(Received for publication, August 2, 1996, and in revised form, November, 22, 1996)
From the § Cardiology Section, Given the central position of the focal adhesion
complex, both physically in coupling integrins to the interstitium and
biochemically in providing an upstream site for anabolic signal
generation, we asked whether the recruitment of non-receptor tyrosine
kinases to the cytoskeleton might be a mechanism whereby cellular
loading could activate growth regulatory signals responsible for
cardiac hypertrophy. Analysis revealed cytoskeletal association of
c-Src, FAK, and Hypertrophy is one of the major compensatory mechanisms available
to the heart for sustaining chronic hemodynamic overloads. There are
two types of hemodynamic overloads: (i) pressure overload (systolic
stress), in which a normal blood volume is pumped during each cardiac
cycle against an increased impedance; and (ii) volume overload
(diastolic stress and strain), in which an increased blood volume is
pumped during each cardiac cycle against a normal impedance (for
review, see Ref. 1). The hypertrophic response to these hemodynamic
overloads consists of both qualitative changes in the expression of
specific proteins and a quantitative increase in total cardiocyte
protein. It is well documented that compensatory hypertrophy of cardiac
muscle in specific hemodynamic settings eventually results in a
decrease in contractile performance, even prior to the onset of heart
failure (2). Indeed, recent studies from this laboratory have shown
that there is a cellular basis for contractile dysfunction of
pressure-hypertrophied cardiac muscle involving changes in the
cardiocyte cytoskeleton (2, 3). However, a central question has not yet
been answered: what are the mechanisms by which changes in cardiac load
are coupled to the modulation of intracellular signals that are
responsible for changes in cardiocyte mass and phenotype?
Since we know that the terminally differentiated adult cardiocyte is
directly responsive to load input in terms of growth regulation (1), we
have chosen to focus this study on potential mechanisms whereby
cellular load input could activate growth regulatory signaling
pathways. In this context, it is known that receptor-mediated protein
tyrosine phosphorylation of various intracellular substrates plays a
major role during both growth induction and the maintenance of
differentiation. In addition to these established receptor-mediated regulatory pathways, there is recent evidence for synergistic growth
regulation by integrin-mediated non-receptor tyrosine kinase pathways.
Integrins are transmembrane proteins that provide tight adhesion of
cells to extracellular matrix proteins at sites referred to as focal
adhesions and that connect the extracellular matrix proteins to
intracellular cytoskeletal proteins (4-10). Although integrins are
transmembrane proteins bearing short cytoplasmic domains, they do not
exhibit intrinsic tyrosine kinase activity. Therefore, a recently
identified non-receptor tyrosine kinase known as FAK ( We know that a specific cytoskeletal change involving tubulin
up-regulation is one phenotypic consequence of cardiac hypertrophy (2,
3). In view of this fact and the information given above, we are now
asking whether load-induced integrin-cytoskeleton interactions may be a
specific mechanism causing cardiac hypertrophy. As a first step toward
answering this question, we sought to define any redistribution of
non-receptor tyrosine kinases in pressure-overloaded myocardium.
These studies, using the detergent-insoluble actin-rich cardiac
cytoskeletal fraction from the feline right ventricular pressure overload model, show for the first time the following. (i) Cytoskeletal association of c-Src, FAK, and Cats with two types of right
ventricular pressure overload (RVPO),1
long-term and acute, were prepared as we have described before (17,
18). Long-term pressure overload of the right ventricle (RV) was
induced by partially occluding the pulmonary artery with a 3.2-mm
internal diameter band (17). Short-term RVPO (4 h) was induced by
partial occlusion of the pulmonary artery by transvenous insertion of a
balloon-tipped catheter (18). In both cases, RV pressure was more than
doubled, while systemic arterial pressure remained unaltered. Controls,
as appropriately specified, consisted of normal cats, sham-operated
cats submitted to thoracotomy and pericardiotomy without hemodynamic
intervention, or the normally loaded left ventricle (LV) from each
animal having either short-term or long-term RVPO. All operative
procedures were carried out in cats weighing 2.6-3.9 kg under full
surgical anesthesia with meperidine (2.2 mg/kg intramuscularly),
acepromazine maleate (0.25 mg/kg intramuscularly), and ketamine HCl (50 mg/kg intramuscularly).
