©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Microtubule-associated Protein (MAP2) Kinase Restores Microtubule Motility in Embryonic Brain (*)

(Received for publication, October 6, 1994; and in revised form, January 11, 1995 )

Luis A. López (§) Michael P. Sheetz

From the  Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Motility driven by the microtubule motors, kinesin and cytoplasmic dynein, is inhibited by MAP2 (López, L. A., and Sheetz, M. P.(1993) Cell Motil. Cytoskeleton 24, 1-16). The MAP2 inhibition is reversed by a kinase that is co-purified with chicken embryonic MAP2, completely releasing MAP2 from the microtubules. We have identified this activity with a kinase, embryonic MAP2 kinase (M(r) = 100,000), which phosphorylates MAP2 at serine amino acid residues. This kinase is c-AMP independent and inhibited by potassium fluoride and glycerol 2-phosphate. Only the phosphorylation produced by embryonic MAP2 kinase can change the affinity of MAP2 by microtubules. Bovine MAP2 kinase, Cdc2 kinase, mitogenic activated protein kinase, and the NIMA kinase are able to phosphorylate MAP2 but do not change the affinity for microtubules. In vivo, embryonic MAP2 kinase could play a major role in the regulation of motility and positioning of membranous organelles within the cells even at substoichiometric levels.


INTRODUCTION

Brain microtubule (MT)^1(^1) preparations normally contain a number of microtubule-associated proteins (MAPs) (1-5). MAP2 is a fibrillar protein found in neuronal cells on dendrite microtubules. It is phosphorylated in vivo and in vitro by several kinases (6, 7, 8, 9, 10) . Two kinases are found in complex with MAP2 (11, 12) . Several reports have indicated that phosphorylation of MAP2 will change its affinity for MTs (6, 8, 13) .

The possible functions of MAP2 include cross-linking between MTs and organelles (14, 15) , MTs and actin filaments (16) , and between MTs themselves (17) .

The ability of MAP2 to inhibit cytoplasmic transport has been reported in a recent paper (18) . We showed that the presence of MAP2 on microtubules restricts the gliding of MTs and MT-dependent organelle transport. It is reported in this paper that when microtubules are decorated with embryonic chicken MAP2, the presence of ATP restores the transport of MTs by kinesin and cytoplasmic dynein. We attributed this ATP effect to a new serine kinase (embryonic MAP2 kinase, M(r) = 100,000) which co-purifies with MAP2 from embryonic chicken brain causing release of MAP2 from MT and restoring motility.

If MAP2 binding to MTs has a role similar to the troponin-tropomyosin complex in muscle in inhibiting motility, then phosphorylation may be the modulator. The cell can use this mechanism to regulate position and transport of organelles in the cytoplasm.


EXPERIMENTAL PROCEDURES

Materials

Taxol was provided by Dr. Nancita Lomax at the National Cancer Institute. Reagents for polyacrylamide gel electrophoresis and the protein assay were purchased from Bio-Rad. ATPS was provided by Boehringer Mannheim, and all other reagents were purchased from Sigma.

SDS-PAGE and Protein Assay

Discontinuous SDS-PAGE was performed as described by Laemmli (19) . The protein concentrations of the samples were determined by assay with the Bio-Rad protein assay kit (Bio-Rad) using bovine serum albumin as a standard. The quantitation of the molar ratio of MAP2 to microtubules was performed as shown in a previous paper (18) .

Microtubule Protein Preparation

Tubulin (PC-tubulin) was purified from bovine brain tubulin using a phosphocellulose column (20). MAP2 purification was performed by two methods: separation by sucrose gradient sedimentation or heat treatment. In the case of sucrose gradient sedimentation, 30 g of 12-day-old chicken embryo brain or 100 g of bovine brain was homogenized in homogenization buffer (PMEE` buffer (35 mM Pipes, 5 mM MgSO(4), 1 mM EGTA, 0.5 mM EDTA, pH 7.4) with protease inhibitors) (18) . MAPs were prepared using the Taxol, salt-dependent procedure (21) . MAP2 was separated from other MAPs by differential sedimentation, applying the crude extract to a 5-20% sucrose gradient centrifuged at 37,000 rpm for 16 h at 4 °C in an SW 40 rotor (Beckman). To purify bovine MAP2 by heat treatment, we used the procedure described previously (18, 22) .

