1 University of British Columbia, Faculty of Dentistry, Department of Oral
Biological and Medical Sciences, 2199 Wesbrook Mall, Vancouver, BC V6T 1Z3,
Canada
2 Kinexus Bioinformatics Corporation, Suite 401, 2389 Health Sciences Mall,
Vancouver, BC V6T 1Z4, Canada
3 University of Toronto, Faculty of Dentistry, Department of Biological and
Diagnostic Sciences, 150 College Street, Toronto, ON M5S 3E2, Canada
* Author for correspondence (e-mail: larjava{at}interchange.ubc.ca)
Accepted 28 May 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Fibronectin, Wound healing, Calcium, EGF, Migration
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Structures resembling the cytoplasmic extensions of keratinocytes in wounds
have been observed previously in cultured mouse epidermal cells and
megakaryoblastic leukemia cells following exposure to the protein kinase
inhibitor, staurosporine (Sako et al.,
1988; Yamazaki et al.,
1999
). Similarly, rat pheochromocytoma PC12 cells exhibit neurite
outgrowth after stimulation with staurosporine
(Rasouly et al., 1994
;
Rasouly et al., 1996
).
Although the signaling pathways underlying these phenomena are not defined,
there may be a common mechanism that also controls the formation of cellular
extensions in keratinocytes. Accordingly, staurosporine may provide a useful
tool to study the cellular signaling events that regulate extended
lamellipodia formation.
Staurosporine is a broad-spectrum protein-serine/threonine kinase inhibitor
whose function is dependent on interactions with the ATP-binding site of its
target kinases. As staurosporine can either stimulate or inhibit multiple
protein kinases in a cell-type specific manner, multiple potential targets may
be involved in the formation of cytoplasmic extensions. For example, molecular
targets of staurosporine that can regulate cell shape include protein kinases
C (PKC1), A, G and B/Akt as well as
Ca2+/calmodulin-dependent kinase, myosin light chain kinase,
p21-activated kinase 2, 3'-phosphoinositide-dependent protein kinase 1
and Rho-associated kinase (Tamaoki et al.,
1986; Herbert et al.,
1990
; Feng et al.,
1999
; Masure et al.,
1999
; Zeng et al.,
2000
; Hill et al.,
2001
). Moreover, the effects of staurosporine appear to be
strongly influenced by the cell type that is studied, the composition of the
matrix and the concentration of staurosporine. Consequently, different studies
employing staurosporine often present contradictory conclusions. For example,
staurosporine can either inhibit or activate PKC in keratinocytes, depending
on the nature of the experimental model
(Sako et al., 1988
;
Dlugosz and Yuspa, 1991
;
Matsui et al., 1992
;
Matsui et al., 1993
;
Jones and Sharpe, 1994
;
Sudbeck et al., 1994
;
Stanwell et al., 1996
).
In this study, several different protein-serine/threonine kinase inhibitors, including staurosporine, promoted dose-dependent spreading and extended lamellipodia (E-lam) formation in keratinocytes seeded on fibronectin. Our major finding is that staurosporine and epidermal growth factor can induce E-lam formation, and that ultimately, the activity of glycogen synthase kinase-3 (GSK-3) is required for epithelial cell migration and E-lam formation.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Electron microscopy of 3-day-old wounds
Wounds were created in the palatal gingiva of human volunteers with a
scalpel (2-3 mm deep, 2 mm wide and 8 mm long). The protocol was approved by
the Clinical Research Ethics Board of the University of British Columbia.
Tissue biopsies from 3-day-old wounds were fixed in 2% glutaraldehyde for 2
hours, impregnated with 1% osmium tetraoxide for 30 minutes, 2% uranylacetate
for 30 minutes, alcohol-dehydrated and embedded in Epoxy-resin (Epon 812). For
transmission electron microscopy, 500 nm thick sections were cut with a glass
knife (Microtome MT 6000 Sorvall, Dupont, Wilmington, DE) and stained with 1%
Toluidine Blue/1% borax for 5 minutes on a hot plate. Sections (80 nm thick)
were made with a diamond knife and placed on nickel grids, stained with 5%
uranylacetate for 20 minutes and with lead citrate for 5 minutes, and then
viewed and photographed with a transmission electron microscope (Philips 300,
Philips, Eindhoven, Holland).
Cell culture
The immortalized epidermal keratinocyte cell line HaCaT (a generous gift
from Dr Norbert Fusenig, German Cancer Center, Heidelberg, Germany) was
maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL Life
Technologies, Rockville, Maryland, USA) supplemented with 23 mM sodium
bicarbonate, 20 mM Hepes, antibiotics (50 µg/ml streptomycin sulfate, 100
U/ml penicillin) and 10% heat-inactivated fetal calf serum (FCS; Gibco BRL).
The HaCaT cell line models many of the properties of normal epidermal
keratinocytes, is not invasive and can differentiate under appropriate
experimental conditions (Boukamp et al.,
1988). Cells were always subcultured for 2 days before experiments
to maintain consistency throughout the study. Primary mouse keratinocytes were
isolated and maintained as described previously
(Häkkinen et al.,
2002
).
To study whether growth factors could induce lamellipodia formation in
HaCaT keratinocytes, cells were seeded on 6-well plates (30,000 per well) in
complete growth medium. The next day, cells were changed to DMEM containing 1%
FCS and epidermal growth factor (EGF; Invitrogen), transforming growth factor
ß1 (TGFß1; Chemicon, Temencula, CA, USA), tumor necrosis factor
(TNF
; Chemicon) or keratinocyte growth factor (KGF; Upstate,
Lake Placid, NY, USA). The morphology of the cells was recorded by digital
photography after 48 hours in culture.
Cell spreading assays
Cell spreading experiments were done essentially as described before
(Koivisto et al., 1999). Cell
culture plates were coated with fibronectin (2 µg/cm2; from
bovine plasma; Chemicon). Unoccupied sites were blocked with 1% heat-denatured
bovine serum albumin. HaCaT cells (30,000/cm2) were seeded on
plates in serum-free DMEM containing 50 µM cycloheximide to prevent de novo
protein synthesis. Inhibitors were added either at the moment of seeding, or
cells were allowed to attach and spread for 120 minutes prior to adding the
agents, depending on the nature of the experiment to be performed. To quantify
the effect of the drugs on cell spreading and E-lam formation, cells were
fixed by carefully adding formaldehyde (8% formaldehyde and 10% sucrose in
phosphate-buffered saline (PBS) containing Ca2+ and
Mg2+). The wells were filled with distilled water to the top and
covered with a glass plate for observation by phase-contrast microscopy. Four
representative fields from each replicate well were studied. The percentages
of spread cells and cells with E-lams of total cell number in each field were
counted. Cell with lamellar cytoplasm were considered as spread.
