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Originally published In Press as doi:10.1074/jbc.M706596200 on April 18, 2008

J. Biol. Chem., Vol. 283, Issue 27, 18873-18882, July 4, 2008
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Glycogen Synthase Kinase (GSK) 3β Directly Phosphorylates Serine 212 in the Regulatory Loop and Inhibits Microtubule Affinity-regulating Kinase (MARK) 2*

Thomas Timm1, Kiruthiga Balusamy1, Xiaoyu Li, Jacek Biernat, Eckhard Mandelkow, and Eva-Maria Mandelkow2

From the Max-Planck-Unit for Structural Molecular Biology, Notkestrasse 85, 22607 Hamburg, Germany

Received for publication, August 8, 2007 , and in revised form, April 17, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MARK/Par-1, a kinase family with diverse functions particularly in inducing cell polarity, can phosphorylate microtubule-associated proteins in their repeat domain and cause their detachment from microtubules, and thereby microtubule destabilization. Because of its role in abnormal phosphorylation of the Tau protein in Alzheimer disease, we searched for regulatory kinases. MARK family kinases can be activated by phosphorylation of a conserved threonine (Thr-208 in MARK2), and inactivated by phosphorylation of a serine (Ser-212), both in the activation loop of the catalytic domain. Activation is achieved by the kinases MARKK/TAO1 or LKB1, although the inactivating kinase was unknown. We show here that GSK3β serves the role of the inhibitory kinase. Because GSK3β can also phosphorylate Tau at sites outside the repeat domain, the activation of GSK3β, and concomitant inactivation of MARK can shift the pattern of pathological phosphorylation of Tau protein in Alzheimer disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The kinase microtubule-associated protein/microtubule affinity regulating kinase (MARK)3/Partitioning defective 1 (Par-1) was initially discovered because of its ability to phosphorylate the Tau protein at the KXGS motifs located in the microtubule binding domain (1, 2). Tau can be phosphorylated at various sites by other kinases (e.g. mitogen-activated protein kinases (MAPK), glycogen synthase kinase 3β (GSK3β), cyclin-dependent kinase 5 (Cdk5), cAMP-dependent protein kinase (PKA)), but the phosphorylation at KXGS motifs results in the strongest reduction of Tau binding to microtubules, with the consequence that microtubules, which serve as tracks for axonal transport, are no longer stabilized and fall apart. The detached Tau accumulates in the cytosol and gradually polymerizes into paired helical filaments that bundle up into neurofibrillary tangles, forming one of the two pathological aggregates in Alzheimer disease (for reviews, see Refs. 3, 4).

MARK/Par-1 kinases belong to the AMPK/Snf1 subfamily (adenosine monophosphate-activated protein kinase/sucrose non-fermenting 1) of the Ca2+/calmodulin-dependent kinase II (CaMK) group (5). Homologous genes have been found in eukaryotes ranging from yeast to mammals. In mammals, the MARK family consists of four members (MARK1–4) with a conserved domain organization (Fig. 1A). An N-terminal header (N) precedes the catalytic protein kinase domain (6), which is linked to a putative common docking domain (CD, Ref. 7) as found in kinases of the extracellular signal-regulated kinase (ERK) family (8). This is followed by a ubiquitin-associated domain (UBA), a spacer domain, and a tail domain which includes the kinase-associated domain (KA1; Ref. 9). The UBA and KA1 domains are conserved among the AMPK-related protein kinases and MARKs; their functions are poorly understood but may be related to ubiquitin-dependent signaling or supramolecular folding with an impact on activity (10).

One important function of MARK/Par-1 is the regulation of the microtubule dynamics by altering the affinity of the Tau protein and its functional relatives MAP2c and MAP4 toward the microtubules (11, 12, 13). MARK/Par-1 plays a pivotal role in the establishment and maintanance of cell polarity in different organisms, e.g. asymmetric distribution of P-granules in the Caenorhabditis elegans zygote (14), axis formation in the Drosophila melanogaster embryo (15), asymmetric organization of polarized epithelial cells (16), polarized neurite outgrowth, and neuronal polarity in neuroblastoma cells and hippocampal neurons (17, 18, 19). However, other AMPK-related kinases may also be involved in neuronal polarization as recently shown for the SAD-(synapses of amphids-defective) kinases (20, 21).

The significant role of MARK/Par-1 in polarity development requires a tight regulation of its activity. Some regulatory aspects have been elucidated (reviewed in Refs. 22, 23). The kinases MARKK/TAO1 (thousand and one amino acids) and the serine/threonine kinase LKB1 activate all four MARK isoforms by phosphorylation of a threonine residue (Thr-208 in MARK2) in the activation loop of the catalytic domain (24, 25). This threonine is conserved in many eukaryotic serine/threonine kinases and often regulates their activity. Fig. 1B displays the activation loops of several serine/threonine kinases where phosphorylation sites are highlighted. Another modulator of the activity of MARK is the kinase p21-activated kinase 5 (PAK5): The two kinases interact via their kinase domains so that MARK (but not PAK5) is inhibited (26). The scaffold protein 14-3-3 binds to MARK/Par-1 at two sites, either in the catalytic domain (27) or in the spacer domain after phosphorylation by atypical protein kinase C, leading to relocalization and inactivation of MARK (28, 29, 18).

