 |
INTRODUCTION |
Protein-tyrosine phosphorylation plays a critical role in
regulating various cellular events, including cell proliferation and
differentiation (1). Protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs)1 function
dynamically to maintain cellular homeostasis (2, 3). PTPs catalyze the
hydrolysis of phosphoryl groups on Tyr residues in proteins. Two
classes of PTPs have been identified, the receptor-like,
membrane-spanning PTPs, which may act as receptors, and the cytosolic
PTPs characterized as intracellular low molecular weight enzymes (1).
PTP
is a member of the receptor-like PTP family with a relatively
short (123-amino acid) extracellular domain that is highly
glycosylated (4). Like most transmembrane PTPs, PTP has two
homologous cytoplasmic catalytic domains: the domain proximal to the
cell membrane, which exhibits the majority of protein-tyrosine
phosphatase activity, and the second domain, which has low but
detectable phosphatase activity (5, 6).
PTP is widely expressed in mammalian tissues and has been implicated
in a variety of signaling pathways. It has been suggested that PTP
plays an important role in regulating insulin signaling. In baby
hamster kidney cells, PTP appeared to be a negative regulator of the
insulin receptor tyrosine kinase, which transmits its signal by
tyrosine phosphorylation of insulin receptor substrates-1 and -2 (7).
In GH4 pituitary cells, PTP blocked the effect of insulin to
increase prolactin gene expression (8). Rat adipose cells
overexpressing PTP showed significantly lower insulin-stimulated levels of cell surface GLUT4 and a 3-fold decrease in insulin sensitivity (9). Although, in the first study (7), PTP overexpression decreased insulin receptor Tyr phosphorylation, the
demonstration that the insulin receptor is a substrate of PTP has
not been a consistent finding (8, 10).
PTP has also been suggested to be involved in Grb2-mediated
signaling (11). Thus PTP can be tyrosine-phosphorylated in its
carboxyl-terminal domain (Tyr-789) forming a binding site for the SH2
domain of Grb2 (6, 12). Because PTP·Grb2 complexes have
been isolated but are not associated with the exchange factor mSOS, the
function of this binding is not clear (6). PTP may also participate
in the activation of MAPK and the c-Jun transcription factor (13).
Recently, p130cas, which is localized to focal adhesions,
has been shown to interact with and be a substrate for PTP (14).
The most clearly defined substrate of PTP is the protein-tyrosine
kinase Src (pp60c-Src) (15, 16). PTP activates Src
in vitro and in vivo, and overexpression of
PTP leads to the dephosphorylation and activation of cytoplasmic Src
(15, 16). PTP was found to be overexpressed in late-stage colon
carcinoma (17) where Src is commonly found to be activated. Furthermore, PTP null cells (PTP
/ cells) derived
from PTP knock-out mice have greatly reduced Src kinase activity and
are defective in cell adhesion and spreading, all of which are restored
upon ectopic expression of PTP (18, 19). Regulation of Src activity
by PTP is believed to occur by the dephosphorylation of the
carboxyl-terminal negative regulatory Tyr site (Tyr-527) of Src (16).
Recently, it has been demonstrated that the binding of the Src SH2
domain to the phosphorylated carboxyl-terminal Tyr-789 in PTP
results in displacement of Src pTyr-527 from the SH2 domain, thus
allowing PTP to dephosphorylate pTyr-527, and thereby specifically
activating Src (20).
PTP has been shown to play a role in both cellular differentiation
and cellular transformation. Thus overexpression of PTP in rat
embryo fibroblasts caused transformation, whereas in P19 embryonal
carcinoma cells this induced neuronal differentiation (15, 16). Both
actions were associated with Src pTyr-527 dephosphorylation and
increased Src activity (21, 22). In addition, overexpression of PTP
enhanced the development of neurotransmitter response during neuronal
differentiation (23), and an up-regulated expression of PTP was
observed during differentiation of embryonic stem cells into a
neuronal phenotype (24).
Differentiation of skeletal muscle precursor cells is a highly ordered
multistep process initiated in response to a number of environmental
signals (25). Extensive studies of this process have demonstrated that
it involves expression of muscle regulatory factors, withdrawal from
the cell cycle, induction of muscle-specific gene expression, and
changes of the cytoskeleton into specialized structures.
Morphologically, muscle differentiation is characterized by cell
alignment, elongation, fusion, and formation of multinucleated myotubes. This process is accompanied by an increase in the expression of muscle-specific proteins such as myogenin, myosin heavy chain, and
creatine kinase (25). Some hormones and growth factors, in particular
IGF-1 (26, 27) and insulin (28), appear to stimulate differentiation
through various signal transduction pathways. Thus, activation of both
phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase
is associated with and appears to be required for muscle
differentiation (29-31). On the other hand, the specific
noncompetitive inhibitor of MAPK/extracellular signal-regulated kinase
kinase (MAPK kinase), PD098059, partially inhibited the fusion of
myoblasts to multinucleated myotubes without affecting the expression
of muscle-specific markers (32).