Alternatively, cells were collected and lysed at certain time points and
analyzed for protein phosphorylation.
To examine the more detailed morphology of spreading keratinocytes, HaCaT
cells were allowed to spread on fibronectin-coated coverslips in the presence
or absence of staurosporine followed by fixation with glutaraldehyde (2.5% in
0.1 M PBS) and preparation for scanning electron microscopy
(Aoki and Tavassoli, 1981;
Firth et al., 1997
).
Immunoblotting
Cells were allowed to spread on fibronectin and treated with staurosporine
as described above and lysed at designated time points in Tris-buffered saline
(pH 7.6) containing 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin,
5 mM EGTA, 1 mM sodium orthovanadate, 5 mM sodium fluoride, 100 mM n-octyl
ß-D-glucopyranoside and 0.5% Nonidet P-40. Cell lysates were separated by
SDS-PAGE, transferred onto Hybond ECL membrane (Amersham, Little Chalfont,
Buckinghamshire, UK) and immunoblotted with antibodies recognizing
phospho-GSK-3/ß (Y279/Y216; BioSource
International, Camarillo, CA, USA or S21/S9; Cell
Signaling Technology, Beverly, MA, USA) or total GSK-3 (0011-A; Santa Cruz
Biotechnology, Santa Cruz, CA, USA). Peroxidaseconjugated IgGs (Santa Cruz
Biotechnology) were used as secondary antibodies. Detection was performed with
Amersham's ECL kit. Cell lysates with or without staurosporine treatment were
also probed for the quantitative expression of 33 known phosphoproteins using
validated commercial antibodies in the KinetworksTM Phospho-Site Screen
(KPSS 1.0), immunoblotting analysis provided by Kinexus Bioinformatics
Corporation (Vancouver, Canada). For this screen, cells were treated with
staurosporine either for 10 minutes, which preceding E-lam formation, or for
60 minutes, by which time the E-lams have formed. Untreated control samples
were collected at the same time points.
Immunocytochemistry
Cells were allowed to spread on fibronectin-coated SonicSeal SlideTM
wells (Nunc, Naperville, IL, USA), treated with staurosporine as described
above, formaldehyde-fixed and permeabilized with PBS containing 0.5% Triton
X-100. Immunolocalization was performed as described previously
(Larjava et al., 1990) using
antibodies recognizing phospholipase C
-1 phosphorylated on Tyr-783
(Sigma) or GSK-3 (0011-A; Santa Cruz Biotechnology). Species-specific Alexa
FluorTM 546 IgG molecules (Molecular Probes, Eugene, OR, USA) were used
as secondary antibodies. Alternatively, anti-mouse Vectastain® ABC and
Vector® VIP peroxidase substrate kits (Vector laboratories Inc.,
Burlingame, CA, USA) were used for immunodetection of GSK-3. Rhodamine-labeled
phalloidin (Sigma) was used to detect actin filaments in acetone-fixed
samples.
Measurement of intracellular Ca2+
Cells were plated and allowed to spread on fibronectin-coated coverslips as
described above and loaded with fura 2/AM (2 µM; Molecular Probes). Single
cells were measured with an inverted microscope optically interfaced to a
Photon Technology Incorporated (London, ON) ratio fluorimeter. The slit widths
were set at 3 nm and excitation was set at alternating wavelengths (100 Hz) of
346 and 380 nm. Measurements were conducted at room temperature in serum-free
PBS containing 20 mM Hepes buffer to maintain the pH at 7.4. A stable baseline
Ca2+ ratio was obtained for 50 seconds after which 50 nM
staurosporine was added. All cells were measured for 500 seconds.
Cell migration
For scratch wound migration assays, HaCaT cells were seeded on 24-well
plates (1.7x105 cells per well) in DMEM containing 10% FCS.
The cells were grown to confluence for 2 days after which the cultures were
scratch-wounded with a pipette tip. Loose cells were removed by washing with
PBS, and the cells were allowed to migrate for 24 hours in Minimum Essential
Medium (EMEM; Bio-Whittaker, Walkersville, MD, USA) supplemented with 1% FCS
in the presence of staurosporine (50 nM) or EGF (10 ng/ml) and inhibitors. The
inhibition in cell migration produced by GSK-3 inhibitors was quantified by
comparing the number of cells within the wound area of inhibitor-treated
samples with the number of cells in control wounds. Eight representative
fields from each wound were studied.
Statistical analysis
All experiments were repeated at least three times. Each of the spreading
assays used two to four replicate wells, and four fields of cells for each
well were counted (n=8-16). Statistical significance was set at
P<0.05, and the difference between two groups was calculated using
unpaired, two-tailed Student's t-test.
![]() |
Result |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Staurosporine induces extended lamellipodia and cell spreading in
human keratinocytes on fibronectin
To focus on a model that exhibited more rapid induction of E-lams and that
would permit study of key signaling events in E-lam formation, we tested
whether the protein-serine/threonine kinase inhibitor staurosporine could
accelerate E-lam formation in keratinocytes. Staurosporine has been shown to
promote lamellipodia extension in other cell types
(Sako et al., 1988;
Rasouly et al., 1994
;
Rasouly et al., 1996
;
Yamazaki et al., 1999
).
Accordingly, HaCaT cells were seeded on to fibronectin-coated substrata in the
presence of staurosporine, a treatment that induced keratinocytes to form
morphologically similar E-lams as seen in EGF-treated keratinocytes. The
ability of staurosporine to induce formation of E-lams was not restricted to
HaCaT keratinocytes as the same phenomenon was observed with primary mouse and
human epidermal keratinocytes (data not shown). In contrast to EGF,
staurosporine induced E-lams within 60 minutes in the absence of de novo
protein synthesis (Fig. 2Ab).
Staurosporine, therefore, possibly bypassed the requirement for EGFR
signaling, as neither EGF (30 ng/ml) nor tyrphostin AG1478 (2 µM) exerted
any effect on staurosporine-induced E-lam formation (data not shown).