In MARK, the regulatory threonine in the activation loop is followed by a serine (Ser-212 in MARK2), which is conserved in many kinases. Changing it into a glutamate yielded an inactive kinase, which suggests that MARK phosphorylated at Ser-212 is inactive (24). However the kinase responsible for this regulatory event is a matter of debate. A recent report claimed that GSK3β phosphorylates this site and activates MARK2 (30). By contrast, we report here data showing that GSK3β in fact inhibits MARK by phosphorylating this site. Because both kinases are involved in several interconnected regulatory pathways, this finding has important consequences for interpreting the roles of GSK3β and MARK.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
MARK2 Preparation from Escherichia coli and MARKK Preparation from Sf9 Cells—Recombinant kinases were expressed as fusion proteins with a polyhistidine tag in E. coli (DE3 pLys) or Sf9 cells using the baculovirus system BaculoGoldTM (BD Pharmingen), respectively. Cells were lysed in buffer A (50 mM Tris-HCl pH 8.5, 100 mM NaCl, 50 mM imidazole, 5 mM CHAPS, 1 mM benzamidine, 1 mM β-mercaptoethanol, 1 mM PMSF) with a French Press. The supernatant was loaded onto a Ni-NTATM column (Qiagen). After washing with buffer A, the protein was eluted with a short gradient of 50–500 mM imidazole. The eluted protein was dialyzed against buffer B (50 mM Tris-HCl pH 7.5, 200 mM NaCl, 1 mM benzamidine, 1 mM dithiothreitol, 1 mM PMSF, 50% glycerol) and stored at -20 °C.

GSK3β Preparation from E. coli and Sf9 Cells—Recombinant GSK3β was expressed as a fusion protein with a polyhistidine tag in E. coli (DE3 pLys) and in Sf9 cells using the baculovirus system BaculoGoldTM. Cells were lysed in buffer C (50 mM sodium phosphate, pH 7.9, 100 mM NaCl, 50 mM imidazole, 5 mM CHAPS, 1 mM benzamidine, 1 mM β-mercaptoethanol, 1 mM PMSF) with a French Press. The supernatant was loaded onto a Ni-NTATM column (Qiagen). After washing with buffer C, the protein was eluted with a short gradient of 50–500 mM imidazole. Fractions containing GSK3β were pooled and dialyzed against buffer D (50 mM sodium phosphate, pH 7.9, 100 mM NaCl, 1 mM benzamidine, 1 mM β-mercaptoethanol, 1 mM PMSF). The sample was loaded onto a Mono STM column (Amersham Biosciences/GE-Healthcare) and eluted in a gradient of 100–1000 mM NaCl. The eluted protein was dialyzed against buffer B (see above) and stored at -20 °C.

Kinase Assay—Kinase activities were assayed in buffer E (50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 2 mM EGTA, 0.5 mM PMSF, 0.5 mM dithiothreitol, 0.5 mM benzamidine) for 120 min at 30 °C. Final concentrations of [32P]ATP (3.7 x 107 MBq/mol) and substrate peptide were 100 µM. As substrate, we used a peptide derived from the first repeat of the Tau protein containing Ser-262 in the KXGS motif (TR1-peptide NVKSKIG-STENLK; Ref. 11). Reactions were stopped by the addition of half the volume of 30% (w/v) trichloroacetic acid. After centrifugation, the supernatant was applied to phosphocellulose paper discs, washed with phosphoric acid (0.1 M), dried by air, and radioactivity was measured in a scintillation counter (Tricarb 1900 CA, Packard). Data show averages of three experiments (bars are S.E.). Alternatively, reactions were stopped by the addition of sample loading buffer and subjected to SDS-PAGE. After staining with Coomassie Blue (RothiBlueTM), gels were dried, and radioactivity was detected with a BAS3000 phosphorimaging system (Raytest). Recombinant p38/SAPK was expressed in Sf9 cells, ERK1/2 was prepared from porcine brain (52), Cdc2/cyclin B and Cdk5/p35 were obtained from New England Biolabs, and CKI was a generous gift from L. A. Pinna, University of Padova, IT.

Cell Culture—Sf9 (Spodoptera frugiperda) cells were grown in a 27 °C incubator in monolayer culture with Grace's medium supplemented with 10% FCS and 100 µg/ml penicillin/streptomycin mixture. Confluent monolayers were subcultured by scraping the cells and diluting in the ratio of 1:4 in complete medium. For the expression of proteins in Sf9 cells, the actively growing cells (80% confluence) were infected with recombinant baculovirus. The MOI (multiplicity of infection) was 1–3. The cells were incubated with the virus at 27 °C for 66–72 h.

Neuroblastoma N2a/F113 cells stably expressing htau40 were grown in a medium containing MEM (minimal Earle's medium), 10% FCS, 1% L-glutamine, 1% nonessential amino acid, and 600 µg/ml gentamycin 418. The cells were maintained in a humidified atmosphere containing 5% CO2 at 37 °C. For Western blot analysis, 1.5–3 x 106 cells/well were grown in a 6-well plate for 24 h, and the cells were transiently transfected with appropriate plasmids using Effectene (Qiagen). 24 h after transfection, the cells were differentiated with 2 ml of differentiation medium (MEM, 0.1% FCS, 0.1% nonessential amino acids, 2 µM retinoic acid) for 6 h. The cells were washed once with 3 ml of PBS. Then 1 ml of PBS was added, and the cells were scraped and centrifuged at 14,000 rpm for 10 s. The cell pellet was used immediately or frozen in liquid nitrogen and stored at -20 °C.

