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INTRODUCTION |
The sterile 20 (STE20)1-like kinases are a
large group of proteins whose catalytic domains have homology to the
STE20 kinase of Saccharomyces cerevisiae involved in
mitogen-activated protein kinase (MAPK) signaling of the pheromone
response in yeast (1). More than 30 mammalian STE20-like kinases have
been identified, and these proteins divide into two subfamilies
according to their structure and regulation (reviewed in Ref. 2). The
p21-activated kinases (PAKs) have a C-terminal catalytic domain and an
N-terminal Cdc42/Rac interacting domain (CRIB), whereas the germinal
center kinase (GCK)-like kinases possess an N-terminal catalytic domain and no CRIB. Most STE20s can act as MAPK kinase kinase kinases, upstream of the c-Jun N-terminal kinases (JNK), p38 and/or
extracellular signal-regulated kinases (ERK) MAPK signaling pathways.
Recent work has demonstrated that some STE20s can also
regulate the cell cytoskeleton. PAKs interact with Rac or Cdc42 GTPases via the CRIB domain and act as downstream effectors for these small GTP
binding proteins to regulate the actin cytoskeleton (reviewed in Refs.
4, 5). Rac and Cdc42 stimulated morphological rearrangements are
blocked by mutated PAK, and activated PAK down-regulates actin stress
fibers and focal complexes (6-8). Some of the effects of PAK1 can be
attributed to the phosphorylation of downstream kinases, such as myosin
light chain kinase, which reduces its activity toward myosin light
chain, and LIM kinase 1, which phosphorylates and inactivates the actin
depolymerizing protein cofilin to generate actin clusters (9, 10).
X-PAK5 co-localizes to actin and microtubules (MTs) and produces
stabilized MTs that are associated in bundles, and PAK1 accumulates at
the MT-organizing center and along mitotic spindles during mitosis (11,
12).
Although GCK-like STE20s lack a CRIB domain, recent work has
demonstrated that members of this subfamily of STE20s can also regulate
the actin cytoskeleton. Proline- and alanine-rich STE20-related kinase
associates with actin, and prostate-derived STE20-like kinase (PSK),
Traf2- and Nck-interacting kinase, and STE20-like kinase (SLK)
decrease actin stress fibers and focal adhesions and inhibit cell
spreading (3, 13-15). SLK has recently been shown to associate with
MTs at the cell periphery (16).
There is increasing evidence that actin filaments and MTs are
coordinately regulated during the establishment and maintenance of cell
polarity, division, and motility. The ability of MTs to undergo
transitions between growth, shrinkage and pause are crucial for their
function and the regulation of these processes. MT dynamics and
stability are controlled by multiple factors, which include MT-associated proteins (MAPs), MT-affinity regulated kinases, severing
factors (e.g. katanin), and catastrophe proteins
(e.g. stathmin/OP18 and XKCM1) (reviewed in (17)). MAPs
provide a focal point for MT regulation and act as structural proteins
that lack enzymatic activity but promote the assembly of tubulin and MT
stability. The affinity of MAPs for MTs is controlled by their phosphorylation, which causes the detachment of MAPs from MTs and
results in MT instability. A number of kinases can phosphorylate MAPs
and thereby modulate MT stability (reviewed in Ref. 17). In contrast,
phosphorylation of the MT-destabilizing protein stathmin inactivates
the protein and stabilizes MTs (reviewed in Ref. 18). Several kinases,
including PAK, modulate the phosphorylation of stathmin and may
therefore regulate tubulin polymerization and MT stability (19). The
signaling pathways and components that regulate MT dynamics remain to
be determined, but small GTP binding proteins and their downstream
targets, which include STE20-like kinases, seem to play crucial roles
in controlling MT function.
Here we show that a recently identified member of the GCK-like family
of STE20s, PSK (3), colocalizes with MTs and produces stabilized
perinuclear MT cables that are nocodazole-resistant and contain
increased levels of acetylated
-tubulin. These findings suggest that
a major function for PSK is to transduce signals to the MT network.