Staurosporine also induced a dose-dependent increase in spreading of
keratinocytes on fibronectin. Maximal induction of spreading was achieved with
10 nM staurosporine. E-lam formation was, however, typically initiated at
concentrations above 30 nM staurosporine
(Fig. 2B). Staurosporine was
able to induce E-lams to the same extent in spreading cells and in cells that
had already spread (Fig. 2C).
This characteristic allowed us to investigate signaling mechanisms of E-lam
formation that are independent of cell spreading. Scanning electron microscopy
was used to visualize staurosporine-induced morphological changes at higher
resolution. In addition to E-lam formation, cells treated with 50 nM
staurosporine exhibited fine filopodia that projected from the lamellipodia
(Fig. 2A).
|
To confirm that E-lam formation was associated with the inhibition of protein-serine/threonine kinases, two other broad-spectrum protein-serine/threonine kinase inhibitors, H7 and K252a, were tested for their ability to induce E-lam formation in cells plated on fibronectin. Both inhibitors were able to induce cell spreading and E-lam formation, although they were less potent than staurosporine, indicating that E-lam formation was indeed related to inhibition of protein-serine/threonine kinases. For H7, maximal cell spreading was induced at 400 nM, whereas E-lams did not start to form until concentrations above 2 µM were used (data not shown). For K252a, both maximal cell spreading and the onset of E-lam formation were reached at 50 nM (data not shown). The maximal incidence of E-lam formation with H7 and K252a never exceeded 20% of the cells (data not shown). At 50 nM staurosporine, E-lams that were morphologically similar to those induced by EGF occurred in 15-25% of cells. Higher concentrations of staurosporine increased the proportion of cells exhibiting E-lams but their morphology became more irregular (Fig. 2B and data not shown). Accordingly, this 50 nM concentration was used in further studies that focused on the signal transduction mechanisms regulating lamellipodia formation in keratinocytes.
To determine whether the formation of E-lams also required changes in
tyrosine phosphorylation in addition to the inhibition of
protein-serine/threonine kinases, cells were allowed to spread on fibronectin
and then treated with staurosporine (50 nM) in combination with the
nonselective protein-tyrosine kinase inhibitors (herbimycin A; 10 µM,
genistein; 200 µM) or a protein-tyrosine phosphatase inhibitor (sodium
orthovanadate; 500 µM). Both herbimycin A
(Fig. 3E,F) and genistein
potently inhibited E-lam formation (Fig.
3G,H), indicating that E-lam formation was dependent on tyrosine
phosphorylation that is mediated by protein-tyrosine kinases other than EGFR
tyrosine kinase. As indicated above, E-lam formation was unresponsive to the
inhibition of EGFR tyrosine kinase. In agreement with the importance of
tyrosine phosphorylation in E-lam formation, sodium orthovanadate increased
the proportion of cells forming E-lams from 20% to
60%
(Fig. 3B,D). In control cells
without staurosporine, sodium orthovanadate caused cell retraction
(Fig. 3A,C).
|
Staurosporine induces GSK-3 activation
To examine changes in protein phosphorylation that are involved in E-lam
formation, cells treated with 50 nM staurosporine were analyzed by Kinexus
KinetworksTM Phospho-Site Screen (data not shown). The results indicated
that staurosporine may increase GSK-3 activation. Notably, GSK-3 is a major
regulator of the cytoskeleton in other cell types
(Wagner et al., 1996). The
activity of GSK-3
/ß is mainly regulated by inhibitory
phosphorylation on serine residues (S21/S9)
(Grimes and Jope, 2001
).
Accordingly, we found that staurosporine strongly induced dephosphorylation of
GSK-3
/ß on serine 21/9 (Fig.
4A,B). The reduction of GSK-3 serine phosphorylation was
detectable within 1-5 minutes after incubation with staurosporine
(Fig. 4A,B). Both
and
ß GSK-3 isoforms were dephosphorylated at serine residues by
staurosporine treatment (Fig.
4A,B). Tyrosine phosphorylation of GSK-3
/ß on residues
279/216 is a prerequisite for its activity
(Hughes et al., 1993
).
Tyrosine phosphorylation of GSK-3 was not markedly altered by staurosporine
(Fig. 4C). Thus, staurosporine
induced changes in GSK-3 phosphorylation that were consistent with its
activation.
|
Extended lamellipodia formation requires the activity of GSK-3
The direct involvement of GSK-3/ß in E-lam formation was tested
by treating cells with staurosporine in the presence of the GSK-3 inhibitors
LiCl2 and SB-415286 (Klein and
Melton, 1996
; Coghlan et al.,
2000
). Both of these GSK-3 inhibitors caused a specific and
concentration-dependent reduction of the incidence of E-lams without a change
in general cell morphology. This experiment indicated that GSK-3 activity is
specifically involved with the extension of E-lams but does not affect
lamellar spreading (Fig. 5A-C).
In agreement with this function, immunostaining for GSK-3 was restricted to
the extended part of the E-lam in staurosporine-treated cells and was not
present in lamellae (Fig.
6B,C). In EGF-treated cells, the localization of GSK-3 was more
diffuse, but it was still more abundant away from cellular perimeters (data
not shown).
|
|
Intracellular free Ca2+ is essential for E-lam
formation
GSK-3 is activated in response to an increase in intracellular
Ca2+ (Hartigan and Johnson,
1999), and staurosporine can induce a long-lasting increase of
intracellular free Ca2+ in primary human keratinocytes
(Jones and Sharpe, 1994
). We
found that in HaCaT cells, 50 nM staurosporine induced a slow and steady
increase in cytoplasmic free Ca2+ that was detectable within 30
seconds after adding the staurosporine and reached a plateau in 300 seconds
(in 6 out of 15 cells measured; 40%). A typical response is shown in
Fig. 7A. None of the cells
treated with vehicle only showed any Ca2+ increase (data not
shown). To test the importance of this Ca2+ influx for E-lam
formation, we treated spread HaCaT cells with either BAPTA/AM (100 nM-10
µM) or EGTA (2 mM) to chelate intracellular or extracellular
Ca2+, respectively, together with staurosporine. Chelation of
extracellular Ca2+ by EGTA slightly increased cell spreading but
did not affect E-lam formation (Fig.
7B). The effect of BAPTA/AM on cell morphology, however, was
dependent on staurosporine (Fig.