CHO wild-type cells and stably transfected with hTau40 were grown in HAM's F12 medium with 10% FCS and incubated in a humidified atmosphere containing 5% CO2 at 37 °C. For stable transfection, we added G418 (600 µg/ml, Invitrogen) to the culture medium. Cells were seeded at 70% confluency in 24-well plates (1.8 cm2) on coverslips and transfected with DOTAP (Roche Applied Science), followed by immunofluorescence analysis at various time points.

PC12 cells (2 x 104 cells) were grown in a 24-well plate (2 cm2) precoated overnight with poly-D-lysine. The cells were incubated at 37 °C with 5% CO2. Cells were grown in a medium containing DMEM (Dulbecco's modified Eagle's medium), 4500 mg/liter glucose, 1% L-glutamine, 10% FCS, 15% HS, and (100 µg/ml) penicillin/streptomycin. The differentiation of PC12 cells was carried out in differentiation medium (DMEM/F12, 1:1) containing 0.1% serum and 100 ng/ml NGF for 24–72 h.

Cortical Neurons—Cortex tissue dissected from E18 rat embryos was digested with 0.1% trypsin for 30 min. Plating medium was then added, and the dissociated cells were gently centrifuged and resuspended in plating medium. The dissociated neurons were plated at a density of 100 neurons/mm2 on a 24-well plate (2 cm2) precoated overnight with poly-D-lysine. After culturing for 4 h, the medium was changed to neuronal culture medium (Neurobasal medium with B-27 and L-glutamine), and the cells were grown for 3–4 days.

Immunofluorescence—Cells were fixed with 3.7% formaldehyde for 15 min at room temperature. Then the cells were washed with PBS three times. Permeabilization was carried out by adding 80% ice-cold methanol and incubated for 5 min at -20 °C. Cells were washed three times with PBS and blocked with 10% goat serum at 37 °C for 45 min. After blocking, the cells were treated with primary antibody (β-actin 1:1000, Sigma; tubulin 1:250, Sigma; 12E8 1:200, gift of P. Seubert, Elan Pharma; ABpT208 1:200, Ref. 24; GSK3β(pS9) SA-310 1:100, Biomol) for 1 h or overnight at 37 °C and then washed three times with PBS. The appropriate secondary antibody was added, and the cells were incubated at 37 °C for 1 h followed by washing three times with PBS. Finally, the coverslips were mounted for microscopy.

Western Blotting—Proteins were separated on SDS gels and electrotransferred to polyvinylidene difluoride membranes by semi-dry blotting (1 mA/cm2, 1 h). The membranes were blocked with 5% nonfat dry milk in 1x Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at room temperature and then treated with the selected primary antibody (ABpS212 1:500, Ref. 24; 12E8 1:1000, Elan Pharmaceuticals; A0024 (K9JA) 1:10000, DAKO; PHF1 1:500; Davies; HA 1:1000, Cell Signaling; β-actin 1:1000, Sigma; GFP 1:2000, Clontech; MARK1/2 SA4632 1:2000, Eurogentec; GSK3βpS9 1:1000, Biomol) in TBST at 37 °C for 1 h. The membranes were washed three times with 1x TBST. The corresponding secondary antibody in TBST was added, and the membranes were incubated at 37 °C for 45 min followed by washing with 1x TBST (three times). The substrate reaction was carried out with ECL detection reagents (GE Healthcare) and visualized using the LAS 3000 system (Raytest). Densitometric analysis was performed with the software AIDA V3.42 (Raytest Isotopenmessgeraete GmbH).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
GSK3β Phosphorylates MARK2 at Serine 212 and Reduces Its Activity in Vitro—MARK isolated from porcine brain is in part phosphorylated at both Thr-208 and Ser-212 (numbering according to MARK2; see Fig. 1) in the activation loop (11). Phosphorylation of the first threonine (Thr-208) by MARKK leads to the activation of all four MARK isoforms (24). This activation is possible only if the subsequent serine (Ser-212) is present: changing Ser-212 to Ala or Glu abolishes the kinase activity completely, despite the activating phosphorylation of Thr-208 (24). This suggests that the phosphorylation of this residue might also lead to inactivation of MARK. Because the Ser-212 in the activation loop is followed by a proline, we tested several proline-directed kinases as candidates. As seen in Fig. 2, different kinases such as the stress- or mitogen-activated protein kinases p38/SAPK, ERK1, ERK2, the cyclin-dependent kinases Cdc2, Cdk5, and GSK3β were tested. Casein kinase I (CKI) was also tested because the serine in the activation loop of MARK resembles the substrate recognition site of CKI. Only GSK3β exhibited an effect on MARK by reducing its activity (Fig. 2A). This lead us to focus on GSK3β, which is also of great interest in the context of AD because it phosphorylates Tau efficiently at Ser/Thr-Pro motifs that are elevated in AD (31, 32). In addition, we could show that MARK and GSK3β co-purified through several steps of purification (11, 33).


Figure 1
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  		FIGURE 1.
Diagram of MARK2. A, kinases of the MARK family comprise five basic domains, shown here for MARK2. The N-terminal header sequence (N) is followed by the catalytic domain. Adjacent are the common docking site (CD) and the ubiquitin-associated domain (UBA). A spacer domain connects it with the tail domain which contains the kinase-associated domain (KA1). MARK can be activated by phosphorylation of the conserved Thr-208 in the activation loop (24, 25). The conserved Ser-212 was found to be phosphorylated in brain (11), although the responsible kinase was unknown. As shown in this report, the site is phosphorylated by GSK3β, causing an inhibition of MARK2. B, activation loops of selected Ser/Thr-kinases. The conserved threonines or serines corresponding to Thr-208 of MARK2 are crucial for activation. Putative phosphorylation sites are underlined, known sites are boxed in gray. The residue equivalent to Ser-212 is conserved, too. Numbers on the right are the GenBankTM accession numbers.