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EXPERIMENTAL PROCEDURES |
Plasmids--
pRK5PSK and pRK5PSK (K57A) were made previously
(3). PSK (1-940) and PSK (745-1235) were prepared by PCR
amplification using appropriate oligonucleotides and pRK5PSK as
substrate, and DNA products were ligated into the pRK5 vector, which
provides a 5'-MYC-epitope tag.
Microinjection--
Cells were grown in 10% fetal calf
serum/Dulbecco's modified Eagle's medium. For microinjection, cells
were seeded on glass coverslips and incubated for 2 days. Where
appropriate, cells were serum-starved for 16 h before
microinjection. Endotoxin-free plasmid DNA (100 ng/µl in
phosphate-buffered saline; Qiagen) was microinjected into the nuclei of
~100 cells per coverslip. After 3-4 h, cultures were fixed for 20 min with 4% paraformaldehyde, 10% Me2SO, and 1% glucose
in phosphate-buffered saline (37 °C) and permeabilized with 0.2%
Triton X-100 for 5 min.
Immunolocalization--
To detect MYC-tagged PSK, cells were
incubated with 1:400 rabbit anti-MYC tag antibody (Santa Cruz
Biotechnology) followed by 1:400 fluorescein isothiocyanate-conjugated
goat anti-rabbit IgG (Jackson Immunoresearch Laboratories). To
visualize tubulin, cells were incubated with either 1:100 mouse
Cy3-conjugated anti-
-tubulin antibody (TUB2.1; Sigma) or 1:100 mouse
anti-
-tubulin antibody (DM1A; Sigma), followed by 1:400
TRITC-conjugated goat anti-mouse IgG (Jackson Immunoresearch
Laboratories). To detect acetylated
-tubulin, cells were incubated
with 1:100 mouse anti-acetylated
-tubulin antibody (6-11B-1; Sigma)
followed by 1:400 Alexa633-conjugated goat anti-mouse IgG (Molecular
Probes). All antibodies were incubated in 20% fetal calf
serum/phosphate-buffered saline. Cells were imaged with a confocal
laser scanning microscope (Bio-Rad 1024). Image processing was
performed using Adobe Photoshop 5.5.
Transfection--
Human embryonic kidney 293 (1.3 × 106) or COS-1 (5 × 105) cells were seeded
onto 60-mm Petri dishes and grown for 16 h before the indicated
plasmids (3 µg) were transfected into cells with Lipofectin
(Invitrogen). After 5 h, transfected cultures were returned
to growth medium and incubated for 36 h.
Immunoblotting--
Cells were lysed in lysis and binding buffer
(1% Nonidet P-40, 130 mM NaCl, 1 mM
dithiothreitol, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 10 mM NaF, 0.1 mM Na3VO4,
1 mM phenylmethylsulfonyl fluoride, and 20 mM
Tris, pH 7.4). 100 µg of each sample was analyzed by SDS-PAGE before
proteins were transferred to nitrocellulose (Schleicher & Schuell).
Samples were processed as described previously (3), except that rabbit
anti-PSK serum was used as the primary antibody (raised against the
amino acid sequences GTLAGRRSRTRQSRALPPWR and EEEGAPIGTPRDPGDGC for
PSK; Sigma Genosys).
Immunoprecipitation--
Cells were lysed as described above,
and 400 µg of protein were incubated with rabbit anti-PSK serum for
1 h at 4 °C. Protein A-Sepharose beads (Sigma) were added to
each sample, and after 1 h, beads were pelleted by centrifugation
and washed three times in lysis and binding buffer.
In Vitro Kinase Assays--
Beads were washed once in kinase
buffer (20 mM Tris-HCl pH 7.4, 4 mM
MgCl2) and placed in 30 µl of kinase buffer containing 40 µM ATP, 5 µCi of
-[32P]ATP (Amersham
Biosciences) and 1 µg of bovine
- and
-tubulin (Cytoskeleton),
which predominantly form unpolymerized tubulin dimers under these
conditions. After 60 min at 37 °C, kinase assays were terminated in
gel sample buffer and processed as described previously (3).