7B): the spread morphology of control cells was unaffected by
BAPTA/AM. The cells exposed to staurosporine, however, underwent
concentration-dependent collapse in response to BAPTA/AM, and E-lam formation
was completely abolished at 10 µM BAPTA/AM. Staurosporine thus sensitized
the cells to chelation of intracellular Ca2+. The Ca2+
concentration-dependent disappearance of E-lams indicates that intracellular
Ca2+ may play a crucial role in staurosporine-induced cell
spreading and E-lam formation.
|
Treatment with staurosporine induces relocalization of tyrosine
phosphorylated phospholipase C-1 to E-lams and localized actin
assembly
As phospholipase C- (PLC-
) is a key mediator in the pathway
that releases Ca2+ from intracellular stores
(Berridge, 1993
), we tested the
involvement of PLC-
in E-lam formation. Treatment of keratinocytes with
the specific PLC-
inhibitor U-71322 induced a concentration-dependent
loss of cell spreading both in control and staurosporine-treated cells, as
well as inhibition of E-lam formation (Fig.
8A). Thus PLC-
activity was involved in the spreading
behavior of keratinocytes, but a direct role for PLC-
in E-lam
formation could not be ascertained from inhibitor studies. As the activity of
PLC-
1 depends on phosphorylation on Tyr-783
(Kim et al., 1991
), indirect
evidence for the importance of PLC-
1 in E-lam formation was obtained by
immunocytochemistry. These experiments demonstrated localization of active,
tyrosine phosphorylated PLC-
1 at the tips of E-lams and co-localization
with actin filaments (Fig. 8B).
Consistent with the importance of PLC-
in staurosporine-induced
morphological change, disturbance of cellular Ca2+ gradients by
thapsigargin (a sarcoplasmicendoplasmic reticulum ATPase (SERCA) inhibitor;
2 nM) blocked cell spreading (inhibition by 63%, n=8,
P<0.001) and E-lam formation in staurosporine-treated cells.
Thapsigargin also reduced the spreading of control cells but less potently
(inhibition by 25%, n=8, P<0.001).
|
Agents that interfere with cellular Ca2+ signaling
modulate GSK-3/ß serine phosphorylation
Because both the activity of GSK-3 and the intracellular Ca2+
signaling participated in E-lam formation, we tested whether these two
pathways were connected. Notably, the activity of GSK-3 has been reported to
be upregulated by increased intracellular Ca2+
(Hartigan and Johnson, 1999).
Therefore, we tested whether disturbance of cellular Ca2+
metabolism by PLC-
inhibitor U-71322 (4 µM), SERCA inhibitor
thapsigargin (100 nM) or intracellular Ca2+ chelator BAPTAAM (10
µM) that all blocked E-lam formation affected staurosporine-induced serine
dephosphorylation of GSK-3. None of the agents had any significant effect on
tyrosine phosphorylation of GSK-3 (data not shown). Thapsigargin prevented
staurosporine-induced GSK-3 serine dephosphorylation
(Fig. 9A). U-71322 did not
completely prevent but significantly delayed GSK-3 dephosphorylation: in cells
treated with staurosporine only, the dephosphorylation of GSK-3 was evident
after a 5-minute incubation (Fig.
9G), whereas in cells treated with a combination of U-71322 and
staurosporine, the reduction in serine phosphorylation was only evident after
30 minutes incubation (Fig.
9C). In contrast, BAPTA-AM that buffers cytoplasmic
Ca2+ greatly accelerated serine dephosphorylation of GSK-3
(Fig. 9E). Thus, agents that
affect cellular Ca2+ signaling seem to modulate the degree of
serine phosphorylation of GSK-3 in keratinocytes, but the GSK-3
dephosphorylation does not always lead to E-lam formation.
|
Staurosporine-induced cell migration is dependent on GSK-3
activity
To determine if E-lam formation induced by staurosporine is indeed
associated with the migratory phenotype in keratinocytes, cells were allowed
to migrate in the presence of staurosporine in scratch-wounded cultures.
Staurosporine potently induced cell migration resulting in wound closure
within 24 hours (Fig. 10B). Staurosporine also induced the migration of primary mouse keratinocytes,
although these cells were more sensitive to staurosporine and a smaller
concentration was required (data not shown). To investigate whether
keratinocyte migration was dependent on GSK-3 activity, staurosporine- and
EGF-treated HaCaT cells were allowed to migrate in scratch wound assays in the
presence of GSK-3 inhibitors. Consistent with the importance of GSK-3 in E-Lam
formation in keratinocytes, staurosporine-induced migration was completely
blocked by 30 µM SB-415286 and by 30 mM LiCl2
(Fig. 10E,H). EGF-induced cell
migration was also blocked when the wounded cell cultures were incubated with
GSK-3 inhibitors before the addition of EGF to the cells
(Fig. 10F,I), indicating that
GSK-3 activity also contributes to EGF-induced cell migration. If the GSK-3
inhibitors were added to the cells simultaneously with EGF, the cell migration
was still strongly reduced although not totally prevented: SB-415286 and
LiCl2 inhibited the number of cell within wounds by about 35% and
by 45%, respectively, compared to cells treated with EGF only (data not
shown).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
GSK-3/ß catalytic activity is positively regulated by tyrosine
phosphorylation (Y279/Y216) and is inhibited by
phosphorylation on Ser-21/9 (Hughes et
al., 1993
; Grimes and Jope,
2001
). GSK-3 is a serine/threonine kinase, and staurosporine can
inhibit GSK-3 activity in vitro (Leclerc
et al., 2001
). However, this inhibition has not been shown to
occur in living cells. In contrast, Bhat et al.,
(Bhat et al., 2000
) reported an
increase in GSK-3 tyrosine phosphorylation and activity in neuronal cells
treated with staurosporine. In the present study, staurosporine did not,
however, markedly regulate tyrosine phosphorylation of GSK-3. The
protein-tyrosine kinase inhibitor genistein can induce GSK-3 dephosphorylation
and inactivation, and this inhibition can be reversed by the proteintyrosine
phosphatase inhibitor sodium orthovanadate
(Yu and Yang, 1994
;
Yu et al., 1997
). In agreement
with these previous results, we found that treatment of HaCaT cells with
genistein and herbimycin A inhibited E-lam formation whereas sodium
orthovanadate enhanced E-lam formation. In addition to GSK-3, tyrosine
phosphorylation regulates the activities of PLC-
1 and Rac1
(Kim et al., 1991
;
Sastry et al., 2002
),
molecules that are required for E-lam formation. In the present study, we also
demonstrated that treatment of keratinocytes with staurosporine potently
reduced serine phosphorylation of GSK-3, an alteration that has been linked to
its inactivation. Staurosporine has been reported to inhibit cAMP-dependent
protein kinase, PKB/Akt and several isoforms of PKC, all of which can
potentially phoshorylate GSK-3 on Ser-21/9
(Goode et al., 1992
;
Cross et al., 1995
;
Fang et al., 2000
;
Li et al., 2000
). These
findings suggest one potential mechanism for GSK-3 activation by
staurosporine. Based on the relative degree of serine dephosphorylation in
HaCaT cells, GSK-3
shows greater relative activity than GSK-3ß. We
have not, however, been able to discriminate the functions of GSK-3
and
GSK-3ß as both LiCl2 and the specific inhibitor, SB-415286,
inhibit both isoforms to the same extent
(Klein and Melton, 1996
;
Coghlan et al., 2000
).