After completion of pressure overloading, the animal was heparinizied
and placed on oxygen. A left thoracotomy was performed, and the heart
was rapidly removed, placed in cold buffer solution, and weighed. The
aorta was then cannulated; the coronary arteries were perfused with
phosphate-buffered saline; and the free walls from the LV and RV were
removed for immediate processing. Cardiocytes from normal and
pressure-overloaded myocardia were isolated as we have described before
(19).
Cytoskeletal preparations
were made as described for platelets by Fox et al. (16),
with minor modifications. Briefly, 100 mg of LV or RV free wall tissue
obtained from either control or pressure-overloaded hearts was minced
and transferred into a tube of 2 ml of ice-cold Tris/Triton extraction
buffer containing 100 mM Tris-HCl, pH 7.4, 2% Triton
X-100, 1 mM Na3VO4, 0.5 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 2 µg/ml pepstatin, 2 µM E-64
(trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane), 200 µg/ml p-aminobenzamidine, and 10 mM EGTA.
The tissue was immediately homogenized using a Tekmar Tissuemizer at a
50% power setting for 40 s; the Triton X-100-insoluble pellet
(cytoskeletal fraction rich in actin filaments) was obtained by
centrifugation at 15,000 × g for 5 min. The insoluble
cytoskeletal pellet was re-extracted once with 1 ml of the same buffer
and centrifuged as described above. To the pellet was then added 500 µl of Laemmli SDS sample buffer, after which it was vortexed and
boiled for 5 min. After centrifugation, the supernatant, referred to as
the Triton X-100-insoluble low-spin fraction, was saved for further
analysis. The Triton X-100 supernatant from the original low-speed
centrifugation was put in a 10-ml Beckman tube and centrifuged at
100,000 × g for 2.5 h in a Ti-50 rotor. The
pellet obtained after centrifugation was extracted in 1 ml of SDS
sample buffer and processed as described above. This sample contains
largely membrane skeleton (16). The supernatant, referred to as Triton
X-100-soluble material, was mixed with equal volumes of SDS sample
buffer and boiled for 5 min. The volumes of material obtained from 100 mg of tissue were as follows: 4 ml of Triton X-100-soluble material,
500 µl of Triton X-100-insoluble low-spin fraction (cytoskeleton),
and 1 ml of Triton X-100-insoluble high-spin fraction (membrane
skeleton).
For immunoblot analysis of Triton
X-100-lysed fractions, 30 µl of samples boiled in SDS buffer was
resolved by SDS-PAGE and transferred to Immobilon-P membranes. The
blots were blocked for 2 h with 10% milk protein in TBST buffer
(10 mM Tris, pH 7.4, 0.1 M NaCl, and 0.1%
Tween) and incubated with primary antibodies. The following primary
antibodies were commercially obtained. Monoclonal antibodies were from
Upstate Biotechnology, Inc. for c-Src (GD11) and Immunoprecipitation and detection of
tyrosine-phosphorylated c-Src were carried out as follows. 50 mg of
ventricular tissue or cytoskeletal pellet was extracted with 0.5 ml of
Laemmli SDS sample buffer containing 1 mM vanadate and
boiled immediately for 5 min. After centrifugation, 100 µl of the
supernatant was diluted in radioimmunoprecipitation assay buffer (20 mM Tris, pH 7.0, 150 mM NaCl, 1% Nonidet P-40,
1% sodium deoxycholate, 100 µg/ml aprotinin, 100 µg/ml
phenylmethylsulfonyl fluoride, 1 mM EDTA, and 1 mM sodium orthovanadate, with the final SDS concentration being adjusted to 0.2%). The samples were then mixed with or without anti-c-Src monoclonal antibody GD11 (1 µg) and incubated overnight at
4 °C. Prewashed protein G beads (10 µl; Pierce) were then added, and incubation was continued for an additional 1 h. The immune complexes were washed three times with radioimmunoprecipitation assay
buffer and boiled with 15 µl of SDS sample buffer. The proteins were
separated by SDS-PAGE before Western blot analysis with
anti-phosphotyrosine antibody as described above. For
immunoprecipitating tyrosine-phosphorylated proteins, a similar
protocol was followed to extract the proteins with SDS sample buffer,
followed by dilution in radioimmunoprecipitation assay buffer. 100 µl
of total lysate or Triton X-100-lysed subfractions was diluted as
described above, and biotin-conjugated anti-phosphotyrosine antibody
(10 µl; Transduction Laboratory) was added and incubated for 1 h
at 4 °C. The immune complex then was precipitated with streptavidin-conjugated agarose beads (Upstate Biotechnology, Inc.) for
30 min and washed with radioimmunoprecipitation assay buffer. After
boiling the immune complex with SDS sample buffer, the proteins were
separated by SDS-PAGE and analyzed for c-Src as described above.