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RESULTS |
PSK Localizes to Microtubules--
We have reported previously
that PSK, a GCK-like kinase, activates the JNK MAPK pathway and
regulates the actin cytoskeleton, and it showed a punctate or
filamentous distribution when expressed in fibroblasts (3). To
investigate the subcellular distribution of PSK in more detail,
expression vectors encoding MYC-epitope tagged PSK were microinjected
into the nuclei of Swiss 3T3 cells, and PSK was found to localize to a
filamentous network in the cytoplasm of injected cells that closely
resembled MTs (Fig. 1A). The
localization of PSK to MTs was confirmed by costaining injected cells
for PSK and the MT component
-tubulin (Fig. 1B). The
immunofluorescence from PSK was more intense from MTs positioned around
the nucleus than from MTs at the periphery of the cell, which appeared
fragmented (Fig. 1, A and B). The staining
patterns for both PSK and
-tubulin were disrupted by addition of the
MT-depolymerizing agent nocodazole (5 µM), demonstrating
that PSK and
-tubulin are closely associated (Fig. 1, C
and D). Moreover, the fixation of cells in formaldehyde, not
paraformaldehyde/Me2SO, resulted in the disruption of MTs and a tubulovesicular staining pattern for PSK (3). Costaining of
injected cells for PSK and markers for other cytoskeletal structures (vimentin and actin) demonstrated that PSK localized specifically to
the MT network (data not shown).

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Fig. 1.
PSK localizes to MTs. Growing Swiss 3T3
cells were microinjected with pRK5-MYC-PSK (wild type) and after
3.5 h, cells were treated with (C and D) or
without (A and B) 5 µM nocodazole
for 30 min and then fixed and costained to detect MYC-PSK (A
and C) and MTs (B and D). Cells become
vesiculated under these conditions. Scale bar, 20 µm.
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PSK Localizes to MTs via its C Terminus and Alters MT
Organization--
To determine whether the catalytic activity and/or
specific sequences outside of the kinase domain of PSK are involved in localizing PSK to MTs, we prepared expression vectors encoding MYC-tagged full-length kinase-defective PSK (K57A) and the C terminus of PSK (amino acids 745-1235) for comparison with wild-type PSK and
the N terminus of PSK (amino acids 1-940) containing the kinase domain
(Fig. 2). PSK and kinase-defective PSK
(K57A) both colocalized with
-tubulin (Fig.
3, A-D) showing
that the catalytic activity of the protein is not required for its
localization. PSK (745-1235), which lacks the entire kinase domain,
also localized to MTs (Fig. 3, E and F), whereas
PSK (1-940), containing the kinase domain, failed to localize to MTs
and was cytoplasmic (Fig. 3, G and H). Taken
together, these results demonstrate that the C terminus of PSK is both
necessary and sufficient for PSK to localize to the MT network, and
this process occurs independently of the protein's catalytic
activity.

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Fig. 2.
PSK constructs prepared for functional
characterization. The kinase domain is shown in dark
gray and the amino acids are indicated by
numbers.
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Fig. 3.
PSK localizes to MTs via its C terminus and
generates perinuclear MT cables. Growing Swiss 3T3 cells were
microinjected with pRK5-MYC-PSK (A and B),
pRK5-MYC-PSK (K57A) (C and D), pRK5-MYC-PSK
(745-1235) (E and F), or pRK5-MYC-PSK (1-940)
(G and H); after 3.5 h, cells were fixed and
stained to detect MYC-PSK (A, C, E,
and G) and MTs (B, D, F,
and H). Perinuclear MT cables were observed in ~75% of
cells injected with PSK or PSK (K57A), 100% of cells injected with PSK
(745-1235), but not in uninjected cells or cells containing PSK
(1-940). Scale bar, 20 µm.