In this study, L6 cells were transfected with either PTP antisense,
with full-length human or mouse wild type, or double catalytic site Cys
to Ala phosphatase-dead mutant PTP cDNA with the ultimate aim of
examining the effects of PTP on insulin metabolic effects. During
these studies we observed unexpected alterations in cell growth and
myogenic differentiation. Thus, although cells harboring PTP
antisense displayed diminished PTP protein and exhibited a slowed
growth rate, overexpressing PTP enhanced cell growth. More
significantly, the PTP antisense myoblasts were incapable of fully
differentiating into myotubes, progressing only to alignment in low
serum-containing medium that promoted wild-type L6 cells to fully
differentiate. In contrast, overexpression of PTP accelerated
myotube formation and differentiation. Overexpression of mutant
phosphatase-dead PTP resulted in a phenotype similar to that of
antisense consistent with a dominant-negative effect. As reported
previously, the extent of PTP expression and activity correlated
with dephosphorylation of pTyr-527 and activation of Src. Moreover,
inhibition of Src kinase activity with an Src kinase family-specific
inhibitor, PP2, completely blocked the formation of myotubes in L6
cells and in the cells overexpressing PTP. These data indicate that
a PTP-c-Src signaling pathway is involved in the process of myogenesis.
| |
EXPERIMENTAL PROCEDURES |
Materials--
The anti-PTP antibody was prepared by
injecting rabbits with a GST-PTP cytoplasmic domain fusion protein.
The Myogenin (M-225), SRC2, Src (N16), and FAK antibodies were obtained
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), Src (GD11) was
from Upstate Biotechnology, Inc. (Lake Placid, NY), and anti-myosin
heavy chain (MF-20) was obtained from the Developmental Studies
Hybridoma Bank, University of Iowa (Iowa City, IA). Nitrocellulose
membrane Hybond-C, anti-mouse IgG-horseradish peroxidase, anti-rabbit
IgG-horseradish peroxidase, and the ECL reagents were obtained from
Amersham Biosciences. [3H]Thymidine (70-90
Ci/mmol) was purchased from PerkinElmer Life Sciences. G418 was
obtained from Invitrogen. Inhibitors PP2, SU6656, SB203580, and PD98059
were purchased from Calbiochem-Novabiochem. Human insulin was a kind
gift from Eli Lilly (Indianapolis, IN).
Construction and Transfection of L6 Cell Lines--
The
spontaneously fusing rat skeletal muscle cell line L6, a kind gift from
Dr. Amira Klip (The Hospital for Sick Children, Toronto, ON, Canada),
was used as the parent strain for all transfection experiments.
Full-length rat PTP (kindly provided by Dr. B. Goldstein, Thomas
Jefferson University, Philadelphia, PA) (33) was cloned into PKK233-2
(Clontech) and cleaved with EcoRI and
BamHI yielding two fragments of 697 bp (bp 722-1419) and
917 bp (bp 1419-2336). The blunt-ended fragments were cloned into the
EcoRV site of the mammalian expression vector pcDNA3.1
containing the SV40 promoter and neomycin resistance gene. The
orientation of the inserts was determined by restriction digestion and
sequencing. PTP underexpression was accomplished by transfecting
cells with the plasmids containing antisense 
917 cDNA using the
calcium phosphate precipitation procedure, and colonies were selected
with G418. PTP overexpression was achieved by co-transfecting cells
with pMJ30 that contained full-length mouse or human PTP cDNA
(34) and pcDNA3.1. Cells were transfected with pcDNA3 alone or
untransfected (referred to herein as L6) and used as controls. The
12 cell line, PTP overexpressing L6 cells in which L6 were
obtained from ATCC, have been described previously (12).
Dominant-negative (DN)-Src cDNA containing two point mutations,
K296R and Y528F, in pUSEamp Upstate Biotechnology Inc. or
empty vector was co-transfected with a cytomegalovirus promoter-driven
-galactosidase expression vector (Clontech)
using the Effectene kit (Qiagen) into parental L6 cells and L6 cells overexpressing PTP according to the manufacturer's instructions.
Cell Culture--
L6 cells were grown and maintained as
myoblasts in -minimal essential medium (MEM) supplemented with 10%
fetal bovine serum (FBS) and 1% antibiotic/antimycotics in an
atmosphere of 5% CO2 at 37 °C as described (35).
Transfected cells were maintained as above with the addition of 400 µg/ml G418. Cell differentiation into myotubes was induced by
reducing the serum concentration in the medium to 2% FBS. For cell
growth rate and cell differentiation studies, cells were plated in
six-well plates and grown in medium with or without addition of
inhibitors. Cell numbers at various days after cell seeding were
counted after trypsinization. Cell differentiation was observed after
Giemsa staining under a microscope (LEITZ LABORLUX S) linked to a JVC
color video camera head (model TK-1280U) and photographed with a
computer program, Leica Q500MC. Fusion indices were measured as
described previously (36). The extent of myoblast fusion was expressed
as the number of fused, multinucleated cells as a percentage of the
total number of cells in ten randomly chosen fields. Cells containing
more than two nuclei were regarded as fused cells.
[3H]Thymidine
Incorporation--
[3H]Thymidine incorporation was
performed as described previously (37). Briefly, myoblasts of L6 and
transfected lines were grown in -MEM supplemented with 10% FBS to
30-40% confluence. Cells were then incubated for 24 h in -MEM
containing 0.1% FBS (serum-deprived) followed by addition of 20% FBS
or 107 M insulin for 24 h.
[3H]Thymidine (0.67 µCi/ml) was added, and cells were
incubated for 18 h. At the end of the incubation period, the
medium was removed, and the cell monolayers were washed three times
with ice-cold PBS. The cells were solubilized with 200 µl of 0.1%
SDS, and the lysates were collected and precipitated with 10% ice-cold trichloroacetic acid. After centrifugation (10,000 × g
for 30 min) the incorporation of [3H]thymidine into DNA
was determined by liquid scintillation counting.