 
To exclude the influence of impurities, especially co-purifying kinases, we used bacterially expressed GSK3β and MARK2 for an in vitro kinase assay. The effect of GSK3β on MARK2 was monitored by its activity toward the peptide TR1, which is derived from the first repeat of Tau containing Ser-262. This peptide is only phosphorylated by MARK2 and not by GSK3β. Fig. 2B shows the influence of GSK3β on MARK2 activity with time. MARK2 was preincubated with GSK3β for different times, then the peptide was added, and the reaction continued for 30 min. The black curve describes the constant activity of MARK2 alone (1 unit = 55 nmol/min/mg). Under the influence of GSK3β, this activity is reduced to one-fourth after 2 h of preincubation. The axis at the bottom depicts the total assay time; note that the two curves meet at 0 min as expected. We next examined the effect of GSK3β on MARK2T208A and MARK2S212A mutants. Similar to the wild-type kinase, MARK2T208A is reduced in its activity by GSK3β (Fig. 2C, compare lane 1 with 2 and 3 with 4). The mutation of the Ser-212 to Ala renders the kinase inactive (see also Ref. 24); hence there is no effect of GSK3β (lanes 5 and 6). The autoradiography in Fig. 2D shows that the inactivation of MARK by GSK3β is accompanied by its phosphorylation. Only the wild-type and the T208A mutant are phosphorylated, but not the S212A mutant (lanes 2 and 4, compare with 6). We conclude that GSK3β phosphorylates MARK2 on Ser-212 to inactivate the kinase. This is further confirmed by a Western blot of equivalent samples shown in Fig. 2E (lanes 1–3). An antibody specific for phosphorylated Ser-212 of MARK2 (AbpS212, rabbit antibody raised against a peptide from the activation loop phosphorylated at Ser-212; see Ref. 24) gives a signal in the cases of wild-type and T208A mutants phosphorylated by GSK3β (lanes 2 and 3), but not with the S212A mutant (lane 4). Lane 1 confirms that the antibody reacts with MARK phosphorylated at Ser-212. This lane shows MARK, a partially purified kinase from porcine brain identified as a mix of MARK1 and 2 (11). Note that the apparent molecular mass is significantly higher than expected for MARK1 or MARK2 (110 kDa versus 88 or 81 kDa), probably due to post-translational modifications, especially phosphorylation. This enzyme is easily degraded by proteases, as indicated by lower Mr fragments (2, 11). Furthermore, GSK3β inhibits MARK2 even when it is activated by MARKK or by the activating mutation T208E (Fig. 2F). The activity of MARK2, activated by MARKK (compare lane 1 with 2) is reduced upon addition of GSK3 (lane 3). As expected, the inactive mutant S212A is neither activated by MARKK (lanes 4 and 5), nor inhibited by GSK3β (lane 6). The active mutant T208E (lane 7) is markedly reduced in its activity by incubation with GSK3β (lane 8). These results clearly indicate that GSK3β inhibits MARK2 by phosphorylating Ser-212. The same results were obtained with GSK3β expressed and purified from Sf9-cells.


Figure 2
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  		FIGURE 2.
GSK3β phosphorylates MARK and reduces its activity in vitro. A, effect of selected kinases on MARK activity. Recombinant MARK2 was incubated with different kinases, and the activity toward the MARK-substrate peptide TR1 measured. Only the kinases MARKK and GSK3β had an influence on MARK activity. While MARKK activated MARK 10-fold (1 unit is 55 nmol/min/mg), GSK3β significantly reduced MARK activity (Student's t test; p < 0.01 for MARKK and GSK3β compared with buffer). Note that the y-axis is not linear for better clarity. B, recombinant MARK2 and GSK3β were preincubated for various times (upper x-axis), followed by incubation with the MARK-substrate peptide TR1 for 30 min (total incubation time on bottom x-axis). As a result, the activity of MARK2 is reduced to 20% within 2 h (gray curve). In contrast, MARK incubated alone does not change its basal activity of 55 nmol/min/mg (1 unit, black line). C and D, MARK2 wild type and mutants were incubated in the presence or absence of GSK3β. GSK3β reduces the activity of MARK2 (compare lanes 1 and 2). The lower basal activity of the T208A mutant, which cannot be activated by MARKK or LKB1, is also reduced by GSK3β (lanes 3 and 4). Mutation of Ser-212 in the activation loop to Ala yields an inactive kinase which is not affected by GSK3β (lanes 5 and 6). Asterisks indicate statistical significance (Student's t test; *, p < 0.05; **, p < 0.01; =, p >> 0.2). The SDS gel shows that equal amounts of MARK proteins were used in this assay. The autoradiograph (D, bottom panel) of the SDS gel shows that GSK3β can phosphorylate MARK2wt and MARK2T208A but not MARK2S212A. Note the increased signal in lanes 2 and 4 (MARK and GSK3β) compared with lanes 1 and 3 where MARK2 is only autophosphorylated (outside the catalytic domain). E, Western blot of GSK3β-treated MARK2wt and mutants with a phospho-Ser-212-specific antibody (AbpS212). Lane 1, partially purified MARK (MARKp110) from porcine brain, which was found to be phosphorylated on residues corresponding to both Thr-208 and Ser-212 in MARK2 (11). Note that this protein shows a higher molecular mass of ~110 kDa than MARK2 because of post-translational modification. It is also prone to proteolytic degradation, which is easily visible in this Western blot (2, 11). Samples in lanes 2-4 are aliquots of the samples shown in C lanes 2, 4, and 6. As expected, the S212A mutant is not detected by this antibody whereas the wild type and the T208A mutant give clear signals. F, GSK3β inhibits MARK2 (independently of its activation by MARKK) or constitutively active MARK2T208E. MARK is strongly activated by MARKK (lane 2), but this activation is counteracted by co-incubation with GSK3β (lane 3). As expected, there is no effect on the S212A mutant of MARK2, which has no basal activity and cannot be activated by MARKK (lanes 4–6). Mutating Thr-208 in the activation loop to glutamic acid increases the activity of MARK significantly (lane 7) but not as strongly as by phosphorylation. However, GSK3β is able to reduce the activity of this mutant, too (lane 8). All experiments were performed in triplicate, graphs show mean values with standard errors.