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The localization of PSK to MTs resulted in a significant change in MT
organization. PSK generated MT cables around the nucleus that were
absent in uninjected surrounding cells (Fig. 3, A and B). PSK (K57A) and PSK (745-1235), which both localize to
MTs, also produced perinuclear MT cables (Fig. 3,
C-F), and PSK was predominantly localized on
these structures. Moreover, MTs appeared to be thicker in the presence
of PSK, and co-staining for injected PSK and pericentrin, a marker for
the MT-organizing center, demonstrated that the MT-organizing center
was retained in cells expressing PSK (data not shown). PSK (1-940),
which is catalytically active, failed to produce these MT structures,
demonstrating that PSK must directly associate with MTs via its C
terminus for their reorganization to occur (Fig. 3, G and
H).
PSK Stabilizes MTs and Increases Acetylation of
-Tubulin--
The ability of PSK to reorganize MTs into perinuclear
cables raised the possibility that PSK might also alter the stability of MTs. To determine whether PSK could affect MT stability, PSK was
microinjected into cells, and nocodazole (0.5 µM) was
added to the culture medium for 30 min. PSK stabilized MTs in the
presence of nocodazole and prevented the loss of MT structures observed in surrounding uninjected cells (Fig. 4,
A and B). The two other forms of PSK that
localize to MTs, kinase-defective PSK (K57A) and PSK (745-1235), also
produced nocodazole-resistant MTs, and PSK was found wherever MTs
remained (Fig. 4, C-F). MT stabilization by PSK therefore
requires protein localization but not catalytic activity, and this was
again illustrated by the inability of PSK (1-940) to protect MTs from
nocodazole (Fig. 4, G and H).

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Fig. 4.
PSK stabilizes MTs against disruption by
nocodazole. Growing Swiss 3T3 cells were microinjected with
pRK5-MYC-PSK (A and B), pRK5-MYC-PSK (K57A)
(C and D), pRK5-MYC-PSK (745-1235) (E
and F), or pRK5-MYC-PSK (1-940) (G and
H); after 3.5 h, cultures were treated for 30 min with
0.5 µM nocodazole. Cells were fixed and stained to
detect MYC-PSK (A, C, E, and
G) and MTs (B, D, F, and
H). Scale bar, 20 µm.
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Tubulin undergoes several post-translational modifications, including
the acetylation of
-tubulin on lysine residue 40; although this
modification is a consequence, not a cause, of stabilization, the
acetylation of
-tubulin is frequently used to distinguish between
stable and dynamic forms of MTs (reviewed in Ref. 20). We therefore
examined the effects of PSK on the levels of acetylated
-tubulin
using anti-acetylated
-tubulin antibodies. The three forms of PSK
that localize to MTs (PSK, kinase-defective PSK (K57A), and PSK
(745-1235)) each increased levels of acetylated
-tubulin above
those in neighboring uninjected cells (Fig.
5, A-F). The N-terminal
kinase domain of PSK (1-940), which was unable to associate with MTs
or generate nocodazole-resistant MTs, failed to stimulate the
acetylation of tubulin (Fig. 5, G and H). Serum
starvation of Swiss 3T3 cells seemed to result in a more cytoplasmic
localization for full-length PSK, whereas C-terminal PSK (745-1235)
remained tightly associated with the MTs under identical conditions
(Fig. 5, A and E). Nocodazole-resistant and
acetylated MTs are increased in Swiss 3T3 fibroblasts after serum
stimulation (21, 22); however, the PSK effects observed here occured in
the absence of serum.

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Fig. 5.
PSK stimulates the acetylation of
-tubulin. Quiescent Swiss 3T3 cells were
microinjected with pRK5-MYC-PSK (A and B),
pRK5-MYC-PSK (K57A) (C and D), pRK5-MYC-PSK
(745-1235) (E and F), or pRK5-MYC-PSK (1-940)
(G and H). After 3.5 h, cells were
fixed and stained to detect MYC-PSK (A, C,
E, and G) and acetylated MTs (B,
D, F, and H). Scale bar, 20 µm.
