Identification of Phosphorylation Sites for PKA In Vitro and In
Vivo
Specific studies of MAP2 phosphorylation and its role in regulating
dendritic architecture first require the identification and functional
characterization of specific amino acid residues where PKA or other
cellular kinases exert regulation of the MAP2 interaction with
microtubules and actin filaments.
Phosphorylation of purified recombinant MAP2c (rMAP2c) by purified PKA
in vitro reaches saturation at a maximal stoichioimetry of ~3 mol
phosphate/mol MAP2c after 1 h (Figure
A). A 2-D phosphopeptide map of MAP2c
phosphorylated to saturation by PKA contained multiple phosphopeptides
with varying intensities, suggesting phosphate incorporation at many
sites to varying stoichiometry. Very early in the time course, the
kinase displayed greater apparent specificity. After only 30 s,
the 2-D phosphopeptide map contained fewer spots with less variation in
intensity. These early targets are likely to represent the preferred
substrates for PKA on the MAP2c molecule and we therefore focused our
attention on these sites.
Purified rMAP2c was briefly phosphorylated (30 s) in the
presence of PKA, digested with trypsin, and the resulting mixture of
peptides was subjected to MALDI mass spectrometry. Multiple
phosphopeptides were observed, as well as nonphosphorylated peptides,
corresponding to the majority of the MAP2c molecule. Identification of
specific phosphorylation sites on each phosphopeptide was complicated
in most cases by the presence of multiple potential phosphorylation
sites on each peptide fragment. Phosphoamino acid analysis on rMAP2c
phosphorylated for 30 s with PKA revealed no detectable levels of
phosphothreonine or phosphotyrosine (our unpublished results). This
allowed the unambiguous identification of phosphorylation at single
serine (S) residues present on three of the phosphopeptides. S319,
S350, and S382 were thus identified as preferred targets of PKA on
MAP2c (Table ). These three residues
constitute the serines of the conserved KXGS motif found in each
microtubule-binding repeat.
| Table 1MAP2c phosphopeptides observed by MALDI mass
spectrometry
|
Mass spectrometry does not provide a quantitative measure of the
components of a peptide mixture and thus does not address the relative
stoichiometry of phosphorylation at specific sites. We therefore
evaluated this issue by using 2-D phosphopeptide maps of wt versus
mutant rMAP2c phosphorylated by PKA in the presence of
[γ-32P]ATP. Mutation of S350 to alanine
resulted in a 41% reduction in total phosphate incorporation after
phosphorylation by PKA for 30 s (Figure B). In protein
phosphorylated to saturation by PKA, the mutation of S350 to alanine
reduced total phosphate incorporation by 66% (our unpublished
results). Mutation of S319 or S382 to alanine resulted in 10–22%
reduction in phosphate incorporation after phosphorylation by PKA for
30 s (Figure B).
To clarify whether S319, S350, and S382 are significant early targets
of PKA in vitro, 2-D phosphopeptide mapping was performed on purified
rMAP2c phosphorylated for 30 s. Mutation of S319 to alanine
resulted in the elimination of one spot from the 2-D phosphopeptide map
(Figure B). Mutation of either S350 or S382 to alanine resulted in the
elimination of two spots in the central cluster of the map, suggesting
either that both residues are contained within two or more peptide
fragments or that mutagenesis of these residues results in altered
kinase specificity at other sites.
To examine whether phosphorylation occurs at S319, S350, and S382 in
vivo, antisera were raised against purified rMAP2c. Both
high-molecular-weight MAP2b and low-molecular-weight MAP2c are
expressed in juvenile rat hippocampus. Antiserum 4170 recognized both
isoforms, similar to the commercially available monoclonal antibody
HM-2 (Sigma; our unpublished results). MAP2 was
immunoprecipitated from juvenile rat hippocampus by using antiserum
4170. After separation by SDS-PAGE, native MAP2c was excised from the
gel and subjected to the proteolytic digestion and MALDI mass
spectrometry protocol described above for recombinant MAP2c.