Staurosporine is commonly used to induce apoptosis; the putative functions
of GSK-3 are often discussed in this context. In concentrations significantly
higher than that used in this study, staurosporine (at 100-500 nM) induces
nuclear translocation of GSK-3ß in neuronal cells, a phenomenon linked to
the onset of apoptosis (Bhat et al.,
2000; Bijur and Jope,
2001
). The narrow window of staurosporine concentrations that
induce E-lam formation indicates a high level of regulation of cell signaling
by staurosporine. At optimal concentrations of staurosporine, GSK-3 localizes
to the nascent E-lam whereas at higher concentrations GSK-3 may associate with
the nucleus. Conceivably, one of the factors that distinguishes normally
regulated wound healing from malignant epithelial invasion is the
susceptibility of the migrating cells to undergo programmed cell death. The
mechanisms that regulate cell migration and apoptosis may be linked, the end
result being determined by the degree of activation and/or cellular location
of regulatory molecules such as GSK-3.
A pool of GSK-3 is active in resting cells, and it can be further activated
by transient Ca2+ influxes
(Hughes et al., 1993;
Hartigan and Johnson, 1999
).
Our findings suggest that an increase in intracellular Ca2+
concentration was involved in staurosporine-induced changes in cell morphology
and probably participated in GSK-3 activation. Staurosporine induced an early
but relatively slow increase in the total level of intracellular free
Ca2+ in HaCaT cells. Similar results demonstrating a slow
Ca2+ increase were obtained previously with primary human
keratinocytes exposed to staurosporine
(Jones and Sharpe, 1994
). The
increase in cytoplasmic Ca2+ influx is likely caused by the release
of Ca2+ from intracellular storage compartments
(Jones and Sharpe, 1994
) and
is consistent with our finding that chelation of extracellular Ca2+
with EGTA did not inhibit E-lam formation whereas the intracellular
Ca2+ chelator BAPTA-AM did. BAPTA-AM induced a selective rounding
of the staurosporine-treated keratinocytes and an inhibition of E-lam
formation but did not affect the untreated control keratinocytes.
The increase of intracellular Ca2+ by staurosporine was probably
mediated by PLC-1, a PLC-
isoform that is expressed by HaCaT
cells (Haase et al., 1997
).
Notably, PLC-
1 is activated by tyrosine phosphorylation
(Kim et al., 1991
). In the
present study, we demonstrated that tyrosine phosphorylated PLC-
1
colocalized with actin filaments at the tips of lamellipodia, in agreement
with previously published data (Yu et al.,
1998
). Conceivably, PLC-
1 activity may also be spatially
restricted in this keratinocyte model system. The importance of PLC-
1
activity in E-lam formation was also suggested by the blockade of E-lam
extension following a disturbance of cellular Ca2+ gradients by
thapsigargin. The treatment of cells with PLC-
1 inhibitor or
thapsigargin also delayed or prevented the staurosporine-induced
dephosphorylation of GSK-3. It appears, therefore, that the localized and
controlled release of Ca2+ by the activity of PLC-
1 is
essential for formation of long lamellipodia and may also contribute to
dephosphorylation of GSK-3 by an unknown mechanism in keratinocytes.
Surprisingly, treatment of HaCaT cells with intracellular Ca2+
chelator BAPTA-AM, which inhibited E-lam formation, accelerated rather than
inhibited GSK-3 serine dephosphorylation. The effects of BAPTA-AM on cellular
Ca2+ are, however, complex, and by buffering intracellular
Ca2+, BAPTA-AM may actually increase the amount of Ca2+
in cells in a high-calcium medium such as DMEM
(Hofer et al., 1998
).
Ca2+ is a ubiquitous intracellular messenger that regulates a
number of cellular processes, and the amplitude, speed and spatiotemporal
patterning of Ca2+ signals affect the way cells interpret them
(Berridge et al., 2000
).
Therefore, these results suggest that intracellular Ca2+ also
regulates other aspects in E-lam formation than just GSK-3 activity, as
witnessed by the coincident enhanced serine dephosphorylation of GSK-3 and
inhibition of E-lam formation by BAPTA-AM. In addition keeping in mind the
broad effect of staurosporine on cellular signaling events, while GSK-3
activity is required for E-lam formation, GSK-3 activation is unlikely the
sole cause of E-lam formation, but it also requires other coordinated changes
in cellular signaling.
As demonstrated in the present and previous studies, long cellular
extensions are found in keratinocytes migrating into the wound provisional
matrix (Odland and Ross, 1968;
Larjava et al., 1996
;
Woodley, 1996
). Long
lamellipodia seen in vitro may represent simple forms of lamellipodia seen
inside three-dimensional matrix in vivo. EGF is a natural wound cytokine that
may regulate the formation of long lamellipodia in vivo. Stimulation of cells
with EGF activates multiple downstream signaling pathways in a complex and
cell-type-specific manner that is not currently completely understood
(Bogdan and Klämbt, 2001
;
Yarden and Sliwkowski, 2001
;
Danielsen and Maihle, 2002
).
It is commonly believed that EGFR activation leads to the activation of
PKB/Akt followed by inactivation rather than activation of GSK-3
(Burgering and Coffer, 1995
).