 
MARK2 Wild Type and Mutants Phosphorylate Tau Differentially—The activity of MARK2wt and mutants was checked in cellular conditions by transfecting the kinases into N2a/F113 cells stably expressing htau40 (human Tau, longest isoform in CNS). After 24 h, the cells were differentiated with retinoic acid for 6 h. The results were monitored on Western blots with the pan Tau antibody K9JA and the pS262-specific Tau antibody 12E8. As expected, wild-type MARK2 exhibited high activity toward Tau at Ser-262, compared with untransfected cells (Fig. 3A, lanes 1 and 2). The non-activable MARK2T208A mutant had only low activity (lane 3), and the S212A mutant as well as the T208A/S212A mutant resulted in no 12E8 reactivity (lanes 4 and 5). The level of MARK2wt and mutants and the level of Tau are similar in all transfections (shown by the loading control of β-actin staining). These data on cells correlate closely with the in vitro data, arguing that Ser-212 must be present for the activity of MARK2 (i.e. it cannot be changed by mutation or phosphorylation), and that the phosphorylation of Thr-208 is needed for activation.


Figure 3
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  		FIGURE 3.
Co-expression of active GSK3βS9A with MARK2wt or active MARK2T208E reduces the phosphorylation of Tau at Ser-262. A, N2a/F113 cells stably expressing htau40 were transiently transfected with HA-tagged MARK2wt, MARK2T208A, MARK2S212A, and MARK2T208A/S212A. 24 h after transfection, the cells were differentiated with retinoic acid for 6 h. A Western blot of the lysates was probed with antibodies against Tau phosphorylated at Ser-262, total Tau, MARK2, and actin as a marker for loading of similar protein amounts. The level of Ser-262-phosphorylated Tau is strongly increased in cells transfected with MARK2wt (lane 2) compared with non-transfected cells (lane 1). In cells with the non-activable T208A mutant, 12E8-staining is only slightly elevated (lane 3). Cells expressing the inactive S212A or T208A/S212A mutants show no 12E8 immunoreactivity. B, N2a/F113 cells stably expressing htau40 were transiently transfected with HA-MARK2wt, active ECFP-MARK2T208E alone and in the presence of active ECFP-GSK3βS9A. 24 h after transfection, the cells were differentiated with retinoic acid for 6 h. On a Western blot, the lysates were probed with antibodies against Tau phosphorylated at Ser-262, total Tau, MARK2, GSK3β, and actin as a marker for equal protein amounts. Expression of either MARK2wt or active MARK2T208E increases the phosphorylation of Tau at Ser-262 (lanes 2 and 3, compare non-transfected cells in lane 1). In contrast, co-expression of MARK2wt or active MARK2T208E with active GSK3S9A results in the loss of 12E8 immunoreactivity (lanes 4 and 5). Note that the level of active MARK2T208E is 3-fold lower during co-transfection with GSK3β (lane 5) than without GSK3β (lane 3), but the reduction of Tau phosphorylation at Ser-262 is more than 10-fold as determined by densitometric analysis. Therefore, this is a result of inactivation (as shown for the wild-type MARK in lanes 2 and 4), not of the reduced protein level.

 
Co-expression of Active GSK3βS9A with MARK2wt or Active MARK2T208E in Cells Reduces the Phosphorylation of Tau at Ser-262—To investigate the effect of GSK3β on MARK in cells, we transfected N2a/F113 cells with either MARK2wt or the constitutively active mutant MARK2T208E alone or together with constitutively active GSK3βS9A. After 24 h of transfection, the cells were differentiated with retinoic acid for 6 h. The activity of MARK was monitored by the phosphorylation of Tau at Ser-262 using the 12E8 antibody in Western blots. Comparable amounts of kinases in single and double transfections are depicted by HA antibody for MARK2wt and with GFP antibody for MARK2T208E and GSK3βS9A, which are both fused to ECFP. As seen in Fig. 3B, the expression of MARK2wt and constitutively active MARK2T208E increased the phosphorylation of Tau at Ser-262 compared with untransfected cells (compare lanes 2 and 3 with lane 1). But upon co-transfection with active GSK3βS9A, this phosphorylation strongly decreased (lanes 4 and 5). Although we see a reduction in the expression level of MARK2T208E when co-transfected with active GSK3βS9A (lane 5) the phosphorylation of Tau at Ser-262 is reduced to a much greater extent. These results demonstrate that GSK3β inhibits MARK2 not only in vitro but also in cells.