Peptides were observed corresponding to the majority of the MAP2c
primary sequence and phosphorylation was observed at fewer sites than
on MAP2c phosphorylated after brief incubation with PKA in vitro. Some
phosphorylated residues were observed on multiple overlapping peptides,
facilitating identification of specific phosphorylation sites. A
phosphopeptide encompassing the sequence C348-K364 contained only one
possible phosphorylation site, S350 (Table ). The additional
phosphopeptide I339-K352 was also detected, confirming that
phosphorylation of S350 is observed in native MAP2c. The phosphopeptide
L302-K328 contained two phosphorylated residues. An additional peptide
was observed with a single phosphorylated residue between Q299-K316.
These data suggest that the second phosphorylation site in the
L302-K328 region falls within the sequence I317-K328. Earlier work
observed no detectable tyrosine phosphorylation on native MAP2 in
hippocampus (
Halpain and Greengard, 1990 
), eliminating Y325 as a likely
candidate and thereby narrowing the candidate residues to either S319
or T320. Although this is not a conclusive detection of phosphorylation
at S319 in the native MAP2c molecule, it is consistent with our
detection of S319 phosphorylation on rMAP2c phosphorylated in vitro.
PKA Activity Regulates MAP2c Interactions with Microtubules
and Actin in Living Cells
To enable observation of MAP2c localization in living cells, a
plasmid was constructed encoding MAP2c as a fusion protein with GFP
attached at the N terminus of MAP2c, distant from the
microtubule-binding region. The GFP-MAP2c fusion protein expressed in
HeLa cells colocalized with tubulin and stabilized microtubules,
resulting in apparent bundles of microtubules at the periphery of the
cell (Figure A). In mammalian cell
lines, expression of MAP2 remodels endogenous microtubule structure,
leading to the formation of bundles of microtubules near the perimeter
of the cell (
Weisshaar et al., 1992 ). MAP2 is not thought to
physically cross-link microtubules (
Burgin et al., 1994 ).
Rather, the bundles are comprised of microtubules that have become
hyperstabilized and aligned in parallel arrays. The cell membrane and
associated cortical actin structure restrain these stabilized
microtubules, causing them to bend and conform to the cell perimeter
(
Edson et al., 1993 ;
Tucker et al., 1993 ). The
data shown in Figure A are consistent with previous observations of
GFP-MAP2c expressed in mammalian cell lines (
Kaech et al.,
1996 ), confirming that the GFP fusion did not disrupt the
MAP2c–microtubule interaction.
Although many studies have reported that phosphorylation reduces the
binding of MAP2 to microtubules in vitro, there have been no previous
demonstrations of stimulus-dependent distribution of MAP2c in situ. To
test whether endogenous protein kinase activity regulates the
interaction of MAP2c with microtubules or actin filaments, we assayed
the effects of PKA stimulation on GFP-MAP2c localization in live HeLa
cells.
Brief incubation of HeLa cells in the presence of 20 μM forskolin, an
activator of adenylyl cyclase, significantly decreased colocalization
of MAP2c with microtubules and increased GFP-MAP2c visible in the
cytoplasm (Figure A). Quantitative image analysis of numerous cells in
each treatment group (n ≥ 50) established that the effect was
statistically significant (Figure B). Because forskolin caused an
apparent reduction in MAP2–microtubule binding in HeLa cells, we
examined the hypothesis that phosphorylation at the KXGS motifs
mediates this inhibition of the MAP2c–microtubule interaction.
Mutation of S350 to alanine, eliminating phosphorylation at this site,
was sufficient to abolish any significant effect of forskolin treatment
on MAP2 localization (Figure , A and B). This is consistent with the
in vitro data suggesting that S350 is a major target of PKA on the MAP2
molecule and suggests that S350 is likely to be a major target of PKA
in living cells.
1The majority of the GFP-MAP2 fluorescence was retained in microtubule
bundles after the 10-min forskolin treatment. This suggested that only
a subpopulation of MAP2c was affected by this brief treatment. No
further change in GFP-MAP2c localization was observed with incubations
up to 30 min (our unpublished results). We hypothesized that MAP2c
already sequestered in microtubule bundles was poorly accessible to
endogenous kinases. By initiating treatments to elevate cAMP before the
accumulation of MAP2c into microtubule bundles, we expected to alter
the phosphorylation state of a larger fraction of overexpressed MAP2c.