EGFR activation, however, also leads to activation of Fyn, a member of Src
family tyrosine kinases (Mariotti et al.,
2001
). Interestingly, Fyn has been shown to be involved in GSK-3
activation (Lesort et al.,
1999
). GSK-3ß is rapidly and transiently activated followed
by its inhibition by extracellular stimuli including insulin
(Lesort et al., 1999
). It is
speculated that even this rapid and transient GSK-3ß activation plays a
significant role in modification of cytoskeletal structures in neuronal
development by modulating cellular plasticity and by inducing neuronal
outgrowth (Lesort et al.,
1999
). In fibroblasts, GSK-3
appears to be transiently
deactivated downstream of EGFR activation
(Shaw and Cohen, 1999
),
whereas in human epidermoid carcinoma A431 cells, EGF caused cellular
translocation of active GSK-3
without affecting its overall activity
(Yang et al., 1989
). Induction
of long lamellipodia (E-lams) by EGF in keratinocytes in vitro appears to be a
slow process probably involving several second messenger signals that may
eventually lead to GSK-3 activation. Similarly, keratinocyte migration induced
by EGF may involve cyclic transient activation and inactivation of GSK-3 as
well as modulation of its cellular localization.
Based on the results and the data discussed above, we propose that E-lam
formation involves the activation and membrane translocation of PLC-1.
PLC-
1-induced elevation of intracellular Ca2+ may contribute
to the activation of GSK-3, which mediates E-lam formation by modulating the
organization of the cytoskeleton. We suggest, therefore, that GSK-3 is
potentially a key molecule in re-epithelization during wound repair.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aoki, M. and Tavassoli, M. (1981). OTO method for preservation of actin filaments in electron microscopy. J. Histochem. Cytochem. 29, 682-683.[Abstract]
Berridge, M. J. (1993). Inositol trisphosphate and calcium signalling. Nature 361, 315-325.[CrossRef][Medline]
Berridge, M. J., Lipp, P. and Bootman, M. D. (2000). The versatility and universality of calcium signalling. Nat. Rev. Mol. Cell. Biol. 1, 11-21.[CrossRef][Medline]
Bhat, R. V., Shanley, J., Correll, M. P., Fieles, W. E., Keith,
R. A., Scott, C. W. and Lee, C. M. (2000). Regulation and
localization of tyrosine216 phosphorylation of glycogen synthase kinase-3beta
in cellular and animal models of neuronal degeneration. Proc. Natl.
Acad. Sci. USA 97,
11074-11079.
Bijur, G. N. and Jope, R. S. (2001).
Proapoptotic stimuli induce nuclear accumulation of glycogen synthase kinase-3
beta. J. Biol. Chem.
276,
37436-37442.
Bogdan, S. and Klämbt, C. (2001). Epidermal growth factor signaling. Curr. Biol. 10, R292-R295.[CrossRef]
Boukamp, P., Petrusevska, R. T., Breitkreut, D., Hornung, J., Markham, A. and Fusenig, N. E. (1988). Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte line. J. Cell Biol. 106, 761-771.[Abstract]
Burgering, B. M. and Coffer, P. J. (1995). Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 376, 599-602.[CrossRef][Medline]
Coghlan, M. P., Culbert, A. A., Cross, D. A., Corcoran, S. L., Yates, J. W., Pearce, N. J., Rausch, O. L., Murphy, G. J., Carter, P. S., Roxbee Cox, L. et al. (2000). Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription. Chem. Biol. 7, 793-803.[CrossRef][Medline]
Cohen, P. and Frame, S. (2001). The renaissance of GSK3. Nat. Rev. Mol. Cell. Biol. 2, 769-776.[CrossRef][Medline]
Cross, D. A., Alessi, D. R., Cohen, P., Andjelkovich, M. and Hemmings, B. A. (1995). Inhibition of glycogen synthase kinase-3 by insulin is mediated by protein kinase B. Nature 378, 785-789.[CrossRef][Medline]
Danielsen, A. J. and Maihle, N. J. (2002). The EGF/ErbB receptor family and apoptosis. Growth Factors 20, 1-15.[CrossRef][Medline]
Dlugosz, A. A. and Yuspa, S. H. (1991). Staurosporine induces protein kinase C agonist effects and maturation of normal and neoplastic mouse keratinocytes in vitro. Cancer Res. 51, 4677-4684.[Abstract]
Fang, X., Yu, S. X., Lu, Y., Bast, R. C. Jr, Woodgett, J. R. and
Mills, G. B. (2000). Phosphorylation and inactivation of
glycogen synthase kinase 3 by protein kinase A. Proc. Natl. Acad.
Sci. USA 97,
11960-11965.
Feng, J., Ito, M., Kureishi, Y., Ichikawa, K., Amano, M., Isaka,
N., Okawa, K., Iwamatsu, A., Kaibuchi, K., Hartshorne, D. J. et al.
(1999). Rho-associated kinase of chicken gizzard smooth muscle.
J. Biol. Chem. 274,
3744-3752.
Fenteany, G., Janmey, P. A. and Stossel, T. P. (2000). Signaling pathways and cell mechanics involved in wound closure by epithelial cell sheets. Curr. Biol. 10, 831-838.[CrossRef][Medline]
Firth, J. D., Putnins, E. E., Larjava, H. and Uitto, V. J. (1997). Bacterial phospholipase C upregulates matrix metalloproteinase expression by cultured epithelial cells. Infect. Immun. 65, 4931-4936.[Abstract]
Fulga, T. A. and Rørth, P. (2002). Invasive cell migration is initiated by guided growth of long cellular extensions. Nat. Cell Biol. 4, 715-719.[CrossRef][Medline]
Goode, N., Hughes, K., Woodgett, J. R. and Parker, P. J.
(1992). Differential regulation of glycogen synthase kinase-3
beta by protein kinase C isotypes. J. Biol. Chem.
267,
16878-16882.
Grimes, C. A. and Jope, R. S. (2001). The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling. Prog. Neurobiol. 65, 391-426.[CrossRef][Medline]
Grinnell, F. (1992). Wound repair, keratinocyte activation and integrin modulation. J. Cell Sci. 101, 1-5.[Medline]
Haase, I., Liesegang, C., Henz, B. M. and Rosenbach, T. (1997). Retinoic acid attenuates phospholipase C-mediated signaling in HaCaT keratinocytes. Arch. Dermatol. Res. 289, 533-539.[CrossRef][Medline]
Häkkinen, L., Koivisto, L. and Larjava, H. (2002). An improved method for culture of epidermal keratinocytes from newborn mouse skin. Methods Cell Sci. 23, 189-196.[CrossRef]
Hartigan, J. A. and Johnson, G. V. (1999).
