous studies, we found that during apoptosis HMW tau was phosphorylated within the Tau-1 epitope and microtubule binding was impaired, as it was for tau (Davis and Johnson, 1999b). In this study, we found that during apoptosis HMW tau was phosphorylated at many of the same epitopes as tau; however, there were some differences. For example, phosphorylation of HMW tau was increased at the AT180 epitope in apoptotic cells, but no change at this epitope was observed for tau. Further, by mass spectrometric analysis, three phosphopeptides, Ser122-Ser131, Thr145-Arg163, and Glu164-Arg179 [numbering based on rat HMW tau (Goedert et al., 1992) (see Table 3)], found only in HMW tau were hyperphosphorylated during apoptosis. The functional effects of phosphorylation within exons 4a and/or 6 that are found exclusively in HMW tau are unknown. However, because these epitopes are not in tau, phosphorylation at these epitopes in HMW tau is likely to have unique effects on the function of this protein.

In conclusion, this study demonstrates for the first time that tau is hyperphosphorylated at numerous specific sites during apoptosis, there is a selective increase in cdk5 and cdc2, and there is an increase in the association between these protein kinases and tau during apoptosis. These data suggest that the phosphorylation of tau at specific sites during apoptosis is due in part to the dysregulation of certain protein kinases and the increase in tau phosphorylation likely contributes to the cytoskeletal rearrangements that occur during apoptosis. Further, because many of the sites on tau that are hyperphosphorylated during apoptosis are also phosphorylated in Alzheimer's disease and result in impaired microtubule binding, it is tempting to speculate that apoptotic-like processes may contribute to the hyperphosphorylation of tau in Alzheimer's disease.

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