Cytoskeleton-associated kinase activity was assayed
after suspending the Triton X-100-insoluble low-spin pellet
(cytoskeleton) in kinase assay buffer (50 mM
Ventricular tissue
samples obtained from either control cats or RVPO cats were homogenized
in Triton X-100 buffer and centrifuged to obtain low-spin (15,000 × g) and high-spin (100,000 × g) pellets (actin-rich cytoskeleton and membrane skeleton) as well as high-spin supernatant (soluble fraction) as described under "Materials and Methods." Fox et al. (16) have shown that both the Triton
X-100-insoluble low-spin fraction (cytoskeleton) and the high-spin
fraction (membrane skeleton) are rich in actin, released from long and
short actin filaments, respectively. Western blots displaying the
distribution of actin, c-Src, FAK, and
The low-spin pellets (cytoskeleton) contained higher amounts of actin
relative to the high-spin pellets (membrane skeleton) or soluble
fractions. This was true even after taking into account the volume of
samples loaded for each Triton X-100-lysed subfraction. More important,
in all of the experimental cats, the actin present in each set of LV
and RV samples showed no observable differences, suggesting that the
net amount of actin per unit mass of ventricular tissue is not changed
by pressure overloading. Furthermore, it suggests that the extraction
procedure was performed uniformly for both normal and
pressure-overloaded ventricles. The presence of very low amounts
of actin in the Triton X-100-soluble fractions indicates that the
cytoskeletal and membrane skeletal proteins were not significantly
disrupted during the isolation procedures.
The majority of c-Src, FAK, and When a similar analysis was done for a 4-h RVPO cat (Fig.
1B), the Triton X-100-soluble fraction showed no significant
difference in the levels of c-Src, FAK, and Changes associated with 4-h RVPO were found to be more pronounced when
RV pressure overloading was extended for 48 h (Fig. 1C). c-Src was significantly increased in the cytoskeletal
fraction and was present as a double band (lane 4), probably
representing variable sizes due to phosphorylation or partial
proteolysis (22). Based on semiquantitation of the blots and the sample
volume used for Western analysis, the cytoskeleton-associated c-Src in
RVPO represents at least 30-40% of the total c-Src present in
ventricular tissue. FAK association with the cytoskeleton also was
found to be significantly increased upon pressure overloading for
48 h (Fig. 1C, compare lanes 4 and
3 for FAK), which represents 25% of the total ventricular
FAK. However, in the case of The above experiments with RVPO for various time periods indicate that
c-Src and FAK (and to a lesser degree, Both c-Src and FAK are members
of the non-receptor tyrosine kinase group, and their cytoskeletal
redistribution would be expected to cause phosphorylation of several
proteins. Therefore, we examined the levels of tyrosine-phosphorylated
proteins in the Triton X-100-lysed subfractions of a 48-h RVPO cat. The
detergent-soluble fractions obtained from the normally loaded LV and
the pressure-overloaded RV (Fig. 2, lanes 1 and 2, respectively) contained several
tyrosine-phosphorylated proteins, although no significant changes
between these two lanes were noticed. However, when such comparisons
were made in the Triton X-100-insoluble low-spin fractions
(cytoskeleton), the pressure-overloaded RV fraction showed several
tyrosine-phosphorylated proteins of varying molecular sizes that were
either completely absent or present at low levels in the control LV
fraction (compare lanes 4 and 3). Two of the
protein bands, indicated by arrows at 60 and 36 kDa, which
were not present in detectable amounts as tyrosine-phosphorylated
proteins in the Triton X-100-insoluble low-spin samples of the normally
loaded LV, exhibited substantial amounts of tyrosine phosphorylation
following 48 h of pressure overloading. A similar comparison in
the membrane skeletal fractions showed no significant difference
between the RV and LV (compare lanes 6 and 5),
although both samples contained several other tyrosine-phosphorylated
proteins.