Transient increases in intracellular calcium result in prolonged
site-selective increases in Tau phosphorylation through a glycogen synthase
kinase 3beta-dependent pathway. J. Biol. Chem.
274,
21395-21401.
Herbert, J. M., Seban, E. and Maffrand, J. P. (1990). Characterization of specific binding sites for [3H]-staurosporine on various protein kinases. Biochem. Biophys. Res. Commun. 171, 189-195.[Medline]
Hill, M. M., Andjelkovic, M., Brazil, D. P., Ferrari, S.,
Fabbro, D. and Hemmings, B. A. (2001). Insulin-stimulated
protein kinase B phosphorylation on Ser-473 is independent of its activity and
occurs through a staurosporine-insensitive kinase. J. Biol.
Chem. 276,
25643-25646.
Hoeflich, K. P., Luo, J., Rubie, E. A., Tsao, M. S., Jin, O. and Woodgett, J. R. (2000). Requirement for glycogen synthase kinase-3beta in cell survival and NF-kappaB activation. Nature 406, 86-90.[CrossRef][Medline]
Hofer, A. M., Landolfi, B., Debellis, L., Pozzan, T. and Curci,
S. (1998). Free [Ca2+] dynamics measured in agonist-sensitive
stores of single living intact cells: a new look at the refilling process.
EMBO J. 17,
1986-1995.
Hughes, K., Nikolakaki, E., Plyte, S. E., Totty, N. F. and Woodgett, J. R. (1993). Modulation of the glycogen synthase kinase-3 family by tyrosine phosphorylation. EMBO J. 12, 803-808.[Abstract]
Hughes, P. E. and Pfaff, M. (1998). Integrin affinity modulation. Trends Cell Biol. 8, 359-364.[CrossRef][Medline]
Jones, K. T. and Sharpe, G. R. (1994). Staurosporine, a non-specific PKC inhibitor, induces keratinocyte differentiation and raises intracellular calcium, but Ro31-8220, a specific inhibitor, does not. J. Cell. Physiol. 159, 324-330.[Medline]
Kim, H. K., Kim, J. W., Zilberstein, A., Margolis, B., Kim, J. G., Schlessinger, J. and Rhee, S. G. (1991). PDGF stimulation of inositol phospholipid hydrolysis requires PLC-gamma 1 phosphorylation on tyrosine residues 783 and 1254. Cell 65, 435-441.[Medline]
Kim, L. and Kimmel, A. R. (2000). GSK3, a master switch regulating cell-fate specification and tumorigenesis. Curr. Opin. Genet. Dev. 10, 508-514.[CrossRef][Medline]
Klein, P. S. and Melton, D. A. (1996). A
molecular mechanism for the effect of lithium on development. Proc.
Natl. Acad. Sci. USA 93,
8455-8459.
Koivisto, L., Larjava, K., Häkkinen, L., Uitto, V.-J., Heino, J. and Larjava, H. (1999). Different integrins mediate cell spreading, haptotaxis and lateral migration of HaCaT keratinocytes on fibronectin. Cell Adh. Commun. 7, 245-257.[Medline]
Larjava, H., Peltonen, J., Akiyama, S. K., Yamada, S. S., Gralnick, H. R., Uitto, J. and Yamada, K. M. (1990). Novel function for ß1 integrins in keratinocyte cell-cell interactions. J. Cell Biol. 110, 803-815.[Abstract]
Larjava, H., Haapasalmi, K., Salo, T., Wiebe, C. and Uitto. V. J. (1996). Keratinocyte integrins in wound healing and chronic inflammation of the human periodontium. Oral Diseases 2, 77-86.[Medline]
Larjava, H., Koivisto, L. and Häkkinen, L. (2002). Keratinocyte interaction with fibronectin during wound healing. In Cell Invasion (ed. J. Heino and V.M. Kähäri), pp. 42-64. Landes Biosciences, Eurecah.com, Georgetown, TX.
Lau, K. F., Miller, C. C., Anderton, B. H. and Shaw, P. C. (1999). Expression analysis of glycogen synthase kinase-3 in human tissues. J. Pept. Res. 54, 85-91.[CrossRef][Medline]
Leclerc, S., Garnier, M., Hoessel, R., Marko, D., Bibb, J. A.,
Snyder, G. L., Greengard, P., Biernat, J., Wu, Y. Z., Mandelkow, E. M. et
al. (2001). Indirubins inhibit glycogen synthase kinase-3
beta and CDK5/p25, two protein kinases involved in abnormal tau
phosphorylation in Alzheimer's disease. A property common to most
cyclin-dependent kinase inhibitors? J. Biol. Chem.
276,
251-260.
Lesort, M., Jope, R. S. and Johnson, G. V. (1999). Insulin transiently increases tau phosphorylation: involvement of glycogen synthase kinase-3beta and Fyn tyrosine kinase. J. Neurochem. 72, 576-584.[CrossRef][Medline]
Levizki, A. and Gazit, A. (1995). Tyrosine kinase inhibition: An approach to drug development. Science 267, 1782-1788.[Medline]
Li, M., Wang, X., Meintzer, M. K., Laessig, T., Birnbaum, M. J.
and Heidenreich, K. A. (2000). Cyclic AMP promotes neuronal
survival by phosphorylation of glycogen synthase kinase 3beta. Mol.
Cell. Biol. 20,
9356-9363.
Mariotti, A., Kedeshian, P. A., Dans, M., Curatola, A. M.,
Gagnoux-Palacios, L. and Giancotti, F. G. (2001). EGF-R
signaling through Fyn kinase disrupts the function of integrin alpha6beta4 at
hemidesmosomes: role in epithelial cell migration and carcinoma invasion.
J. Cell Biol. 155,
447-458.
Masure, S., Haefner, B., Wesselink, J. J., Hoefnagel, E.,
Mortier, E., Verhasselt, P., Tuytelaars, A., Gordon, R. and Richardson, A.
(1999). Molecular cloning, expression and characterization of the
human serine/threonine kinase Akt-3. Eur. J. Biochem.
265,
353-360.