Co-expression of GSK3β with MARK2 Stabilizes the Microtubule Network—In CHOwt cells, the activity of MARK2 leads to drastic morphological changes (13). Because CHO cells do not have Tau, MARK phosphorylates the related MAP4-like protein at its KXGS sites in the repeat domain and abolishes its microtubule-stabilizing function. As a result, the microtubules breakdown, the cells shrink, and finally die. CHO cells were transfected with constitutively active MARK2 (EYFP-MARK2T208E). After 16 h, cells were fixed and stained for microtubules with antibody YL1/2. Untransfected cells have an extended shape with a clear microtubule network. As expected, transfection of active EYFP-MARK2T208E alone leads to loss of microtubules and shrinkage in 55% of the cells (Fig. 4, upper row, arrows) In contrast, co-expression of both active MARK2 and active GSK3β retains the microtubule network in 60% of the cells (Fig. 4, lower row) indicating that MARK2 is inhibited by GSK3β in these cells.

GSK3β Inhibits MARK in PC12 Cells—In contrast to the detrimental effect of MARK on microtubules in CHO cells, overexpression in PC12 cells results in neurite outgrowth. Active MARK (ECFP-MARK2T208E) or active GSK3β (mRFP-GSK3βS9A) or both were transfected into PC12 cells and the cells were differentiated with NGF for 48 h. In transfected cells, ECFP-MARK2T208E is located at the plasma membrane, similar to the endogenous protein (24). These cells exhibit strong 12E8 staining and the formation of neurite outgrowth after addition of NGF (Fig. 5, upper row) showing that the exogenous MARK is not toxic in these cells. In contrast, transfection of active mRFP-GSK3βS9A leads to a lower level of phosphorylated Ser-262-Tau than in untransfected cells, and after NGF treatment no neurite outgrowth is observed (middle row), indicating that GSK3β inhibits the endogenous MARK. Moreover, upon co-transfection of active GSK3βS9A and active MARK2T208E, the activity of MARK2 is inhibited, and as a result the cells do not form neurites (lower row).

Endogenous Inactive GSK3β and Active MARK Co-localize in the Neurite Tips of PC12 Cells and in the Growth Cones of Rat Cortical Neurons—To further investigate the functional relationship between GSK3β and MARK, we examined the endogenous activities of both kinases in differentiated PC12 cells and in stage three cortical neurons (34). To monitor the activation state of MARK, we probed the cells with an antibody against phosphothreonine 208 in the activation loop of MARK, labeling the activated enzyme (AbpT208, Ref. 24). Furthermore we probed the cells with an antibody against phosphoserine 262 of Tau as an indicator for MARK activity. Both signals co-localize strongly in the tips of the cell processes and at the membranes (Figs. 6 and 7, D, E, and F). At the same time, inactive GSK3β (detected by an antibody against phosphoserine 9) is also predominantly found at the tips, co-localizing with active MARK (Figs. 6 and 7, A, B, and C). The tips also show a high level of co-localization of actin and active MARK, consistent with our previous observations (24) (Figs. 6 and 7, G, H, and I). These results indicate a zone of a highly dynamic actin and microtubule cytoskeletons where active MARK and inactive GSK3β are involved in the growth of an axon.


Figure 4
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  		FIGURE 4.
Co-expression of GSK3β with MARK2 stabilizes the microtubule network. CHO cells were transfected with either wild-type MARK (EYFP-MARK2) or co-transfected with wild-type MARK (EYFP-MARK2) and active GSK3β (mRFP-GSK3βS9A). In cells only transfected with MARK, the expression leads to the loss of the microtubule network. The cells round up and become smaller (upper row, arrows). In contrast, co-expression of EYFP-MARK2 and active mRFP-GSK3βS9A retains the microtubule network, indicating that GSK3β inhibits MARK2 in cells (lower row, arrows). The graphs on the right show the classification of cells transfected with MARK alone (upper graph) or co-transfected with MARK and GSK3β (lower graph). Around 55% of the cells transfected with MARK alone lost their microtubule (MT) network, became round and small, whereas only 10% of the cells co-transfected with GSK3β exhibited that phenotype (white columns). On the other hand, only 15% of the MARK-transfected cells appeared normal; this was increased to 60% upon co-transfection with GSK3β (dark gray columns). The number of cells with reduced size but normal cytoskeleton did not change (30%; light gray columns). (Three independent experiments, 132 cells per series were counted.)

 


Figure 5
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  		FIGURE 5.
GSK3β inhibits MARK2 in PC12 cells. PC12 cells were transiently transfected with either active mutants of MARK (ECFP-MARKT208E) or GSK3β (mRFP-GSK3βS9A) or both. They were then differentiated with NGF for 48 h. After fixation, the cells were stained with phospho-Ser-262 Tau antibody (12E8). Cells transfected with active MARK alone show strong 12E8 staining and multiple processes (upper row, see arrows), whereas in cells transfected with active GSK3β the 12E8 staining is strongly reduced (middle row, arrows). In cells overexpressing both active MARK2T208E and active GSK3βS9A, the phosphorylation of Ser-262 in Tau is comparable with untransfected cells, and the transfected cells show no neurite outgrowth (lower row, arrows), indicating that MARK2 activity is inhibited. Upper left, diagram of the effect of MARK2 on microtubule dynamics and neurite outgrowth, which is inhibited by GSK3β.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The microtubule-associated protein Tau stabilizes microtubules in axons as tracks for axonal transport. In degenerating neurons, Tau becomes missorted, hyperphosphorylated, and detaches from microtubules, which leads to the breakdown of the microtubules and the collapse of energy supply and transport in the axons of neurons. In addition, the hyperphosphorylated Tau aggregates into paired helical filaments (PHFs), which deposit as neurofibrillary tangles (NFTs) in the neurons. The binding of Tau protein to microtubules is primarily regulated by phosphorylation of the KXGS motifs located in the repeats of the microtubule binding domain of Tau. MARK has been identified as a kinase family phosphorylating the structural microtubule-associated proteins (MAPs) Tau, MAP2, and MAP4 at the KXGS motifs, thereby reducing their affinity to microtubules and disrupting microtubule stability (11). This type of phosphorylation is enhanced in the early stages of Alzheimer disease (35). Furthermore, it was proposed that in a Drosophila model the activation of MARK primes Tau for the phosphorylation by other kinases like GSK3β and Cdk5, which in turn triggers the aggregation of Tau into PHFs, a hallmark of AD neurofibrillary pathology (36).