Creatine Kinase Assay--
Creatine kinase activity was assayed
spectrophotometrically using a diagnostic kit (Sigma). Briefly, day 4, 6, and 8 cultures grown in differentiation medium were washed twice
with ice-cold PBS and scraped. Cells were spun at 1000 × g and resuspended in PBS containing 0.1% Tween 20 and
incubated on ice for 15 min. The lysates were centrifuged at
10,000 × g for 15 min at 4 °C, and the protein
concentration in the supernatant was determined using the Bio-Rad
protein assay reagent. Protein content in the samples was then adjusted
to 0.4 µg/µl with PBS containing 0.1% Tween 20, and, typically,
100 µl of sample was used for creatine kinase assay according to the
manufacturer's instructions. The activity of the enzyme was determined
by measuring the absorbance at 510 nm and expressed as units/mg
protein. A unit of creatine kinase activity was defined as
(
A510 units/min·1000)/6.22 (extinction coefficient).
Immunoblot Analysis--
Culture plates (160-mm) were washed two
times with ice-cold PBS and scraped. The cell suspension was spun down
and homogenized in cell lysis buffer containing 50 mM Tris,
pH 7.5, 10% glycerol, 1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 7.5 mM sodium
pyrophosphate, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, and 2.5 µM pepstatin A. Insoluble cellular
debris was removed by centrifugation at 500 × g for 10 min. After determination of protein content, extracts were separated by
SDS-PAGE and transferred to nitrocellulose membranes. The membranes
were blocked in Tris buffered saline with Tween (TBST; 25 mM Tris, pH 7.5, 154 mM NaCl, 0.1% Tween 20)
containing 5% skim milk powder for 1 h. After washing with fresh
TBST, membranes were incubated with primary antibodies (1:1000) in TBST
with 5% bovine serum albumin overnight at 4 °C. The membranes were
then washed with TBST for 1 h and subsequently incubated with
secondary antibodies conjugated with horseradish peroxidase for 1 h at room temperature. After washing with TBST the immunoreactive bands were revealed by ECL (Amersham Biosciences) according to the
manufacturer's instructions. Intensity of the bands was determined by
scanning the bands using a Vivitar scanner (model VSF-200) and
appropriate software.
For the study of the association of Src with its substrate, FAK,
FAK was immunoprecipitated from 500 µg of cell lysate with a
polyclonal antibody at 4 °C overnight. Immune complexes were collected by centrifugation and were washed twice with a solution containing 500 mM LiCl3, 10 mM
Tris-HCl, pH 7.6, 0.1% Triton X-100, and 1 mM
dithiothreitol and then washed twice with Buffer A (20 mM
Hepes, pH 7.2, 2 mM EGTA, 10 mM
MgCl2, 1 mM dithiothreitol, and 0.1% Triton
X-100). Protein samples were separated and immunoblotted as above.
Immunofluorescence--
Cells grown on glass coverslips coated
with 1 mg/ml poly-L-lysine were washed with PBS and fixed
for 20 min at room temperature with 4% paraformaldehyde in PBS. The
coverslips were washed twice with PBS for 5 min and permeabilized with
a solution containing 10% normal goat serum (Vector) and 0.05% Triton
X-100 in PBS for 30 min at 4 °C. Cells were incubated with primary
antibody (1:100 in normal goat serum solution) at 4 °C overnight.
After three washes with PBS, cells were incubated for 1 h at
4 °C with secondary antibody, Cy3 anti-mouse (Jackson
ImmunoResearch) (1:400 in normal goat serum solution), and the
coverslips were light-protected by wrapping in foil. After washing with
PBS four times, the glass coverslips were mounted on microscope slides
with fluorescent mounting medium (Dako). Cells were viewed with
appropriate filter blocks for fluorescein, and images were acquired
with use of the DeltaVision deconvolution microscope (Applied
Precision, Issaquah, WA).
Statistics--
Results are expressed as means ± S.E., and
the Student's t test or analysis of variance was used to
evaluate significance, which was taken as a minimum of
p < 0.05.
| |
RESULTS |
Expression of PTP in L6 Cells--
Stable transfectants of L6
cells with underexpression and overexpression of PTP were
established. Underexpression of PTP was achieved by transfection of
wild-type L6 cells with PTP antisense cDNA, whereas
overexpression was obtained by transfecting cells with either a
full-length mouse or human PTP cDNA (38). The efficiency of the
transfection on the expression of PTP was assessed by immunoblotting
of PTP. Whole cell extracts isolated from different lines at day 5 in culture were used. In addition to the PTP band at 130 kDa (34), a
second band with a larger molecular weight, the glycosylated form of
PTP (4), was also detected with our antibodies (Fig.
1, A and B). In
comparison to untransfected L6 cells, densitometric analysis of the
PTP bands from cells transfected with antisense PTP, cell lines
AS14 and AS22, showed that expression of PTP was about 30 and 50%,
respectively, of the level in L6 cells (Fig. 1C). In cell
lines WT5 and M4 from cells overexpressing human or mouse PTP,
PTP protein content was increased about 3- and 5-fold, respectively,
when compared with L6 cells (Fig. 1C). The different
expression of PTP in these cell lines was further examined using
immunofluorescence staining and deconvolution microscopy. Cells
harboring PTP antisense or overexpressing PTP were grown on glass
coverslips and stained with rabbit polyclonal antibody against PTP.