MAP2-Microtubule Interaction

MAP2bulletMTs were prepared in two ways although most of the studies utilized the first. (a) MAP2 was added to preformed microtubules (30 µl of MAP2 fraction (0.04-0.15 mg/ml) in 200-µl Airfuge tubes were mixed with Taxol-stabilized PC-MTs (final concentration of 0.3 mg/ml tubulin), and the mixture was incubated for 15 min at 20 °C). (b) MAP2 was added to tubulin prior to polymerization (MAP2 (0.15 mg/ml) was incubated with PC-tubulin (1.2 mg/ml), 1 mM GTP, and 20 µM Taxol for 30 min at 37 °C). Both preparations of microtubules, a and b, were centrifuged through 25% sucrose cushions (100,000 times g for 5 min) and resuspended with PMEE` buffer, 1 mM GTP, and 20 µM Taxol (PGT buffer). For analysis, the pellets were resuspended with sample buffer (19) , and proteins were identified by SDS-PAGE. For protein phosphorylation assays, the samples were incubated with [P]ATP (0.33 µCi/nmol) containing 0.5 mM ATP for 15 min at 20 °C. The microtubules were pelleted, and supernatant and pellets were incubated with sample buffer (19) . The samples were electrophoresed in an SDS-PAGE followed by autoradiography.

Effect of Nucleotides and Kinase Preparations on the Association of Bovine MAP2 with Microtubules

Bovine MAP2 purified by heat treatment was incorporated into PC-microtubules (see above), centrifuged through a 25% sucrose cushion (100,000 times g for 5 min), and resuspended with PGT buffer. A fraction of the suspension was left untreated or incubated with 1 mM ATP in the presence or absence of native chicken MAP2 (0.1 mg/ml) or the decided protein kinase. After 20 min of incubation at 22 °C, suspensions were pelleted across a 25% sucrose cushion by centrifugation (55,000 times g for 5 min at 22 °C in an Airfuge), and pellets were resuspended in PGT buffer. The samples were analyzed by electrophoresis to quantify the amount of MAPs bound and tested in motility assays.

Motor Protein Preparation and Motility Assay

Kinesin and cytoplasmic dynein were purified from embryonic chicken brain by microtubule affinity and velocity sedimentation through a sucrose gradient (23, 24) . Microtubule motility assay was performed as published previously (18) .

Protein Kinase Preparations

Mitogenic activated protein kinase (p44 ) was provided by UBI, Cdc2 kinase was purified from activated Xenopus oocytes with P conjugated beads (25) . Purified recombinant NIMA enzyme (26) was a gift of Dr. A. R. Means (Duke University Medical Center).

Kinase Assay in SDS-PAGE

Kinase assay in SDS-polyacrylamide gels was performed as published previously (27, 28, 29) with some modifications. Seven percent polyacrylamide minigels were cast with or without 0.09 mg/ml purified bovine MAP2 added to the gel mixture prior to polymerization (29) . After electrophoresis, the SDS was removed from the gel, proteins were denatured and renatured (29) , and the kinase assay was carried out for 60 min at 20 °C in 50 mM ATP, 50 µCi of [P]ATP (3000 Ci/mol, Amersham) in PMEE` buffer. The gels were washed, dried, and exposed to X-OMAT AR film (Eastman Kodak Co.).

ATP-Agarose Chromatography

Four milliliters of embryonic MAP2 fraction (0.1 mg/ml) was loaded on a 1-ml ATP-agarose column (Sigma) equilibrated with PMEE` buffer. After 15 min of incubation at 22 °C the column was washed with 10 ml of PMEE` buffer, dried by centrifugation, and loaded with 400 µl of 10 mM ATP in PMEE` buffer. After 15 min of incubation, the column was centrifuged, and the filtrate was collected and analyzed by SDS-PAGE and for kinase activity.

Phosphoamino Acid Analysis

Phosphoamino acids were identified on a thin layer chromatography plate (30) , with some modifications. The P-labeled embryonic MAP2 was excised from gels and incubated with 50 µg/ml trypsin in 50 mM NH(4)HCO(3) at 37 °C for 18 h. The incubation medium was dried, and the digested protein was treated with 6 N HCl at 100 °C for 90 min. The hydrolysate was dried and solubilized with distilled water containing phosphoamino acid standards and xylene cyanol FF, Orange G, and acid fuschin to standardize the migration distance.

