(Received for publication, October 6, 1994; and in revised form, January 11, 1995 )
From the
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 = 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.
Brain microtubule (MT)(
)
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 = 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.
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.
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. MAP2MT 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.
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.
-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
MAP2MT complexes.
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 = 100,000 band in the gel
(Fig. 6A, filled arrow) and a minor band of
M
= 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 = 100,000 named by us as
embryonic MAP2 kinase.
Due to these differences, it seems logical to propose a new protein with kinase activity on the cytoskeleton.
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).
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
O
, 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.