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PSK Binds Tubulin in Vitro--
Comparison of the C-terminal
regulatory domain of PSK with other sequences present in the GenBank
data base has shown that PSK does not share significant homology with
any other proteins or with the MT-binding domains reported for MAPs,
which contain a proline-rich sequence and three pseudorepeats (reviewed
in Ref. 17). The ability of the C terminus of PSK (745-1235) to
localize to MTs and produce stabilized perinuclear MT cables led us to investigate whether these sequences were able to interact with
- or
-tubulin in vitro. A C-terminal fragment of PSK fused to glutathione-S-transferase (GST) was prepared, and recombinant PSK
(amino acids 1064-1235) was able to bind
- and
- tubulin in vitro, suggesting that PSK may interact directly with MTs
(Fig. 6, lanes 1-3).

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Fig. 6.
GST-PSK (1064-1235) binds
- and -tubulin in
vitro. 3 µg of tubulin was mixed with glutathione
beads (lane 1), glutathione beads coupled to GST (lane
2), or glutathione beads coupled to GST-PSK (1064-1235)
(lane 3), preblocked with 20% goat serum in
phosphate-buffered saline overnight. Samples were incubated in 1 ml of
kinase buffer (1 h, 4 °C), washed three times in kinase buffer, and
separated using SDS-PAGE/6 M urea. - and -tubulin
were detected by immunoblotting and enhanced chemiluminescence.
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Taxol Dissociates PSK from MTs--
The ability of PSK
to bind and stabilize MTs led us to examine the effect of
Taxol-stabilized MTs on PSK. Swiss 3T3 cells expressing PSK were
incubated in the presence of Taxol for 30 min, and cultures were fixed
and costained for PSK and MTs. Fig. 7
shows that the addition of Taxol causes PSK to dissociate from MTs and
become cytoplasmic (Fig. 7, A and B), whereas the
MTs formed a separate ring around the nucleus (Fig. 7, C and
D). These results demonstrate that PSK can respond to
Taxol-induced changes in MT dynamics and that PSK might dissociate from
MTs that are sufficiently stable.

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Fig. 7.
Taxol dissociates PSK from MTs. Growing
Swiss 3T3 cells were microinjected with pRK5-MYC-PSK; after 3 h,
cultures were treated for 30 min with (B and D)
or without (A and C) 5 µM Taxol.
Cells were fixed and stained to detect MYC-PSK (A and
B) and MTs (C and D). Scale
bar, 20 µm.
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PSK Phosphorylates Tubulin--
The localization of PSK to MTs
brings the kinase into the local vicinity of a number of potential
MT-associated substrates that are regulated by phosphorylation, and
these proteins could lead to additional affects of PSK on MTs. Because
PSK binds tubulin, we investigated the ability of PSK to phosphorylate
tubulin. Plasmids encoding PSK or PSK (K57A) were transiently
transfected into cell cultures, and immune complexes of each protein
were assayed for in vitro kinase activity using
- and
-tubulin as substrates. The isoforms of tubulin were separated from
each other using SDS-PAGE/6 M urea, and we found that PSK,
but not kinase-defective PSK (K57A), phosphorylated
- and
-tubulin (Fig. 8A,
lanes 2 and 3).

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Fig. 8.
PSK phosphorylates -
and -tubulin and is down-regulated by Taxol.
A, growing COS-1 cells were transfected with either PSK
(lanes 1 and 2), kinase-defective PSK (K57A)
(lane 3), or pRK5 vector (lane 4). After 36 h, cell lysates were immunoprecipitated with anti-PSK antibodies, and
immune complexes were subjected to an in vitro kinase assay
using - and -tubulin as substrate. Tubulin was omitted from
lane 1 as a control. Tubulin subunits were separated by
SDS-PAGE/6 M urea, and the subunits were identified by
immunoblotting with - or -tubulin specific antibodies (DM1A or
TUB2.1, respectively). B, human embryonic kidney 293 cells
were transfected with either pRK5-MYC vector alone (lanes 1 and 4), pRK5-MYC-PSK (lanes 2 and 5),
or pRK5-MYC-PSK (1-940) (lanes 3 and 6). After
36 h, cells were treated with (lanes 4, 5,
and 6) or without (lanes 1, 2, and
3) 10 µM Taxol for 30 min. PSK was
immunoprecipitated using anti-PSK antibodies, and immune complexes were
subjected to an in vitro kinase assay using bovine - and
-tubulin as substrate. Immunoprecipitated PSK was detected by
Western blotting.