The samples were spotted onto a plastic backed cellulose thin layer chromatography plate (E. M. Laboratories) and electrophoresed, 75 min at 800 V, in a Varsol-cooled TLE apparatus (Savant). The phosphoamino acid standards were visualized with ninhydrin, and autoradiography was used for detection of P-labeled phosphoamino acids from MAP2.

Protein Phosphorylation Assay

After being incubated with [P]ATP, samples were electrophoresed on 5% and 7.5% gels, stained, dried, and exposed to X-OMAT AR film. Quantification of the amount of phosphorylation was determined by cutting out the MAP2 region of the gel and solubilizing it with 10% H(2)O(2) prior to counting with scintillant.

Immunofluorescent Staining of Microtubules

Staining was performed according to Steuer et al.(31) . Microtubules were fixed in 2.0% paraformaldehyde, 0.1% glutaraldehyde in PGT buffer for 10 min at 20 °C, centrifuged onto a coverslip, and postfixed in -20 °C methanol. Samples were incubated for 30 min with 50:50 mixture of a 1:500 dilution of anti-tubulin monoclonal YOL 1/34 antibody (32) and a 1:100 dilution of anti-MAP2 monoclonal AP 14 antibody (33) diluted in phosphate-buffered saline. After three rinses in phosphate-buffered saline, the samples were incubated for 30 min with a 50:50 mixture of a 1:200 dilution of fluorescein isothiocyanate-labeled goat anti-mouse IgG and a 1:200 dilution of rhodamine-labeled goat anti-rat IgG in 1% bovine serum albumin in phosphate-buffered saline. Coverslips were mounted in 50% (v/v) glycerol in phosphate-buffered saline containing 1% N-propyl gallate and observed under a Zeiss Axioplan microscope. Micrographs were taken with TMAX 400 ASA black and white print film (Kodak).


RESULTS

Release of MAP2 and Activation of Motility by an Embryonic Brain MAP2 Fraction

In a recent paper (18) , it was demonstrated that microtubules decorated with bovine MAP2 are unable to be transported by kinesin or cytoplasmic dynein. Now the motility experiments were performed using microtubules decorated with embryonic chicken MAP2. In this new condition, MAP2 was obtained from a sucrose gradient (native form). Contrary to previous experiments (18) , we observed that the native form of MAP2 had no effect on MT gliding when kinesin or dynein were used as motors and ATP was used as a source of energy, but it inhibited gliding when GTP was used in a kinesin assay (Table I). Using immunofluorescence analyses, we observed that native embryonic chicken MAP2 was released from MTs by the addition of ATP (Fig. 1). It is possible that the detachment would be due to an ATPase activity in MAP2 (34) or that there exists a kinase that may phosphorylate and release MAP2. We support the second hypothesis since embryonic chicken MAP2 purified by boiling was as inhibitory as bovine MAP2 in the presence of ATP (Table I). Moreover, we incubated purified bovine MAP2bulletMTs with native embryonic chick MAP2 preparations and found that ATP released 80-90% of the bovine MAP2 from the MTs (Fig. 2A) and activated MT gliding to control levels (Fig. 2B). A factor present in the embryonic fraction, likely a kinase, releases completely bovine MAP2.


Figure 1: Effect of ATP on binding of embryonic chick brain MAP2 to microtubules. The figure shows the double immunofluorescence of MAP2-microtubules with anti-tubulin YOL 1/34 antibody (A and B) and anti-MAP2 AP 14 antibody (C and D). In A and C, there are chick brain MAP2-microtubules only. In B and D, the MAP2-microtubules were incubated with 1 mM ATP. Bar, 10 µm.




Figure 2: Incubation of microtubules with an embryonic chick fraction and movement on glass coverslip. Twenty µl of bovine MAP2-microtubule (0.3 mg/ml) (lane 5) were incubated at 20 °C for 15 min, with 50 µl (lanes 1 and 2), 10 µl (lane 3), and 0 µl (lane 4), respectively, of embryonic chicken MAP2 fraction (0.1 µl/ml) with 1 mM ATP. PC-microtubules (0.3 mg/ml) were used in lane 5). (In lane 1, embryonic chicken MAP2 fraction was boiled before use.) The microtubules were analyzed for protein composition (A) and for motility (B). A, 2.5 µg of samples were run in 7.5% SDS-PAGE and stained with Coomassie Blue. Molecular weight markers are indicated at the right of the figure. MAP2bulletMT are indicated at the left of the figure. B, the graphic shows the number of microtubules moving across the surfaces coated with cytoplasmic dynein (40 µg/ml). The bars represent the average number of microtubules found gliding in a field during 1-min intervals ± S.D.