As shown in Fig. 1D, PTP was distributed widely but more
concentrated in the perinuclear area. There was no nuclear staining.
The intensity of staining of PTP in AS14 was decreased markedly when
compared with that in L6, whereas it was much higher in M4 than that in
L6, consistent with the immunoblot results (Fig.
1D).
View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1.
Expression of
PTP in transfected L6 cells.
A, cells were transfected with PTP antisense
cDNA (AS14, AS18, AS19, and
AS22). L6, untransfected, and 12, PTP overexpressing
cells (12), were used as control. Equal aliquots of protein from the
lysates of different cell lines were separated by SDS-PAGE, and PTP
expression was determined by immunoblotting with PTP antibody.
gPTP represents the glycosylated forms of PTP.
B, cells were transfected with a full-length human PTP
cDNA (WT4 and WT5) or with a full-length
mouse PTP cDNA (M4, M6, and
M7). C, expression of PTP was quantified from
Western blots of underexpressing (left panel) and
overexpressing (right panel) cell lines by densitometry.
D, immunofluorescent images were acquired with the
deconvolution microscope as described under "Experimental
Procedures." Similar results were obtained in three separate
experiments.
|
|
Effects of Expression of PTP on Cell Growth--
To determine
the effects of the expression of PTP on cell proliferation, equal
amounts (105 cells/well) of cells were plated and cultured
in the presence of 10% FBS. The medium was changed at 48-h intervals,
and cells were trypsinized and counted at the indicated times. After a
4-day lag there was a rapid increase in cell number from day 4 to 6 with a plateau at day 7 (Fig.
2A). M4 and 12, both
overexpressing PTP, showed significantly faster proliferation than
L6 cells. Cell numbers in these lines were already increased above
control L6 after 4 days in culture and were more than 2-fold greater by day 6. In contrast, cells harboring either antisense PTP or double mutant PTP, AS14, AS22, and DM8, grew significantly more slowly than
L6, and cell numbers remained at about 50% of L6 from day 5 onwards
(Fig. 2A). The growth rate of L6 cells transfected
with an empty vector PC10 was similar to untransfected L6 cells.
View larger version (16K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of PTP
overexpression and underexpression on proliferation rate of
myoblasts. Equal numbers of cells (105 cells/ml) were
plated in 6-well plates and grown in -MEM supplemented with 10%
FBS. A, cells were trypsinized on days 3, 4, 5, 6, and 7, and cell numbers were determined by microscopy. pc10, L6
cells transfected with vector alone; DM8, L6 cells
transfected with a double mutant PTP. B, cells were
serum-deprived and then stimulated with 20% FBS or 107
M insulin followed by the [3H]thymidine
incorporation assay as described under "Experimental Procedures."
Results are the mean ± S.E. (n = 3-5).
|
|
We further investigated the rate of proliferation by measuring
[3H]thymidine incorporation into DNA, an indirect measure
of proliferation. In comparison with L6 cells, basal levels of DNA
synthesis were significantly lower in cells harboring antisense PTP,
and, in contrast, cells overexpressing PTP exhibited a considerable
increase (data not shown). Stimulation of control L6 cells with either 20% serum or with 107 M insulin caused a
2-fold increase in the level of [3H]thymidine
incorporation. Treatment of AS14 cells with serum or insulin induced an
increase in DNA synthesis significantly lower than in L6 cells (70% of
L6; see Fig. 2B) (p < 0.05). On the other
hand, [3H]thymidine incorporation observed in M4 cells
was ~170% of control L6 in response to serum or insulin (Fig.
2B).
Effect of Expression of PTP on Myoblast Differentiation--
In
conjunction with differences observed in the growth rate of cell lines
harboring antisense PTP and overexpressing PTP, we examined the
capacity of these cells to differentiate into myotubes. Wild-type L6
myoblasts (70-80% confluent) transferred to -MEM containing 2%
FBS began to form multinucleated myotubes after 4-5 days in culture
(Fig. 3) and reached the maximum
(near 100% fusion) by day 9 (Fig. 3B). Cells transfected
with either antisense PTP or with the phosphatase-dead double mutant
PTP showed alignment at day 5 but failed to fuse into myotubes (Fig. 3A). This apparent lack of differentiation was not because
of a lower cell density as myotubes were not observed in these cells even after 10 days. In contrast, formation of myotubes in cells overexpressing PTP began earlier, by 3 days after the shift to low
serum, and reached a maximum at day 8 (Fig. 3B). PC10 showed myotube formation at a rate similar to that of wild-type L6.
View larger version (60K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of PTP
overexpression and underexpression on myogenesis of L6
cells. Cells were grown in 10% FBS in 6-well plates, and at 70%
confluence, cell differentiation was induced by reducing the serum
concentration in the medium to 2% FBS. A, cell morphology
was observed after Giemsa staining and photographed at days 3, 5, and 8 after induction of cell differentiation. Arrowheads indicate
fused multinucleated myotubes. B, the extent of myoblast
fusion was determined by the ratio of fused, multinucleated cells to
total number of cells in percent as described under "Experimental
Procedures." The results (mean ± S.E.) shown are representative
of three separate experiments.
|
|
Muscle differentiation is characterized by an increase in the
expression of muscle-specific proteins such as myogenin and creatine
kinase (25). To determine whether this effect of PTP was simply a
perturbation of the fusion process or a more general effect related to
differentiation, we determined the expression of myogenin in cells
transfected with antisense or overexpressing PTP. Cell lysates were
obtained from confluent cells after 2 days of culture in 2% serum, and
proteins were separated by SDS-PAGE. Myogenin was detected in wild-type
L6 cells with M225, a polyclonal antibody against this protein (Fig.