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Interestingly, addition of the MT-stabilizing agent Taxol rapidly
down-regulated the ability of PSK and, to a lesser extent PSK (1-940),
to phosphorylate tubulin, demonstrating that the catalytic activity of
PSK responds to Taxol-induced changes in MT stability (Fig.
8B, lanes 2, 3, 5, and
6). In addition, transfected PSK, which is normally
expressed in cells as a protein doublet of 185 and 165 kDa (3), was
converted to the smaller form of the protein in the presence of Taxol
(Fig. 8B, lanes 2 and 5). Because 9E10
antibody detects the N-terminal MYC-epitope tag on both forms of PSK
(3), it is likely that the 185-kDa form of the protein is either
truncated at the C terminus to generate a protein of 165 kDa that
potentially lacks MT localization sequences; alternatively, PSK might
undergo a change in post-translational modification. In contrast,
nocodazole had no effect on either the kinase activity or mobility of
PSK (data not shown).
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DISCUSSION |
We have shown that the STE20-like kinase PSK localizes to MTs and
produces stabilized perinuclear MT cables that are nocodazole-resistant and contain increased levels of acetylated
-tubulin. The N-terminal kinase domain of PSK (1-940) is unable to bind MTs or regulate their
organization or stability, demonstrating that the association between
PSK and MTs is required for these MT alterations to occur. The C
terminus of PSK (745-1235) contains the MT localization and regulatory
domain and recombinant PSK (1064-1235) binds
- and
-tubulin
in vitro. The catalytic activity of PSK is not required for
these effects on MTs because kinase-defective PSK (K57A) associates with MTs and regulates their organization and stability. The
MT-stabilizing drug Taxol dissociates PSK from MTs and down-regulates
the protein's catalytic activity, suggesting that PSK is sensitive to
Taxol-induced changes in MT stability.
The stabilized perinuclear MT cables generated by PSK are similar to
the MT structures produced by another STE20, X-PAK5, which binds MTs
and induces stabilized curly MTs that form whorls around the nucleus
(11). X-PAK5, like PSK, binds to MTs via its non-catalytic regulatory
domain, and neither the stabilization nor the reorganization of MTs
require the protein's catalytic activity (11). The Rho effector Rho
kinase
and DCAMKL1 also generate MT clusters around the nucleus
independently of their kinase activity, and another non-enzymatic
protein, mDia, co-localizes and stabilizes MTs orientated toward the
wound edge (23, 24). Interestingly, the ability of X-PAK5 to bind MTs
is negatively regulated by its kinase activity because constitutively
activated X-PAK5 is unable to bind MTs and relocates to the cytoplasm,
where it causes dissolution of actin stress fibers and cell retraction (11). PSK also down-regulates actin stress fibers and induces cell
rounding. and another STE20 SLK associates with MTs located at adhesion
sites and reduces actin stress fibers (3, 16). Interestingly, the
catalytic activity of PSK is required for its effects on actin. and it
is plausible that the kinase activity of PSK might act in a manner
similar to X-PAK5 and regulate its localization and function (3). How
PSK kinase activity is regulated in vivo is not known, but
we have been unable to detect clear differences between the
localization of PSK and kinase-defective PSK (K57A) in injected cells.
PSK does, however, become more cytoplasmic when cells are starved of
serum, whereas the C terminus of PSK (745-1235) lacking the kinase
domain, remains tightly bound to MTs. The identification of upstream
regulators for PSK and its kinase activity will be required to explore
these possibilities further.