Characterization of Releasing Factor as a MAP2 Kinase

It was analyzed whether the release of MAP2 was correlated with kinase activity and, if so, with which of them. Nonhydrolyzable analogs of ATP and other nucleotides were tested, and it was found that inhibition of binding of MAP2 to MTs required ATP hydrolysis (see Table II). Half-maximal release used 0.02-0.05 mM ATP (Fig. 3). If kinase activity was assayed using [P]ATP, MAP2 was the major species phosphorylated in our preparations and there was no phosphorylation of tubulin (Fig. 4, A and B). Thus, a kinase causes MAP2 to release from MTs. Different kinase inhibitors were used to define the relevant kinase activity (9, 35, 36) . Staurosporine and heparin did not inhibit the ATP effect; however, two kinase inhibitors were found to inhibit the ATP-dependent release of MAP2 to MTs (Table III), and the extent of inhibition correlated with the inhibition of MAP2 phosphorylation (Fig. 4). Furthermore, there was no increase in release with cAMP added to ATP (Table II).


Figure 3: Quantification of ATP effect on binding of embryonic chick brain MAP2 to microtubules. Percentage of MAP2 and tubulin that remains in the microtubules after incubation of MAP2-microtubules with ATP for 15 min. The data are the average of two experiments and represent the percent protein in the microtubules considering 100% a sample without ATP.




Figure 4: Protein phosphorylation of embryo chicken MAP2 fraction. A, embryonic chick MAP2 was incubated with 0.2 mM ATP (lane 1) or [P]ATP (0.33 mCi/pmol) (lanes 2-5) in the presence of KF (60 mM) (lane 3), glycerol 2-phosphate (125 mM) (lane 4), and heparin (30 µM) (lane 5). The samples were run on 5% SDS-PAGE (lanes 1, 2, 3, 4, and 5) and exposed to a sensitive film (lanes 1`, 2`, 3`, 4`, and 5`). B, a mixture of embryonic chick MAP2 and microtubules were incubated with [P]ATP (0.33 mCi/pmol) and 0.2 mM ATP, the sample was run in a 7.5% SDS-PAGE (1) and exposed to a sensitive film (lane 1`). MAP2 (M) and tubulin (T) are indicated at the left of the figure, and molecular weight markers are indicated at the left (A) and at the right (B).



The nucleotide dependence of the release, the effect of kinase inhibitors, and the direct measurements of MAP2 phosphorylation all indicate that the release of inhibition of motility was dependent upon the release of MAP2 from MTs by a kinase activity.

Characterization of the Embryonic MAP2 Kinase

A series of kinases has been shown to phosphorylate MAP2. We have tested the effect of several of these proteins in MAP2 binding to MT.

beta-Casein kinase (the NIMA protein) (26) and Cdc2 kinase (37) were able to phosphorylate MAP2 but do not change the affinity for microtubules (see Table IV). Mitogenic activated protein kinase phosphorylates MAP2 (38) . We found that this protein kinase is able to phosphorylate MAP2 in a way higher than embryonic MAP2 kinase, but it is unable to change the amount of MAP2 bound to microtubules (see Table IV) indicating that a specific phosphorylation is necessary to release MAP2. In addition, a native bovine MAP2 fraction presented a MAP2 kinase activity; however, it did not affect MAP2 affinity (see Table IV).

To determine whether the kinase activity was able to reassociate with microtubules, native embryonic MAP2 was incubated with microtubules, and the complex sedimented through a 25% sucrose cushion. We found that the kinase co-sedimented with the complex because MAP2 was released when ATP was added and the same inhibitors blocked the release (Table V). Thus, a kinase causes MAP2 to release from MTs, and that kinase binds to MAP2bulletMT complexes.