4A). In AS14 and AS22 cells, myogenin levels at 2 days were ~10- and 5-fold lower, respectively, than in control L6 cells. In contrast, myogenin expression in M4 cells
was 3-5-fold higher than in L6 (Fig. 4A). It should be noted that myogenin expression was transient in all cell lines and that
no further or delayed increase was observed in AS cells (not shown). We
next examined the activity of creatine kinase, a muscle-specific enzyme
marker, in cell lysates. As shown in Fig. 4B, control L6
cells exhibited an ~10-fold increase in creatine kinase activity
during the transition from early fusing myoblasts at day 4 to fully
differentiated myotubes at day 8. M4 cells showed an ~2-fold greater
creatine kinase activity than L6 by day 6 that persisted through day 8. In contrast, AS14 cells displayed very little creatine kinase activity
at day 4, and no increase in activity was observed up to 8 days of
culture (Fig. 4B). These results suggest that PTP
regulates the myogenic differentiation program of L6 cells rather than
only a specific fusion event.
View larger version (22K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of PTP on the
expression of muscle-specific markers in L6 cells. Cells were
grown in a medium containing 10% FBS until 70% confluent and then
cultured in medium containing 2% FBS. A, cell lysates were
obtained from cells at 2 days after inducing differentiation, and
proteins were separated by SDS-PAGE. Myogenin was detected by
immunoblotting. B, creatine kinase activity was measured on
extracts prepared from cells 2, 4, 6, and 8 days after inducing
differentiation. Results are the mean ± S.E. of three independent
experiments.
|
|
PTP Expression Regulates Src Kinase in L6 Cells--
In
previous studies, PTP has been shown to play an important role in
the regulation of Src kinase activity in the human epidermoid carcinoma
cell line A431 (39) and in PTP-deficient mice (18, 19). The
mechanism of Src activation by PTP appears to be via the
dephosphorylation of the inhibitory carboxyl-terminal Tyr-527 (15, 20).
We therefore examined Src Tyr-527 phosphorylation in L6 cells harboring
antisense PTP and cells overexpressing this enzyme by immunoblotting
with an antibody, SRC2, which recognizes the dephosphorylated
carboxyl-terminal Tyr-527 active form of Src (40). As seen in Fig.
5A, although dephosphorylated
Tyr-527 Src in AS14, AS22 (antisense-containing lines), and DM8 (double mutant phosphatase-inactive expressing line) were reduced significantly (66, 49, and 77% of L6, respectively), the amount of carboxyl-terminal dephosphorylated Src was augmented in WT5 and M4 (PTP-overexpressing lines) (167 and 166% of L6, respectively). Total Src protein, detected
with antibody GD11, was not different among these cell lines (Fig
5A). It has been demonstrated that activation of Src stimulates the Tyr phosphorylation of FAK, a 125-kDa kinase activated in response to integrin-dependent cell adhesion (41).
Moreover, PTP overexpression in A431 epidermoid carcinoma cells was
found previously to increase the association of Src kinase with FAK (39). To further examine the effects of PTP expression on the activity of endogenous Src in our cell lines, FAK was
immunoprecipitated from whole cell lysates, and the immunoprecipitates
were separated by SDS-PAGE and immunoblotted with anti-Src. Increased
quantities of Src were observed in FAK immunoprecipitates from
PTP-overexpressing cells, WT5 and M4 cells (146 and 175% of L6,
respectively) (Fig. 5B). Decreased levels of FAK-associated
Src were detected in cells harboring antisense PTP, AS14 and AS22
(33 and 48% of L6, respectively), and a very low level of
FAK-associated Src was found in cells with the double mutant PTP
(27% of L6). Total FAK protein levels were not altered by either
underexpression or overexpression of PTP (Fig. 5B). These
results indicate that PTP plays an important role in the regulation
of c-Src activity in L6 cells.
View larger version (46K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of expression of
PTP on the activity of Src kinase. Cell
lysates were obtained from myoblast cell lines as indicated.
A, equal aliquots of protein were separated by SDS-PAGE,
transferred to membranes, and immunoblotted with antibodies SRC2
(upper panel), which recognizes the active,
Tyr-527-dephosphorylated form of Src (40), and GD-11, (lower
panel), which stains total Src protein. The intensities of the
immunoblots of active Src were corrected for total (see text for
details) B, total cell lysates (400 µg protein) were
immunoprecipitated with an antibody against FAK overnight and
subsequently separated by SDS-PAGE followed by immunoblotting with
anti-FAK (upper panel) or with anti-Src (lower
panel). Similar results were obtained in three separate
experiments.
|
|
A PTP-c-Src Signaling Pathway Is Required for L6 Cell
Differentiation--
The observations that the level of PTP
expression altered cell growth and differentiation, as well as Src
activity in L6 cells, raised the possibility that Src was involved in
mediating these effects of PTP. To explore this possibility, the
cells were treated with PP2, a specific inhibitor of Src family kinases (42), and cell growth rate and cell differentiation were determined. A
highly selective p38 MAP kinase inhibitor, SB203580, and a specific MAPK kinase inhibitor, PD098059, were also used as controls for these
studies. It has been reported that activation of p38 is required for
myoblast fusion and myotube formation in C2C12 cells (43). In contrast,
although not all reports are consistent (32), the traditional
MAPK/extracellular signal-regulated kinase enzymes are involved in cell
proliferation but not differentiation (44). Cells were plated in 6-well
plates, and inhibitors were added to the medium at day 2 after seeding.