The major Although the results shown in Fig. 1C indicate significant
amounts of FAK being translocated to the cytoskeleton of the 48-h pressure-overloaded RV, there was only a moderate increase in the
125-kDa tyrosine-phosphorylated protein band (Fig. 2,
arrow). Finally, we wished to identify the 36-kDa
tyrosine-phosphorylated band, also indicated by an arrow in
Fig. 2. Annexin II is 36-kDa Ca2+-dependent
phospholipid-binding protein that recent studies (23, 24) have
identified as a cellular substrate for c-Src. Western blot analysis
with anti-annexin II antibody indicated that a small but significant
amount ( Since cytoskeletal
association of c-Src was seen maximally after 48 h of RVPO (Fig.
1), we asked whether the
Immunoprecipitation combined
with Western blot analysis was done in total tissue extracts to confirm
whether c-Src is present in the
Association of tyrosine-phosphorylated c-Src with the cytoskeleton has
been reported in activated platelets (14-16) and in growth
factor-stimulated glioblastoma cells (25). Phosphorylation under these
conditions has been shown to occur primarily at Tyr-416 by
autophosphorylation, which in turn amplifies the kinase activity (26).
However, in several tissues and cells under normal conditions, the
majority of c-Src has been shown to be phosphorylated at Tyr-527 by a
recently identified C-terminal Src kinase (Csk), and such phosphorylation suppresses c-Src kinase activity (27, 28). The absence
of any such phosphorylation in normally loaded ventricular tissue
further suggests that c-Src kinase might be differently regulated in
the heart. These findings are not likely due to any artifact created by
changes in tyrosine phosphatase activity since for the combined
immunoprecipitation and Western blotting experiments described above
(Fig. 4, B and C), tissue samples were prepared by direct extraction in Laemmli SDS sample buffer containing vanadate, and the homogenate was boiled immediately prior to centrifugation. It
is unlikely that phosphatases remained active under these
conditions.
The finding that
cytoskeleton-associated c-Src exhibits a high level of tyrosine
phosphorylation could imply either activation or inactivation,
depending upon whether the phosphorylation is at Tyr-416 or Tyr-527
(for review, Ref. 28). It was therefore important to determine whether
cytoskeleton-bound tyrosine-phosphorylated c-Src is functionally active
and thus potentially able to play a role in mediating hypertrophic
growth. Unfortunately, we could only immunoprecipitate c-Src from the
cytoskeletal fraction under denaturing conditions (see Fig. 4,
B and D) and thus could not directly demonstrate
c-Src kinase activity. Studies shown in Fig. 4 clearly demonstrate that
a significant portion of the cytoskeleton-associated c-Src is
tyrosine-phosphorylated. To determine whether cytoskeleton-associated c-Src is active in pressure-overloaded myocardium, we examined the
tyrosine phosphorylation status of c-Src in normal and hypertrophied hearts. For this purpose, we employed a monoclonal antibody (clone 28)
developed by Kawakatsu et al. (29) that reacts with c-Src only when the kinase is not phosphorylated at Tyr-527. Western blot
analysis utilizing the clone 28 monoclonal antibody revealed that
pressure overloading causes an association of a significant amount of
the Tyr-527 unphosphorylated kinase relative to its low level in the
normally loaded control LV (Fig. 5A, compare lanes 4 and 3). This population represents the
unphosphorylated form of c-Src and/or c-Src phosphorylated at another
tyrosine residue, which includes the other major Tyr-416
phosphorylation site. Therefore, we next attempted to demonstrate
directly that at least a significant portion of the phosphorylated