Matsui, M. S., Chew, S. L. and DeLeo, V. A. (1992). Protein kinase C in normal human epidermal keratinocytes during proliferation and calcium-induced differentiation. J. Invest. Dermatol. 99, 565-571.[Abstract]
Matsui, M. S., Illarda, I., Wang, N. and DeLeo, V. A. (1993). Protein kinase C agonist and antagonist effects in normal human keratinocytes. Exp. Dermatol. 2, 247-256.[Medline]
Mikhailov, A. and Gundersen, G. G. (1998). Relationship between microtubule dynamics and lamellipodium formation revealed by direct imaging of microtubules in cells treated with nocodazole and taxol. Cell Motil. Cytoskeleton 41, 325-340.[CrossRef][Medline]
Nobes, C. D. and Hall, A. (1995). Rho, rac and cdc42 GTPases: regulators of actin structures, cell adhesion and motility. Biochem. Soc. Trans. 23, 456-459.[Medline]
Odland, G. and Ross, R. (1968). Human wound
repair. I. Epidermal regeneration. J. Cell Biol.
39,
135-151.
Osherov, N. and Levizki, A. (1997). Tyrphostin AG 494 bolcks Cdk2 activation. FEBS Lett. 410, 187-190.[CrossRef][Medline]
Rasouly, D., Rahamim, E., Ringel, I., Ginzburg, I., Muarakata, C., Matsuda, Y. and Lazarovici, P. (1994). Neurites induced by staurosporine in PC12 cells are resistant to colchicine and express high levels of tau proteins. Mol. Pharmacol. 45, 29-35.[Abstract]
Rasouly, D., Shavit, D., Zuniga, R., Elejalde, R. B., Unswotrh, B. R., Yayon, A., Lazarovici, P. and Lelkes, P. I. (1996). Staurosporine induces neurite outgrowth in neuronal hybrids (PC12EN) lacking NGF receptors. J. Cell. Biochem. 62, 356-371.[CrossRef][Medline]
Sako, T., Tauber, A. I., Jeng, A. Y., Yuspa, S. H. and Blumberg, P. M. (1988). Contrasting actions of staurosporine, a protein kinase inhibitor, on human neutrophils and primary mouse epidermal cells. Cancer Res. 48, 4646-4650.[Abstract]
Sastry, S. K., Lyons, P. D., Schaller, M. D. and Burridge,
K. (2002). PTP-PEST controls motility through regulation of
Rac1. J. Cell Sci. 115,
4305-4316.
Shaw, M. and Cohen, P. (1999). Role of protein kinase B and the MAP kinase cascade in mediating the EGF-dependent inhibition of glycogen synthase kinase 3 in Swiss 3T3 cells. FEBS Lett. 461, 120-124.[CrossRef][Medline]
Small, V. J., Rotter, K. and Kaverina, I. (1999). Functional design in the actin cytoskeleton. Curr. Opin. Cell Biol. 11, 54-60.[CrossRef][Medline]
Stanwell, C., Denning, M. F., Rutberg, S. E., Cheng, C., Yuspa, S. H. and Dlugosz, A. A. (1996). Staurosporine induces a sequential program of mouse keratinocyte terminal differentation through activation of PKC isoenzymes. J. Invest. Dermatol. 106, 482-489.[Abstract]
Stenn, K. S. and Malhorta, R. (1992). Epithelialization. In Wound Healing: Biochemical and Clinical Aspects (eds. I. K. Cohe, R. F. Diegelmann and W. J. Lindblad), pp. 115-127. WB Saunders Co., Philadelphia.
Sudbeck, B. D., Parks, W. C., Welgus, H. G. and Pentland, A.
P. (1994). Collagen-stimulated induction of keratinocyte
collagenase is mediated via tyrosine kinase and protein kinase C activities.
J. Biol. Chem. 269,
30022-30029.
Tamaoki, T., Nomoto, H., Takahashi, I., Kato, Y., Morimoto, M. and Tomita, F. (1986). Staurosporine, a potent inhibitor of phospholipid/Ca++ dependent protein kinase. Biochem. Biophys. Res. Commun. 135, 397-402.[Medline]
Wagner, U., Utton, M., Gallo, J. M. and Miller, C. C.
(1996). Cellular phosphorylation of tau by GSK-3 beta influences
tau binding to microtubules and microtubule organisation. J. Cell
Sci. 109,
1537-1543.
Woodgett, J. R. (1990). Molecular cloning and expression of glycogen synthase kinase-3/factor A. EMBO J. 9, 2431-2438.[Abstract]
Woodgett, J. R. (1991). cDNA cloning and properties of glycogen synthase kinase-3. Methods. Enzymol. 200, 564-577.[Medline]
Woodley, D. T. (1996). Reepithelization. In The Molecular and Cellular Biology of Wound Repair (ed. R. A. F. Clark), pp. 339-354. NY: Plenum Press.
Yamazaki, Y., Sanokawa, R., Fujita, Y., Zhou, D., Kawasaki, K., Tanaka, H., Komatsu, T., Nagasawa, T. and Oka, S. (1999). Cytoplasmic elongation and rupture in megakaryoblastic leukemia cells via activation of adhesion and motility by staurosporine on fibronectin-bound substratum. J. Cell. Physiol. 179, 179-192.[CrossRef][Medline]
Yang, S. D., Chou, C. K., Huang, M., Song, J. S. and Chen, H.
C. (1989). Epidermal growth factor induces activation of
protein kinase FA and ATP.Mg-dependent protein phosphatase in A431 cells.
J. Biol. Chem. 264,
5407-5411.
Yarden, Y. and Sliwkowski, M. X. (2001). Untangling the ErbB signalling network. Nat. Rev. Mol. Cell. Biol. 2, 127-137.[CrossRef][Medline]
Yu, H., Fukami, K., Itoh, T. and Takenawa, T. (1998). Phosphorylation of phospholipase Cgamma1 on tyrosine residue 783 by platelet-derived growth factor regulates reorganization of the cytoskeleton. Exp. Cell Res. 243, 113-122.[CrossRef][Medline]
Yu, J. S. and Yang, S. D. (1994). Tyrosine dephosphorylation and concurrent inactivation of protein kinase FA/GSK-3 alpha by genistein in A431 cells. J. Cell. Biochem. 56, 131-141.[Medline]
Yu, J. S., Chen, H. C. and Yang, S. D. (1997). Reversible tyrosine phosphorylation/dephosphorylation of proline-directed protein kinase FA/glycogen synthase kinase-3alpha in A431 cells. J. Cell. Physiol. 171, 95-103.[CrossRef][Medline]
Zeng, Q., Lagunoff, D., Masaracchia, R., Goeckeler, Z., Cote, G.
and Wysolmerski, R. (2000). Endothelial cell retraction is
induced by PAK2 monophosphorylation of myosin II. J. Cell
Sci. 113,
471-482.