To strengthen the degree of PKA activation, we used two compounds to
synergistically and specifically elevate intracellular cAMP. In
addition to forskolin, which activates adenylyl cyclase, we added
rolipram to inhibit phosphodiesterase IV. Incubation for 10 h with
a combination of forskolin (10 μM) and rolipram (20 μM), applied
immediately after transfection, reduced GFP-MAP2c localization to
microtubules and concomitantly raised cytoplasmic fluorescence to
levels comparable to those after brief forskolin treatment. The
stronger activation did not eliminate the formation of microtubule
bundles, suggesting that a mixture of phosphorylated and
unphosphorylated MAP2 was maintained in the cell. However, we noted a
significant difference in the localization of MAP2c in these strongly
cAMP-stimulated cells. In many cases, we observed the presence of
GFP-MAP2c in peripheral membrane ruffles, structures that are highly
enriched in actin (Figure B) but
normally devoid of microtubules (see also Figures and ). In the
absence of PKA stimulation, MAP2c was absent from peripheral membrane
ruffles (Figure A).
MAP2 and actin thus appeared specifically enriched and partially
colocalized within the peripheral membrane ruffles (Figure B). One
possibility was that this appearance was due to thicker regions of
cytoplasm, protruding upward perpendicular to the coverslip. This could
cause soluble cytoplasmic proteins artifactually to appear enriched in
specific locales. To determine whether this was the case, we evaluated
3-D reconstructions assembled from a z-dimensional series of
deconvolved single plane images (Figure , video
supplement). Neither MAP2 nor actin filled the cytoplasmic volume
within the membrane ruffle. Both proteins were confined to specific
domains within the ruffle, confirming that the observed colocalization
was not an artifact of increased cytoplasmic volume.
KXGS Motifs Regulate MAP2c–Microtubule Interactions: Biochemical
Studies
Phosphorylation states within cells are maintained via a balance
of protein kinase and phosphatase activities. To clarify the functional
impact of phosphorylation at a specific site, quantitative and stable
modification of that site is desirable. As a mimic of constitutive
phosphorylation, S319, S350, and S382 were mutated singly and in
combination to glutamic acid, within the GFP-MAP2c eukaryotic
expression vector. This approach allows the evaluation of the
functional impact of phosphorylation at specific residues in vitro and
in vivo, without the potential for modification of the sites under
study due to cellular phosphatase and/or kinase activity (
Bibb and da
Cruz e Silva, 1997 ).
The transfection efficiency of all constructs was similar (at ~15%
of cells transfected). Similarly, the expression levels of the S319E,
S350E, S382E, S350A, S319E/S350E, and S350E/S382E GFP-MAP2c mutants
displayed no significant differences from the expression level of wt
GFP-MAP2c, according to quantitative immunoblot analysis
(our unpublished results).
The ability of wt MAP2c and the KXGS glutamic acid mutants to bind to
microtubules in living cells was evaluated biochemically by lysis of
equivalent populations of transiently transfected cells in a
microtubule-stabilizing buffer containing taxol. Centrifugation was
used to separate the microtubule-containing fraction from the soluble
fraction. Previous work demonstrated that this protocol preserves the
in vivo polymerization state of the microtubules, polymer content being
neither enriched nor depleted by the procedure (
Minotti et
al., 1991 ). Consistent with previous observations, ~40% of the
cellular tubulin was observed in the soluble fraction (Figure
). Approximately 60% of wt MAP2c
partitioned with the soluble fraction in this assay. Mutation of any
KXGS site to glutamic acid significantly enriched MAP2c in the soluble
fraction to 80–90% (P < 0.01, n = 5; Figure ). These data
suggest that the binding affinity of the mutants for microtubules is
lower than the wt protein. No statistically significant differences
were observed among the mutants (P > 0.05, n = 5) and no
greater disruption was observed for double mutants than for any single
mutation.