c-Src is due to phosphorylation of residues (e.g. Tyr-416)
other than Tyr-527. The cytoskeletal fractions were first
immunoprecipitated with anti-phosphotyrosine antibody, and then
tyrosine-phosphorylated c-Src was detected by probing with the clone 28 anti-c-Src antibody. Fig. 5B clearly shows the presence of
tyrosine-phosphorylated c-Src in the cytoskeletal fraction of
pressure-overloaded myocardium that is not due to Tyr-527
phosphorylation and therefore most likely represents the active Tyr-416
phosphorylated species (see "Discussion"). In addition, we
performed in vitro kinase reactions directly in the
cytoskeletal pellets of control and pressure-overloaded myocardia. Such
studies showed enhanced phosphorylation of several cytoskeletal
proteins, including a major 60-kDa protein band in pressure-overloaded
myocardium (data not shown). Furthermore, such kinase assays with the
p34cdc2 kinase derived-substrate peptide
(Lys-Val-Glu-Lys-Ile-Gly-Glu-Gly-Thr-Tyr-Gly-Val-Val-Tyr-Lys), a
specific and efficient substrate of Src family tyrosine kinases (21),
showed a 3-fold increase in phosphorylation by the cytoskeletal fraction prepared from pressure-overloaded ventricles (data not shown).
In all these experiments, the cytoskeletal fractions from pressure-overloaded RVs and same-animal control LVs exhibited very
similar levels of actin. Taken together, these data suggest that a
significant proportion of the cytoskeleton-associated c-Src is
active.
Since our earlier experiments revealed that tyrosine-phosphorylated
c-Src was present only in the cytoskeletal fraction of pressure-overloaded myocardium and was completely absent in the normal
heart, we anticipated that the clone 28 antibody would react with the
c-Src present in the Triton X-100-soluble fractions. As shown in Fig.
5A, the detergent-soluble fractions of both normal and
pressure-overloaded myocardia contain substantial amounts of c-Src
recognized by the clone 28 anti-c-Src monoclonal antibody (lanes
1 and 2), and pressure overloading does not appear to
change the level. In addition, this form of c-Src is also present in the Triton X-100-insoluble membrane skeletal fraction (lanes 5 and 6), showing no change due to pressure
overloading.
To demonstrate that the
cytoskeletal association and phosphorylation of c-Src in the
pressure-overloaded ventricles represent changes occurring at the level
of individual cardiocytes, we isolated these cells from both the LV and
RV of a 48-h RVPO cat (19). The cardiocytes were extracted with 2%
Triton X-100 buffer, and the detergent-insoluble low-spin fraction
(cytoskeleton) was obtained for Western blot analysis with
anti-phosphotyrosine antibody. The
The Src kinase family is composed of nine related
non-receptor tyrosine kinases. Thus, we asked whether other Src family
members were also translocated to the cardiac cytoskeleton during
pressure overloading, as has been reported in platelets (16). The
experiments in Fig. 7 show the levels of actin, c-Src,
and Fyn in Triton X-100-soluble and -insoluble cytoskeletal and
membrane skeletal fractions from another 48-h RVPO cat. As shown above,
there was no significant difference in actin levels when comparing sets
of Triton X-100-extracted LV and RV fractions, while there were
substantial amounts of c-Src being recruited to the cytoskeleton during
48 h of pressure overloading. Western blot analysis of Fyn showed
that both the detergent-soluble fractions (lanes 1 and
2) and the insoluble membrane skeletal fractions
(lanes 5 and 6) contained substantial amount of
Fyn. However, in contrast to c-Src, we did not see Fyn association with
the cytoskeleton after 48 h of pressure overloading (Fig. 7,
compare lanes 4 and 3 for Fyn and c-Src).