Embryonic MAP2 Fraction Presents a 100-kDa MAP2 Kinase

The native embryonic MAP2 fraction was electrophoresed in an SDS-polyacrylamide gel containing purified bovine MAP2 as substrate in order to identify the MAP2 kinase activity (39) . A protein band with a relative motility of 100 kDa was identified (see Fig. 5 ). The degree of phosphorylation of this band decreased considerably when KF (60 mM) or glycerol 2-phosphate (125 mM) was added to the solution of kinase assay (data not shown). When the experiment was repeated using a gel without substrate, no band could be identified indicating that the band corresponds to a MAP2 kinase activity and not to an autophosphorylated protein.


Figure 5: Detection of embryonic MAP2 kinase activity in polyacrylamide gels containing purified bovine MAP2 as substrate. A, radioautography of 7.5% gel containing bovine MAP2 as substrate that was loaded with 20 µl of 0.1 mg/ml of embryonic chicken MAP2 fraction (lane 1), 20 µl of sample buffer (lane 2). B, radioautography of 7.5% gel loaded with 20 µl of 0.1 mg/ml embryonic chicken MAP2 fraction (lane 1`). Embryonic MAP2 kinase (arrow) is indicated to the left of the figure, and molecular weight markers are indicated to the right of the figure.



We used another assay to analyze the kinase activity of the fraction. We applied the embryonic MAP2 fraction to an ATP-agarose column. The ATP-release (filtrate) contains a prominent protein corresponding to a M(r) = 100,000 band in the gel (Fig. 6A, filled arrow) and a minor band of M(r) = 85,000 (Fig. 6A, empty arrow). The filtrate is able to phosphorylate MAP2 in a KF and glycerol 2-phosphate dependable way (Fig. 6B). The 100-kDa protein was separated from the 85-kDa protein in a Centricon filter, molecular size cutoffs at 100 kDa (Amicon). The 100-kDa protein (in the upper solution) shows the kinase activity; meanwhile, the 85-kDa protein (in the pass-through solution) shows no kinase activity (data not shown).


Figure 6: Activity of embryonic MAP2 kinase in the filtrate of an ATP-agarose column. A, 2 µl of embryonic MAP2 fraction (lane 1) and 400 µl of filtrate (previously precipitated with 5% trichloroacetic acid) (lane 2) were run in a SDS-PAGE. The molecular weight markers are indicated at the left of the figure. The arrows indicate the position of the 100-kDa band (filled arrow) and the 85-kDa band (open arrow). B, a mixture of 10 µl of filtrate and 10 µl of bovine MAP2 was incubated with [P]ATP (0.33 mCi/pmol) and 0.2 mM ATP (lane 1) in the presence of 60 mM KF (lane 2) and 125 mM glycerol 2-phosphate (lane 3). The samples were run in a 7.5% SDS-PAGE, the gel was stained with Coomassie Blue (CB), and the radioactivity was analyzed by exposition to a sensitive film (R). The bands of MAP2 in the gel are shown in the upper lane (CB), and the radioactivity pattern is shown in the bottom lane (R).



The characteristics of amino acid phosphorylation of this kinase were analyzed. After phosphorylation with embryonic chick fraction, the band of phosphorylated MAP2 was separated from the gel, and the phosphoamino acids were analyzed in a TLC electrophoresis plate (see ``Experimental Procedures''). It was shown that embryonic kinase phosphorylates MAP2 at serine amino acid residues (see Fig. 7).


Figure 7: Phosphoamino acid analysis of P-phosphorylated embryonic MAP2 kinase. A representative autoradiogram of P-labeled phosphoamino acids detected by autoradiography is presented in A. The migration of unlabeled standards (PS, phosphoserine; PT, phosphothreonine; and PY, phosphotyrosine) was detected by ninhydrin spray, and these spot positions are indicated on B.



The evidence that the kinase activity of embryonic MAP2 fraction is generated by a protein of 100 kDa in both procedures, the gel renaturation kinase assay and the ATP-agarose chromatography assay, together with the fact that the kinase activity is inhibited by KF and glycerol 2-phosphate, led us to think that this protein kinase is able to phosphorylate and release MAP2 from the microtubules. We hold that it is a new protein serine kinase with a M(r) = 100,000 named by us as embryonic MAP2 kinase.