Figure 6
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  		FIGURE 6.
Colocalization of endogenous inactive GSK3β and active MARK in the neurite tips of differentiated PC12 cells. PC12 cells were differentiated with NGF for 72 h, fixed, and stained with antibodies against inactive GSK3β (phosphorylated at Ser-9), active MARK (phosphorylated at Thr-208), Tau phosphorylated at Ser-262 and actin. Colocalization of active MARK (A) and inactive GSK3β (B) is seen predominantly at the tips of neurites (arrows) and the cell periphery. Magnified images of the tips labeled by arrows are shown as insets. MARK is highly active at the tips of the neurites, seen by co-staining of active MARK (D) and phospho-Ser-262 Tau (E) (merge in F). Magnified images of the tips labeled by arrows are shown as insets. Note the strong overlap of active MARK and phospho-Ser-262 Tau are at the very tips (F1–F3). The staining for active MARK (G) overlaps with the actin signal (H) at the tips of the neurites (I, magnified images as insets).

 
MARK and its homologues Par-1 are involved in generating and maintaining cell polarity during development in Drosophila and C. elegans (37). In mammalian cells MARK/Par-1 is important for establishment of neuronal polarity in neuroblastoma cells and hippocampal neurons (17–19). Other important functions are linked to cell cycle regulation (38), Wnt signaling (39), and exocytosis (10). As MARK/Par-1 is involved in the regulation of many essential pathways, it is important to understand the control of its activity.

MARK/Par-1 kinases are activated by MARKK/TAO-1 or LKB1 by phosphorylation of a conserved threonine in the activation loop, Thr-208 in MARK2 (see Fig. 1B; Refs. 24, 40). Unlike other kinases, a fraction of MARK2 purified from brain is additionally found to be phosphorylated at a second site Ser-212, which is adjacent to the Thr-208 (11). Site-directed mutagenesis of this residue to the phosphoserine-mimicking Glu suggested that phosphorylation might be inhibitory (24). X-ray analysis of the catalytic domains of MARK1 and MARK2 (41, 7) confirm the important function of this particular serine in stabilizing the activation loop. Fig. 8 displays the structural model of MARK2. It shows that Ser-212 forms hydrogen bonds to the catalytic Asp (Asp-175) and a nearby Lys (Lys-177), as proposed for PKA (Fig. 8B, residues involved in PKA are Thr-201, Asp-166, and Lys-168; Ref. 42). The change of Ser-212 to Ala or Glu disrupts this stabilizing interaction, and the same is achieved when Ser-212 is phosphorylated (Fig. 8C). The data presented here demonstrate that GSK3β is able to phosphorylate MARK2 at Ser-212 in vitro and that this indeed results in an inactive kinase, irrespective of the phosphorylation status of the activating residue Thr-208. Because the activation loops of the four MARK isoforms are similar, these results hold for all isoforms.

To confirm the inhibition of MARK by GSK3β, we expressed combinations of these kinases in several cell lines. Wild type and constitutively active mutants of MARK and GSK3β were expressed in CHO, N2a/F113, and PC12 cells. In all cases, the co-expression of GSK3β with MARK resulted in the inhibition of MARK activity and the preservation of the microtubule network.

Many substrates of GSK3β have to be primed by pre-phosphorylation at a site 4 residues downstream of the phosphorylation site. The classic case is glycogen synthase (primed by casein kinase II), others are Tau (primed by Cdk5/p35), β-catenin (by casein kinase I), or CREB (cAMP-response element-binding protein, by PKA) (for reviews, see Refs. 43, 44). In this respect, MARKs are not typical substrates for GSK3β because no priming phosphorylation is necessary. The activating phosphorylation on Thr-208 by MARKK is N-terminal to the GSK3β target site and has no influence on the phosphorylation of Ser-212 by GSK3β.

Our conclusions are in contrast to those of Kosuga et al. (30) who claimed that MARK could be activated by phosphorylation of Ser-212 by GSK3β. Our explanation of the discrepancy is that these authors have only tested point mutations of MARK in cells without checking their actual kinase activities. For instance, they observed that overexpression of the MARKS212A mutant had no effect on the phosphorylation of Tau at Ser-262 and concluded that Ser-212 of MARK was the residue responsible for activation by phosphorylation, without noticing that the S212A mutant is not active as such because it cannot stabilize the activation loop by H-bonds (see above).