KXGS Motifs Regulate the MAP2c–Microtubule Interactions: Live-Cell
Imaging
Localization of MAP2c in living cells was observed by confocal
fluorescence microscopy of transiently transfected HeLa cells
expressing GFP-MAP2c fusion proteins (Figure
). As before, expression of wt MAP2c
induced the apparent reorganization of endogenous microtubules into
hyperstabilized bundles (Figure A). The S319E mutation resulted in a
substantial reduction of this ability of MAP2c to remodel endogenous
microtubule structure, even though this mutant retained a high degree
of colocalization with microtubules (Figure B). The S350E mutation
resulted in a further reduction of bundled microtubules and caused a
visible increase in cytoplasmic fluorescence, resulting in the
appearance of negatively outlined cytoplasmic vesicles and organelles
from which MAP2c was excluded (Figure C). The S382E mutation also
resulted in reduced microtubule bundling and enrichment of the
cytoplasmic fluorescence (Figure D).
Quantitative image analysis was performed, as described in MATERIALS
AND METHODS, on a large population of cells expressing wt GFP-MAP2c and
each mutant (n ≥ 50 in each group) to compare the level of
cytoplasmic fluorescence, expressed as the coefficient of variation
(SD/mean pixel intensity) (Figure E). Images of cells expressing wt
GFP-MAP2c exhibited high variability in pixel intensity, due to the
restricted localization of MAP2c to microtubules. The S319E mutation
caused a statistically significant reduction in pixel variation
compared with wt (P < 0.001), consistent with the visible
reduction in intensely fluorescent bundles of microtubules. Differences
between the S350E and the S382E mutants were not significant (P >
0.05). Mutation of S350 or S382 resulted in a reduction of pixel
variation to ~50% of the wt value (P < 0.001), consistent with
the diffuse cytoplasmic fluorescence observed. We note that all of the
glutamic acid mutations resulted in significantly more
disruption to the MAP2c–microtubule interaction than an alanine
mutation. Images presented in the figures are representative of
predominant phenotypes observed in cells expressing wt and mutant
GFP-MAP2c constructs. All constructs including wt, however, do induce a
certain range of microtubule phenotypes, prompting us to develop and
use a quantitative assay to objectively evaluate the MAP2c–microtubule
interaction.
All three KXGS motifs are targets of PKA in vitro and both S319 and
S350 were observed to be phosphorylated in native tissue (Table ). We
hypothesized, therefore, that functional phosphorylation states in vivo
may include simultaneous phosphorylation of multiple KXGS motifs. To
evaluate the functional impact of these phosphorylation states,
multiple KXGS motifs were mutated to glutamic acid to mimic specific
constitutive phosphorylation. In contrast to wt GFP-MAP2c (Figure
A), S319E/S382E (Figure B), S350E/S382E
(Figure C), and S319E/S350E/S382E (Figure D) GFP-MAP2c all displayed
reduced microtubule localization, an absence of microtubule bundles,
and increased cytoplasmic fluorescence. Quantitative analysis of the
coefficient of variation demonstrated that all multiple mutations were
significantly different from wt (P < 0.001) (Figure E). Multiple
mutations did not result in additional disruption of the
MAP2c–microtubule interaction beyond that observed with the single
mutations S350E or S382E. In either biochemical- or microscopy-based
assays evaluating microtubule localization, no significant difference
was observed between multiple mutations and the single mutations S350E
and S382E (P > 0.05 in both assays; Figures and ).
KXGS Phosphorylation Promotes Localization to the
Actin-Rich Cellular Periphery
Imaging studies suggested a potentially important difference
between single and multiple KXGS mutants of MAP2c. Double and triple
glutamic acid mutations in the KXGS motifs consistently promoted
GFP-MAP2c localization to membrane ruffles, similar to that seen with
treatments elevating intracellular cAMP (Figure B). This localization
was almost never observed in cells expressing wt GFP-MAP2c without PKA
activation, or any of the single mutant GFP-MAP2c constructs (Figures
A, , and ). Similar results were also obtained in Rat-1
fibroblasts (our unpublished results). The S350E/S382E double mutation
was sufficient to promote GFP-MAP2c localization to ruffles in >95%
of transfected cells (Figure , B–D). No
additional increase in peripheral localization or altered morphology
was observed with the triple mutation S319E/S350E/S382E (Figure D). A
double mutation not including S350, S319E/S382E, also promoted
GFP-MAP2c localization to ruffles in ~50% of transfected cells. wt
GFP-MAP2c, in the absence of PKA stimulation (Figure A), was seen in
cellular ruffles in <5% of transfected cells. In addition to altered
MAP2c localization, the S350E/S382E mutation also occasionally promoted
unusual pseudopod-like morphologies (Figure B), rarely seen in
untransfected HeLa cells, suggesting that MAP2c may enhance
lamellipodial growth. The altered phenotype was not a consequence of
differential expression level; expression level in a large population
of cells was similar for all constructs. Although differences in
fluorescence intensity indicated variation in expression level among
individual cells, S350E/S382E GFP-MAP2c was localized to peripheral
ruffles to the same degree in cells with low versus high expression
levels (cf. cells in Figure , C and D). Combined differential
interference contrast and fluorescence time-lapse imaging confirmed
that S350E/S382E GFP-MAP2c was localized to highly motile ruffle
structures at the periphery of the cell (Figure E).