DISCUSSION

Embryonic MAP2 Kinase Binds to Microtubules of Embryonic Chicken Brain

According to these results, it is considered that embryonic MAP2 kinase (E-MAP2k) is a new kinase with a M(r) = 100,000 that interacts with microtubules, hydrolyzes ATP, phosphorylates MAP2 at serine residues, and provokes detachment from the microtubules. Several assays were used to demonstrate that this enzyme is unrelated to other kinases. Staurosporine, an inhibitor of protein kinase C, cAMP kinase, cGMP kinase, pp60, and myosin light chain kinase (36-40) and heparin, a specific inhibitor of casein kinase II (41) , did not inhibit either phosphorylation activity or the detachment of MAP2 from the microtubules. Two inhibitors, KF and glycerol 2-phosphate, inhibit E-MAP2k activity in the same way that they inhibit MAP2 kinase (9, 10, 42) . MAP2 kinase is the original name of mitotic activated protein kinase (p44 ) (43) . There are several characteristics that allow differentiation of E-MAP2k from this kinase: (i) p44 is not able to release MAP2 from the microtubules (38) and our result, (ii) it phosphorylates serine/threonine residues (45, 46) whereas E-MAP2k phosphorylates only serine residues, and (iii) it has a M(r) = 42,000-44,000 (43) whereas E-MAP2k has a M(r) = 100,000.

Due to these differences, it seems logical to propose a new protein with kinase activity on the cytoskeleton.

Effect of Embryonic MAP2 Kinase on MAP2 Affinity to Microtubules

A surprising finding in these studies is that the release of MAP2 upon phosphorylation by E-MAP2k is nearly complete (70-90% released, see Fig. 1, 2, and 3). Previous analyses of the effect of phosphorylation of MAP2 (previously dephosphorylated in vitro) by a calf brain cAMP kinase reported only a 30% reduction in the binding to MTs under similar ionic conditions to those of the present study (8) or even smaller changes in the amount bound under these ionic conditions with a chicken cAMP-dependent kinase (6, 38) . The present observations suggest that the binding constant is reduced by a factor of 10 or more by phosphorylation with the embryonic kinase preparation.

cAMP-dependent protein kinase is bound to MAP2 in brain (4, 12, 47) , and phosphorylation of MAP2 has been suggested to inhibit its binding to microtubules (7, 8, 13) , but according to the above mentioned results, E-MAP2k is not a cAMP kinase (it is not inhibited by staurosporine and cAMP does not stimulate the release of MAP2).

The Role of MAP2 and Embryonic MAP2 Kinase in Positioning and Movement of Organelles

Inhibition of microtubule-dependent motility by MAP2 and activation by the presence of E-MAP2k are two events that we consider closely related in the cell.

To explain this association, it is necessary to mention the special characteristics of MAP2. (i) MAP2 is a specific protein of neuronal cells with a particular localization on dendrite microtubules which has a relevant function in order to determine the shape of the cell. MAP2 expression is necessary for both neurite extension and cessation of cell division (48) . (ii) MAP2 has an important role in the regulation of MT-dependent organelle motility, particularly for small organelles which could direct cell polarization processes such as dendrite differentiation (18) . (iii) Like kinesin, MAP2 is bound to membranous organelles and can operate as a transitory binding of vesicles to microtubules (15) .

According to this evidence we think, like Severin et al. (15), that MAP2 positions vesicles in the cytoplasm and facilitates the transport by microtubule motors. But, according to our results, it is highly unlikely that this model could explain the mechanism to trigger the transport without a factor that releases MAP2 from the microtubules because of the following. (i) Microtubules decorated with MAP2 cannot transport vesicles in vitro in the presence of a high concentration of motor proteins and they cannot support formation of a normal membranous network formation either (18) . (ii) MAP2-microtubules restricted the transport of latex beads decorated with kinesin when the molar ratio of MAP2 to tubulin was still 1:30 (49) . (iii) Furthermore, in any of these experiments, we can appreciate a significant loss of MAP2 from the microtubules by the action of motor protein coincident with the interpretation that a steric impediment but not a site competition between MAP2 and motor proteins is the origin of transport inhibition (18) .

All these suggest that activation of motility of the vesicles previously positioned by MAP2 should trigger the release of MAP2 from the microtubule.

It was observed that E-MAP2k phosphorylates and releases MAP2 from microtubules, restoring the transport mediated by them. It is suspected that this protein-kinase could trigger the same activity in the cell.

The hypothesis that a factor releases MAP2 from the MTs in the neuron is supported by two facts: (i) MAP2 is present in both axons and dendrites of cultured neurons at the early stages of development and is selectively lost from the axon during subsequent maturation (50, 51, 52) and (ii) exogenous MAP2 injected into the axon is removed faster from axonal than dendrite microtubules (53) .