However, our data are in line with results from other investigators: Jiang et al. (45) showed that GSK3β activity is differentially distributed in the axon versus the dendrites. A constitutively active GSK3β mutant inhibited axon formation, whereas reduction of GSK3β activity by pharmacological or peptide inhibitors or siRNAs resulted in multiple axons. A pool of inactive S9-phosphorylated GSK3β is localized to the tips of axons where the highest dynamics of microtubules is needed, and therefore MARK is expected to be active. We confirmed this by showing that endogenous MARK is active, and endogenous GSK3β is inactive at the tips of neurites in NGF-differentiated PC12 cells and in the growth cones of differentiating rat cortical neurons. Activation of GSK3β at the leading edge of neuronal growth cones by semaphorin 3A inhibits growth cone advance and is followed by growth cone collapse (46), consistent with MARK inhibition. Furthermore, active GSK3β impairs neuronal polarization by phosphorylating and inhibiting CRMP-2 (collapsing response mediator protein 2), which binds to tubulin dimers and promotes MT assembly when unphosphorylated (47). Conversely, in neurospheres, the inhibition of GSK3β results in increased differentiation of neuronal precursors into dopaminergic neurons (48). Partial or complete knock-down of GSK3β by shRNA in cultured neurons and tissue slices (49) showed that inactivation of GSK3β at neurite tips leads to axon elongation, whereas reduction of GSK3β throughout the neuron causes axon branching, and strong suppression of GSK3β results in termination of axon growth.


Figure 7
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  		FIGURE 7.
Colocalization of endogenous inactive GSK3β and active MARK in the growth cones of cortical neurons. Active MARK (A) as well as inactive GSK3β (B) are concentrated in the growth cones of stage three neurons (arrow and arrowhead, magnification of arrow-labeled growth cone below). The activity of MARK is highest at the growth cone as indicated by phosphorylation of Tau at Ser-262 (D–F; see arrow, magnification below). The highest activity of MARK also coincides with strong actin staining in the growth cone (G, H, and I; see arrow, magnification below), the site of the highest microtubule and actin dynamics.

 


Figure 8
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  		FIGURE 8.
Model of conformational changes in the catalytic domain of MARK2. The model is based on our x-ray structure of MARK2 (Ref. 7, PDB-ID 1Y8G). The catalytic loop (gray) is located deep in the cleft between the small and the large lobes of the kinase domain (blue). The UBA domain (red) in the back is linked to the large lobe by a stretch that contains the CD domain (green). A, in the inactive state the catalytic loop (yellow; partly disordered and modeled here as dotted line) is folded back into the cleft and resides underneath the ATP binding loop (P-loop). Both Thr-208 and Ser-212 point to the right and are accessible for kinases, e.g. Thr-208 to MARKK and Ser-212 to GSK3β. B, in the activated state, phosphorylation of the Thr-208 (indicated by red sphere) results in a reorientation of the activation loop. It becomes folded to the right and stabilized by interactions of the pT208 to residues in helix C. Ser-212 is now fixed by hydrogen bonds toward Lys-177 and Asp-175 in the catalytic loop (gray). The catalytic pocket opens up and allows entry of ATP (violet) and substrate (Tau peptide, cyan), which aligns with the catalytic (gray) and the activation loop (yellow). C, phosphorylation of Ser-212, or mutation to Glu or Ala disrupts the stabilizing interaction between the activation loop (yellow) and the catalytic loop (gray) resulting in an inactive kinase. Furthermore it is likely that the phosphate of pS212 (indicated by red sphere) will interfere with the correct alignment of the substrate within the catalytic cleft.

 
Luo and co-workers (18) have shown that reduction of MARK2 activity by siRNA or by the Par3-Par6-aPKC complex leads to a loss of polarity and multiple axons in hippocampal neurons. Conversely, the elevation of MARK activity promotes axons outgrowth, and the same holds for the related SAD kinase (17, 20). This supports the model that for neurite outgrowth MARK has to be largely inactive in the shafts of axons where the cells need stable microtubules. On the other hand, MARK has to be very active in the growth cone where highly dynamic microtubules are required for growth and retraction of the axon tip. Another recent report revealed an additional pathway signaling from LKB1 via SAD kinases to pS262-Tau in cortical neurons (21). To date, it is unclear if GSK3β is also acting on SAD in an inhibitory manner, but it is interesting to note that in Xenopus the kinase LKB1 regulates Wnt signaling by inhibition of GSK3β (50). Kojima et al. (51) showed evidence that phosphorylation of Tau at Ser-262 by MARK2 not only causes suppression of tubulin polymerization but also proteasome-mediated degradation of Tau. This points to a higher degree of complexity in the signaling pathways, which has yet to be elucidated.


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

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Tel.: 49-40-8998-2810; E-mail: mandelkow{at}mpasmb.desy.de.

3 The abbreviations used are: MARK, MAP/microtubule affinity regulating kinase; AD, Alzheimer disease; GSK3β, glycogen synthase kinase 3β; MAP, microtubule-associated protein; MARKK, MARK-activating kinase; Par-1, partitioning-defective mutant 1 kinase; PKA, cAMP-dependent kinase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; FCS, fetal calf serum; PBS, phosphate-buffered saline; AD, Alzheimer Disease; HA, hemagglutinin; GFP, green fluorescent protein; wt, wild type; UBA, ubiquitin-associated domain; NTA, nitrilotriacetic acid. Back


   ACKNOWLEDGMENTS
 
We thank Dr. P. Davies (Albert Einstein College, Bronx, NY) and Dr. P. Seubert (Elan Pharmaceuticals, South San Francisco, CA) for providing the antibodies PHF-1 and 12E8. We thank Edda Thies and Kerstin Skokann for help with cell culture procedures.



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 DISCUSSION
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