The cell growth rate was decreased markedly by PP2 in all cell lines
tested, L6, AS14, and M4 (Fig. 6). Cell
numbers were 50% of their respective untreated controls. To assess
cell differentiation the inhibitors were added at the time of reduction
of serum from 10 to 2%. The inhibitor PP2 completely blocked
differentiation assessed by fusion index of control L6 and M4. In
contrast, inhibition of MAPK kinase (MAPK/extracellular signal-regulated kinase kinase) by PD098059 resulted in inhibition of
cell growth but did not affect myotube formation. Consistent with
previous reports of other muscle cell lines (30, 44), SB203580
suppressed myotube formation but not cell growth (Fig. 6).
View larger version (24K):
[in this window]
[in a new window]
|
Fig. 6.
Inhibition of cell growth and cell
differentiation by PP2, a specific inhibitor of Src kinase. Cells
were plated in 10% FBS in 6-well plates. For growth inhibition
studies, 10 µM PP2, 25 µM PD098059, or 10 µM SB203580 were added to the cultures at day 2 after
seeding. Cell numbers were determined at days 4, 6, and 8 as described
in the legend for Fig. 2. For the differentiation inhibition assay,
cells were grown in 10% FBS, and at 70% confluence, cell
differentiation was induced by reducing FBS to 2%. Inhibitors were
added to medium immediately after the change to 2% FBS. Fusion index
was determined as described in the legend for Fig. 3. Results are the
mean ± S.E. of three independent experiments.
|
|
To be certain that PP2 effects were mediated by inhibition of Src we
co-transfected L6 or M4 cells with an expression vector for DN-Src
(45), along with -galactosidase as an indicator of
transfection. Cells were then grown in differentiation medium for 4 days, myotube formation was observed by microscopy after 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-gal)
staining, and fusion index was determined after Giemsa staining.
-Galactosidase-positive cells in DN-Src-transfected cultures showed
a marked decrease in myotube formation (Fig.
7A). In these cultures most of
the myotubes lacked blue staining. In contrast, myotubes that formed in
the cultures transfected with empty vector (pcDNA3.1) showed -galactosidase staining (Fig. 7B). Determination of cell
fusion as an index of myotube formation demonstrated a significant
reduction in cells transfected with DN-Src (39%) compared with that in
cells transfected with pcDNA3.1 (61%) (Fig. 7C).
Assuming a transfection efficiency of 50% (estimated by
-galactosidase staining) myotube formation was decreased by about
70% by DN-Src. These results were substantiated by the complete
inhibition of myogenesis in L6 and M4 cells achieved by SU6656 (Fig.
7D), a novel and specific Src kinase inhibitor (46). The
effect of this inhibitor on cell growth was similar to PP2 (data not
shown).
View larger version (29K):
[in this window]
[in a new window]
|
Fig. 7.
Inhibition of L6 cell differentiation by DN
Src and SU6656. L6 cells were grown to 70% confluence and
transfected with an expression vector for DN-Src (A) or with
empty vector, pcDNA3.1 (B) and co-transfected with a
-galactosidase expression vector as described under "Experimental
Procedures." Transfected cells were then grown in differentiation
medium for 4 days and photographed after staining for
-galactosidase. Arrowheads indicate fused multinucleated
myotubes. C, similarly transfected M4 cells were grown in
differentiation medium for 4 days, and fusion index was determined
(n = 3); *, p < 0.01. D,
Cells were grown to 70% confluence, and the medium was changed to
differentiation medium with or without SU6656 (1 µM).
Fusion index was determined as described in Fig. 3. Results are
mean ± S.E. of two experiments.
|
|
As these data indicated a role for PTP and Src kinase in muscle cell
differentiation, we investigated whether an enhanced expression of
PTP and activation of Src occurred during myogenesis in
untransfected cells. Parental L6 cells were grown in growth medium and,
at 70% confluence, were transferred to differentiation medium. Whole
cell lysates were obtained at different days of culture from both
myoblasts and myotubes and subjected to immunoblotting analysis for
PTP- and Tyr-527-dephosphorylated Src. As shown in Fig.
8A, expression of PTP was
low in myoblast cultures at day 2 but was enhanced at day 3 as
myoblasts proliferated, and cell density was increased. Upon transfer
to differentiation medium, expression of PTP initially remained high
and declined by day 3 after transfer (day 6 after seeding) when most
myotubes were formed. In contrast, Src activity, determined by SRC2
antibody immunoblots, was not changed in myoblasts but increased and
remained elevated in myotubes (Fig. 8A).
View larger version (29K):
[in this window]
[in a new window]
|
Fig. 8.