Another important aspect of MAP2 and E-MAP2k is the fact that MAP2 is concentrated in the dendrites of neurons where there is a random polarity of the MTs (54) . If MAP2 were to uncoat one population preferentially, due to phosphorylation-detachment of MAP2 by E-MAP2k, then a directional array of MTs would be adequate to support MT-dependent motility and would conduct the transport of vesicles. In the case of a totally random array of microtubules, the motors would not be able to produce directed movement over long distances because of the saltatory nature of the movement.

Our model for the positioning and subsequent transport of vesicles suggests that the coating of MTs by MAP2 is analogous to the troponin-tropomyosin system in striated muscle. The regulator of motility in the MAP2 system would be the E-MAP2k described in this paper, which would remove the MAP2 from the MTs and would allow motility to occur. A cellular function of the E-MAP2k could be to up-regulate motility by phosphorylating and releasing MAP2 or perhaps another inhibitory MAPs from MTs.

It will be important to determine whether the in vitro effect of the MAP2 corresponds to in vivo effects.

  
Table: Characteristics of embryonic chicken MAP2-microtubule gliding using ATP or GTP as energy source


  
Table: Effect of nucleotides on MAP2 binding to microtubules

Microtubules were polymerized with 1 mM GTP and 20 µM Taxol, except in Footnote c.


  
Table: Effect of kinase inhibitors on the release of MAP2 by ATP

Native embryonic MAP2 was incubated with PC-MTs plus 1 mM ATP in the presence or absence of inhibitors. The microtubules were centrifuged through a sucrose cushion (25%). The samples were then centrifuged, and MAP2 was quantified in the pellet by densitometry of the SDS-PAGE gels.


  
Table: Degree of MAP2 phosphorylation and release from microtubules

Tubulin was polymerized with 1 mM GTP and 20 µM Taxol in the presence of MAP2, centrifuged through 25% sucrose, and resuspended in stabilizing buffer. Thirty g of MAP2-microtubules were incubated with the kinase sample in the presence of [P]ATP and 0.5 mM ATP in a final volume of 90 µl at 20 °C 30 min. The microtubles (bound) and the supernatant (free) were quantified by densitometry after SDS-PAGE. The bands containing MAP2 were excised from the gels, the gel was dissolved in H(2)O(2), the radioactivity was determined in a scintillation counter, and the moles of phosphate incorporated into 1 mol of MAP2 were estimated except in Footnote a. The data represent the average ± S. D. of two experiments.


  
Table: Effect of ATP and kinase inhibitors on the release of MAP2 from MAP2-MTs pelleted on sucrose cushion

Native embryonic MAP2 was assembled with tubulin in the presence of 1 mM GTP and 20 µM Taxol. The microtubules were centrifuged through a sucrose cushion (25%) and incubated with ATP in presence or absence of inhibitors. The samples were then centrifuged, and MAP2 was qualified in the pellet by densitometry of the SDS-PAGE gels.



FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants GM-34775 and NS-23345. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Partially supported by Instituto de Ciencias Básicas, Universidad Nacional de Cuyo of Argentina. To whom correspondence and reprint requests should be addressed. Present address: Instituto de Histologa y Embriologa, Facultad de Ciencias Medicas, Universidad Nacional de Cuyo, Casilla Correo 56, 5500 Mendoza, Argentina. Fax: 054-61-380-232.

(^1)
The abbreviations used are: MT(s), microtubule(s); PAGE, polyacrylamide gel electrophoresis; MAPs, microtubule-associated proteins; E-MAP2k, embryonic MAP2 kinase; ATPS, adenosine 5`-O-(3-thiotriphosphate); Pipes, 1,4-piperazinediethanesulfonic acid; AMP-PNP, adenyl-5`-yl imidodiphosphate.


ACKNOWLEDGEMENTS

We thank Dr. Katherin Swenson (Dept. of Cell Biology, Duke University) for her generous help on the preparation of Cdc2 kinase and phosphoamino acid analysis and Dr. A. R. Means (Dept. of Pharmacology, Duke University) for providing NIMA enzyme and Dr. Luis Mayorga (IHEM, Universidad Nacional de Cuyo, Argentina) for helpful discussions and critical reading of the manuscript.


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