Catalytic activity is not required by PSK to re-organize and stabilize
MTs, but the localization of PSK to MTs would be expected to bring the
kinase into the vicinity of a number of potential MT-associated
substrates. PSK could therefore have additional regulatory affects on
MTs that require its catalytic activity. PSK phosphorylates
- and
-tubulin, and others have shown that Syk and Src phosphorylate
tubulin (25, 26). The physiological significance of this modification
and the sites of phosphorylation are unknown, but it has been suggested
that the phosphorylation of tubulin could alter its binding properties
to signaling proteins via SH2 domain-mediated interactions and/or
regulate the accessibility of kinases to their substrates (25, 26).
Although a number of kinases can regulate the activity and function of
MT components, such as MAPs, by phosphorylation, only a few kinases
have been shown to localize to MTs. Interestingly, MT-associated
kinases include several components of MAPK signaling pathways, such as
JNK and ERK (MAPKs), MAP kinase/ERK kinase (MEK), mixed-lineage kinase,
X-PAK5 and SLK (MAPK kinase kinase kinases) (11, 16, 27-29). PSK can
activate JNK (3), as can its rat homolog, Tao2 (30), and it is
therefore possible that this occurs on MTs. Whether JNK is involved in
regulating MT stability remains to be determined, but JNK, ERK, p38,
and glycogen synthase kinase-3
can each phosphorylate MAPs, such as
tau, potentially regulating its affinity for MTs and their stability
(31). Moreover, the MT-stabilizing agent Taxol activates JNK via
mixed-lineage kinases such as MAP kinase/ERK kinase 1 and
apoptosis-regulating kinase (32, 33), but our finding that Taxol
down-regulates the activity, expression, and localization of PSK
indicates that PSK is unlikely to contribute to Taxol-induced JNK
activation. Interestingly, ERK kinase activity has been shown to
decrease the stability of MTs (34). In contrast, phosphorylation and
inactivation of the MT-destabilizing protein stathmin stabilizes MTs,
and the STE20 PAK can modulate the phosphorylation of stathmin (19);
however, we have found that immune complexes of PSK were unable to
phosphorylate stathmin on Ser-16, -25, or -38 (data not shown),
suggesting that PSK does not regulate the stability of MTs via
stathmin. Another MT-associated protein, disheveled,
generates stabilized and acetylated MTs by inhibiting glycogen synthase
kinase-3
and reducing its ability to phosphorylate MAPs (35).
Dishevelled activates JNK (36), as does PSK (2), but the effects of PSK
on glycogen synthase kinase-3
are unknown. PSK does bind directly to
MTs in vitro, whereas glycogen synthase kinase-3
has been
shown to bind MTs indirectly via its association with the neuronal MAP tau, and JNK may interact with MTs via JNK-interacting proteins (37,
38).
The ability of PSK, SLK, and X-PAK5 to regulate MTs and actin filaments
suggests that these STE20 proteins could function in the regulation of
both cytoskeletal networks. Similarly, there is growing evidence that
Rho family members (Rho, Rac, and Cdc42) may coordinately regulate both
networks (39). MT growth activates Rac, itself a MT binding protein,
and promotes actin polymerization and lamellipodial protrusion,
whereas MT disassembly stimulates Rho and the formation of
actin stress fibers and focal adhesions (40, 41). The Rho guanine
nucleotide exchange factors (GEFs), p190 Rho GEF, GEF-H1, and Lfc bind
MTs and may locally activate Rho (42-44). Lfc activates JNK via STE20s
such as PAK or mixed-lineage kinase (43), and it is possible that PSK,
which activates JNK, might also interact with Lfc or another
MT-associated Rho GEF. In addition, X-PAK5, which contains a CRIB
domain, is targeted away from MTs toward actin-rich structures by Rac
and Cdc42, and PSK might be regulated in a similar manner by small
GTP-binding proteins that do not require a CRIB domain (11).
In conclusion, MTs play important roles in the regulation
of cell morphogenesis, division, migration, and vesicle trafficking, and the ability of MTs to undergo changes in their stability and dynamics are crucial for their function. Here we show for the first
time that a member of the GCK family of protein kinases, which lack
CRIB domains, can localize to MTs and regulate their stability and
organization. These findings imply a functional role for PSK, and
perhaps other GCKs, in the regulation of MT dynamics.