Furthermore, we observed no tyrosine-phosphorylated protein band
matching the size of Fyn in the cytoskeletal fraction of
pressure-overloaded myocardium (Fig. 2 and data not shown). When such
studies were performed for Lyn using a monoclonal antibody
(Transduction Laboratory), we detected no protein band in any the
Triton X-100-lysed samples. While a polyclonal antibody (Santa Cruz)
detected low levels of this kinase in the Triton X-100-soluble
fractions, we saw no association with the cytoskeleton of
pressure-overloaded myocardium (data not shown). Similarly, we were
unable to detect Yes in any of the Triton X-100-lysed subfractions when
Western-blotted with a specific monoclonal antibody, although it is
possible that antibody specificity prevented recognition of feline
samples. Nonetheless, of the Src kinase family members that we
examined, c-Src appears to be the only kinase that demonstrated a shift
in cytoskeletal association in pressure-overloaded myocardium.
Both skeletal and cardiac striated muscle cells undergo
developmentally programmed terminal differentiation, with subsequent adaptation to increased demands for mechanical output being met by
hypertrophy. Therefore, hypertrophic growth is considered to be a
fundamental adaptive response to hemodynamic overloads. A series of
studies from these laboratories (1, 2) has established that the
cardiocyte is directly responsive to load input in terms of initiating
and regulating this hypertrophic response. However, a critical
unresolved question is that of how changes in mechanical loading of the
myocardium are transformed into the intracellular chemical signals that
eventually result in hypertrophic growth and any accompanying
phenotypic changes. In this work, we asked whether the focal adhesion
complex could function as a potential mediator through which changes in
mechanical loading might be received from the interstitium and
transmitted into chemical signaling via cardiocyte integrin-mediated
pathways.
There are two broad views proposed for the mechanism of
integrin-mediated signaling (for review, see Ref. 30). Since
cytoplasmic domains of the integrin receptor family interact with
cytoskeletal components (31, 32), one view suggests that integrins
transmit signals by organizing the cytoskeleton and regulating cell
shape. It is believed that such a change in cell shape and cytoskeletal organization might regulate the biosynthetic capabilities of the cell,
thereby controlling cell growth and differentiation (33, 34). In the
second view, integrins are considered to exert biochemical signals
similar to growth factor receptor-mediated signaling pathways (35).
Studies performed in platelets show that thrombin treatment coupled
with mechanical stirring results in the cytoskeletal association of
non-receptor tyrosine kinases, in particular c-Src (14-16, 36, 37).
Under such conditions, Fox et al. (16) have further
demonstrated that in addition to c-Src, glycoprotein IIb-IIIa (a major
form of platelet integrin), FAK, and other Src family members are
recruited to the cytoskeleton. Furthermore, cytoskeletal association of these signaling proteins has been shown to result in the
phosphorylation of several cytoskeletal proteins and is suggested to be
responsible for changes in cytoskeletal structure and thus for overall
platelet shape changes. Interestingly, to show such observations in
nucleated cells, Weernink and Rijksen (25) have recently shown
translocation of c-Src to the cytoskeleton in A172 glioblastoma cells
following treatment with platelet-derived growth factor and epidermal
growth factor. Furthermore, in many of the above reports, ligation of integrins with extracellular matrix proteins has been shown to be
necessary for the translocation of c-Src. Although Src family kinases
are present at significant levels in cardiocytes, present knowledge of
their role in proliferation, differentiation, and signaling processes
as well as of their target proteins is limited. Our studies show for
the first time mechanical load-induced movement of non-receptor
tyrosine kinases (c-Src and FAK) and integrins to the cytoskeleton of
the myocardium. Association of these signaling molecules with the
cytoskeleton could be important for both the qualitative phenotypic
changes and the quantitative increase in mass that constitute cardiac
hypertrophy. While it is difficult to determine using in
vivo models whether integrin-mediated signaling pathways are
necessary for translocation of c-Src in pressure-overloaded myocardium,
comigration of Because phosphorylation of c-Src is relatively high when compared with
that of FAK, we focused our further studies on the cytoskeletal
association and activation of c-Src. Cytoskeletal association of c-Src
in platelets is not due to nonspecific trapping during platelet
aggregation (14). Our study of the cytoskeletal association of c-Src
further supports the idea that this translocation occurs as a specific
event in response to cardiac pressure overloading for the following
reasons. First, a closely related Src family member, Fyn, does not
appear to migrate to the cytoskeleton in pressure-overloaded
myocardium, although substantial amounts of this kinase are present
both in Triton X-100-soluble as well as membrane skeleton fractions.