To evaluate whether these peripheral structures also contained
microtubules, cells expressing wt and S350/S382 mutant GFP-MAP2c were
fixed and immunolabeled for tubulin (Figure
). wt GFP-MAP2c was localized primarily
to areas of the cell also enriched in tubulin and many of the
endogenous microtubules appeared in bundles. In contrast, microtubules
in cells expressing S350E/S382E GFP-MAP2c were not remodeled into
bundles and appeared similar to those in nontransfected cells. Mutant
MAP2c was not restricted to tubulin-rich areas of the cell but was
enriched in peripheral ruffles, structures that typically are highly
enriched in actin filaments.
Association of MAP2c with the Actin Cytoskeleton
The appearance of MAP2c in peripheral ruffles suggested that
phosphorylation events reducing the ability of MAP2 to bind
microtubules might enhance its ability to interact with the actin
cytoskeleton. Such a finding would be consistent with observations that
MAP2 specifically binds actin filaments in vitro (
Sattilaro, 1986 ;
Cunningham et al., 1997 ). Accordingly, we examined the
ability of wt MAP2c and the S350E/S382E mutant to colocalize with actin
by fixing transfected cells and immunolabeling for actin (Figure
). As expected, in both transfected and
untransfected cells, actin was enriched in areas adjacent to the plasma
membrane. In regions of cells not in direct contact with neighboring
cells, membrane ruffles enriched in actin were observed. As in the cAMP
experiments (Figure ), 3-D reconstruction of images of several cells
confirmed the identity of these actin-rich structures as membrane
ruffles (our unpublished results).
Expression of wt GFP-MAP2c did not result in detectable modifications
of actin localization. Actin was not enriched in or around the
microtubule bundle structures and MAP2 was absent from actin-rich areas
at the cell periphery. In contrast, S350E/S382E GFP-MAP2c was enriched
in peripheral areas of the cell that were also highly enriched in
actin. The mutant GFP-MAP2c fusion protein colocalized with actin in
numerous membrane ruffles. This result again was confirmed in 3-D
reconstructions assembled from single-plane images.
To test whether the colocalization of MAP2c and actin was due to
specific interaction, unfixed cells transfected with cytoplasmic GFP,
wt GFP-MAP2c, or S350E/S382E GFP-MAP2c were imaged before and after
extraction with a cytoskeletal-stabilizing buffer containing 0.4%
Triton-X 100 (Figure A). Soluble,
cytoplasmic GFP was almost entirely removed by the extraction and was
not retained in association with any cytoskeletal structure. The
microtubule-like localization of wt GFP-MAP2c was unchanged and the
total fluorescence signal was only slightly diminished by this
extraction procedure, indicating that wt MAP2c was tightly associated
with the microtubule cytoskeleton. In contrast, extraction removed
>90% of the S350E/S382E GFP-MAP2c fluorescence signal from the cell.
Fluorescence was specifically retained in association with both
microtubule and actin-based cytoskeletal structures, including
peripheral ruffles. Interestingly, in some cells we observed the
specific retention of mutant GFP-MAP2c in what appeared to be
actin-based stress fibers (Figure B). Localization of mutant MAP2c to
stress fibers was also seen in unextracted cells, although it was much
more difficult to discern above the background cytoplasmic
fluorescence.
This article has been 