L6 cell differentiation and expression of
myogenin are associated with the expression of PTP
and Src. A, L6 cells were plated in medium
containing 10% FBS, and serum was reduced at the end of day 3. Cell
lysates were obtained from myoblasts (MB) at day 2 and 3 and
from myotubes (MT) at day 4, 5, and 6 after cell seeding
(corresponding to day 1, 2, and 3 after induction of differentiation).
Proteins were separated by SDS-PAGE, and immunoblotting was performed
using an antibody against PTP (upper panel) or antibody
SRC2 (lower panel). B, L6 cells were grown in
10% FBS, and 10 µM PP2 was added to the culture when
serum was reduced at the end of day 3. Myogenin was probed by
immunoblotting of L6 cell lysates at day 5 after seeding. Myogenenin
expression was blocked by PP2.
|
|
Muscle cell differentiation is known to be accompanied by expression of
specific proteins such as myogenin (25) (Fig. 4), and we observed that
inhibition of Src kinase activity resulted in inhibition of myotube
formation (Fig. 6). We thus examined the effect of PP2 on the
expression of myogenin. As expected, myogenin expression was increased
significantly in day 2 myotubes (day 5 after seeding) compared with
that in myoblasts (Fig. 8B). In contrast, myogenin
expression was inhibited completely in cells grown in differentiation
medium treated with PP2. This result strongly supports the concept that
Src activation is required for muscle differentiation and
muscle-specific protein expression.
Src Activation Is Required for C2C12 Cell
Differentiation--
To determine whether the role of Src in skeletal
muscle differentiation was a unique characteristic of the rat-derived
L6 cell line or a more general phenomenon, we tested whether PP2 inhibition of Src family kinases could alter differentiation of the
mouse skeletal muscle cell line, C2C12. C2C12 cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% FBS. Cell
differentiation was induced by changing the medium to Dulbecco's modified Eagle's medium supplemented with 2% horse serum, and PP2 was
added to the differentiation medium. At day 3 after induction of
myogenesis, C2C12 myoblasts began to form multinucleated myotubes that
were blocked in cells treated with PP2 (Fig.
9). These results are consistent with a
critical role for Src kinase signaling in skeletal muscle
differentiation.
View larger version (66K):
[in this window]
[in a new window]
|
Fig. 9.
Inhibition of C2C12 muscle cell
differentiation by PP2. C2C12 cells (obtained from ATCC) were
grown in Dulbecco's modified Eagle's medium supplemented with 10%
FBS and 1% antibiotic/antimycotics. Cell differentiation was induced
when cells reached 50% confluence by changing the medium to a
differentiation medium supplemented with 2% horse serum. PP2 (10 µM) was added to the culture following induction of
myogenesis. Cell differentiation was monitored microscopically and
photographed on day 3 after induction as described in the legend for
Fig. 3.
|
|
| |
DISCUSSION |
The cellular functions of the transmembrane protein-tyrosine
phosphatase, PTP, are not well defined. Several studies have suggested that it acts as an inhibitor of insulin signaling (7, 47,
48), yet other reports have not confirmed these actions (8, 10). We
sought to investigate this hypothesis in skeletal muscle, a major
insulin target tissue, and employed the rat skeletal muscle cell line
L6. Upon the generation of stable lines overexpressing and
underexpressing PTP we made the unexpected observation that a
decrease of PTP expression in PTP AS cells or overexpression of
DM of PTP resulted in a modest alteration of cell growth and a
marked defect in differentiation. Consistent with these findings, the
cell lines overexpressing either human or mouse wild-type PTP showed
an increase in cell proliferation, as well as accelerated differentiation compared with control untransfected L6 cells or L6
cells transfected with empty vector.
The effects on differentiation were first noted microscopically and
characterized by both a lack of formation of multinucleated myotubes
(AS cells and DM cells) and an enhanced rate of fusion (overexpression
cells). Because transmembrane PTPs have been implicated in cell-cell
and cell-extracellular matrix interactions (reviewed in Ref. 3), and
PTP has been found to alter cell adhesion properties, it was
possible that only the fusion process was altered rather than
myogenesis per se. However, other specific markers of
skeletal muscle differentiation, namely myogenin and creatine kinase,
were affected in a similar pattern to myotube formation indicating that
the differentiation process was altered.
The process of myoblast differentiation is complex, and several
signaling mediators have been shown to be involved such as phosphatidylinositol 3-kinase, mammalian target of rapamycin, p70s6k, and p38 MAPK (29-31, 44). Both insulin and the
IGFs have been found to stimulate a short burst of proliferation
followed by differentiation in myoblast cell lines (26-28) and are
capable of activating these signaling pathways. In this study we did
not employ these growth factors to promote differentiation but used a
low serum-containing medium. It was of interest, however, that PTP
overexpression enhanced insulin-stimulated [3H]thymidine
incorporation and enhanced differentiation, another insulin response,
opposite to what would be expected if insulin signaling was inhibited.
Finally, we could not demonstrate any difference in insulin-stimulated
insulin receptor or IGF-1-stimulated IGF-1 receptor Tyr
autophosphorylation in these various cell lines (data not shown)
similar to the findings of Jacob et al. (8) and Arnott
et al. (49).
To investigate the mechanism of the influence of PTP expression on
differentiation we examined the effects on endogenous Src
phosphorylation and kinase activity. A number of studies have demonstrated that PTP overexpression is associated in various cell
lines with activation of Src (13, 16, 17, 20, 39). Recently, studies in
cells and tissues obtained from mice with targeted disruption of PTP
(PTP/) confirmed an important role in regulating Src
and the related Src family kinase, fyn, activation (18, 19). This
activation is mediated by binding of the SH2 domain of Src to the
phosphorylated carboxyl-terminal Tyr-789 of PTP, which competes with
and disengages the SH2 domain from binding to its internal Src
pTyr-527. This is followed by pTyr-527 dephosphorylation by the
membrane proximal PTP catalytic domain (20). Concomitant with Src
activation, previous studies in P19 embryonal carcinoma cells showed
that overexpression of PTP leads preferentially to neuronal
differentiation (15, 16). In our cell lines, we found evidence for
increased Src activation by PTP overexpression and decreased
activity in AS cells. Specifically, this was manifested by a reduced
Tyr-527 phosphorylation, as well as increased association of Src with FAK, a reflection of the amount of endogenous active Src. These findings raised the possibility that Src (or an Src family kinase member) was a significant mediator of myoblast differentiation.
The role of Src in cellular differentiation is complex. Thus, in
studies of neuronal differentiation, expression of v-Src, a
constitutively active oncogenic mutant, in P19 cells, inhibited differentiation into neuronal cells (50). The extent and timing of Src
activation appears to be critical to the differentiation process (21,
50). Similar to some of these findings in nerve cells, expression of
v-Src in skeletal myoblasts inhibited differentiation into myotubes
(51), whereas expression at even later stages repressed the expression
of several muscle-specific genes (52, 53) or disrupted sarcomeres (54).
On the other hand phosphorylation of the acetylcholine receptor by Src
and/or fyn in myotubes appears to be important for receptor aggregation
and post-synaptic differentiation (55). Thus, the role of Src in
skeletal muscle differentiation has not been established clearly.
The findings in our cell lines of a correlation of altered Src activity
with myoblast differentiation allowed us to examine whether these were
related. Inhibition of Src with PP2 blocked differentiation in parental
L6 cells, as well as L6 cells overexpressing mouse or human PTP. The
inhibitor PP2 is specific for the Src kinase family, and although we
cannot rule out a role for other family members, we could not detect
significant expression of fyn in our L6 lines (not shown). In addition
to PP2, a novel inhibitor of Src kinase, SU6656, also blocked
differentiation. Transient transfection of DN-Src just prior to onset
of differentiation inhibited myotube formation by 70%. We also noted
that PP2 and SU6656 blocked the more modest effect of PTP
overexpression on cell proliferation consistent with roles for Src
family kinases in both growth and differentiation (56, 57). As
controls, we noted that inhibition of MAPK/extracellular
signal-regulated kinase kinase, the upstream activator of extracellular
signal-regulated kinases 1 and 2, with PD098059 only blocked cell
proliferation and not differentiation, whereas inhibition of p38 with
SB203580 had opposite effects, blocking differentiation but not
proliferation. These results are consistent with previous studies (30,
44). Taken together, the data indicate that a previously undescribed PTP-c-Src signaling pathway is essential for myoblast differentiation.
Two questions raised by these findings are the mechanism by which
PTP is activated in the early differentiation program to recruit Src
and the precise role or signaling pathway stimulated by Src in this
process. A putative activating ligand for PTP has not been
reported, and in contrast to receptor PTKs, dimerization of PTP
inhibits its activity (58). Recently, in brain and neuronal cells,
PTP has been found to complex with the
glycosylphosphatidylinositol-linked receptor contactin (59).
Complex formation between contactin and the Src family kinase fyn was
demonstrated previously (60), and thus it was proposed that PTP may
provide the link. Contactin participates in the regulation of neuronal
migration in the developing brain (60, 61). One might speculate that a
similar glycosylphosphatidylinositol-linked protein may be involved in
PTP-Src signaling in differentiating myoblasts.
The target of Src in developing muscle remains unclear. In the case of
neuronal cell differentiation, distinct signals mediated by both ras
and Src were shown to be required (62). In skeletal muscle C2C12 cells,
a transmembrane disintegrin and metalloprotease termed ADAM12 was
described recently to be up-regulated during differentiation (63, 64)
and to be associated via its proline-rich cytoplasmic domains with the
SH3 domain of Src (65). This suggests involvement in recruitment or
localization of Src to the cytoskeleton as ADAM12 binds to
-actinin-1 (65) and -actinin-2 (66). A variety of cytoskeletal
proteins may be Tyr-phosphorylated by Src and may be critical in
transmitting the differentiation signal by cell-cell and/or cell-matrix interactions.
In summary, in this study we demonstrate a novel PTP-c-Src signaling
pathway required for differentiation of skeletal muscle. The trigger
and target(s) of this pathway remain to be defined. Using antisense
strategies, Arnott et al. (49) found that in differentiated
3T3-L1 adipocytes insulin action was not regulated by
PTP. We have also found that PTP underexpression or
overexpression did not alter insulin receptor Tyr phosphorylation in L6
muscle cells. Finally, we found that overexpression of the double Cys catalytic site mutant of PTP produced a phenotype similar to low
level expression. This indicates that the DM phosphatase-inactive PTP functions in L6 cells in a dominant-negative manner. These strategies will be useful to further dissect the roles of PTP in
cellular function.
-mediated C-Src Signaling Pathway*
,
,
TOP
ABSTRACT
INTRODUCTION
Ala mutant (DM8) PTP