Second, pressure overload-induced cytoskeletal association of c-Src was
observed to be a slow and prolonged process, seen maximally after
48 h of RVPO and then declining to a normal low level after 1 week
of RVPO. This is quite different from the rapid recruitment of c-Src to
the cytoskeleton seen within minutes after agonist-induced activation
of either platelets (16) or glioblastoma cells (25), suggesting that the association of c-Src with the cardiocyte cytoskeleton might be a
specific process occurring subsequent to changes in cytoskeletal structure and/or in c-Src kinase itself. Interestingly, this slow time
course of c-Src recruitment to the cytoskeleton of the
pressure-overloaded ventricle precisely matches the time frame of the
increase in ventricular mass in response to pressure overloading (3).
Third, tyrosine-phosphorylated c-Src is not present in fractions other than the cytoskeletal fractions of pressure-overloaded cardiac tissue
or isolated cardiocytes. If this association is due to nonspecific
trapping, at least some residual amount of tyrosine-phosphorylated c-Src would be expected to be present in the Triton X-100-soluble fraction, where this kinase is mostly present as a
non-tyrosine-phosphorylated protein. Previous studies of the expression
of the oncogenic form of Src (v-Src) in chicken embryonic fibroblasts
showed a spontaneous association with the cytoskeleton, and such an
association of the constitutively active form of this kinase has been
linked to cellular transformation (40, 41). These studies support the
idea that in pressure-overloaded myocardium, c-Src might play a key
role in mediating changes in cardiac structure, composition, and
function through its movement to the cytoskeleton and phosphorylation of associated proteins.
Interestingly, the complete absence of tyrosine-phosphorylated
c-Src in the normal heart suggests tight cardiac regulation of this
kinase. The C-terminal Src kinase (Csk), which phosphorylates c-Src at
Tyr-527 and negatively regulates its activity, has been shown to be
present in almost all tissues and cells. In addition, its level has
been reported to be sufficient to phosphorylate even high amounts of
overexpressed c-Src (28). Although we found substantial amounts of Csk
in ventricular total lysates, we observed no tyrosine phosphorylation
of c-Src in the normal heart. This is further supported by the fact
that the clone 28 monoclonal antibody specific for the Tyr-527
unphosphorylated form of the kinase detects substantial amounts of
c-Src in the Triton X-100-soluble fraction. Furthermore, the complete
absence of tyrosine phosphorylation of c-Src indicates that in addition
to Tyr-527, another positive regulatory site, Tyr-416, is also not
phosphorylated in the normal heart. In the context of this observation,
a recent study from this laboratory shows the presence of a novel
suppressor activity for Src family kinases in the adult
cardiocyte.2 The presence of
tyrosine-phosphorylated c-Src exclusively in the cytoskeletal fraction
of pressure-overloaded myocardium further suggests that by moving to
the cytoskeletal compartment, c-Src could be liberated from inhibitory
constraints that might prevent its phosphorylation.
Our studies on cytoskeleton-bound c-Src in pressure-overloaded
myocardium strongly suggest that the kinase is present in its active
form. Earlier studies in platelets show that cytoskeleton-bound c-Src
is phosphorylated at Tyr-416 upon stimulation with thrombin (26). In
addition, expressed v-Src, which lacks the Tyr-527 negative regulatory
site of c-Src, binds spontaneously to the cytoskeleton and becomes
phosphorylated (40, 41). Similarly, binding of c-Src with polyoma
middle tumor antigen also results in the activation of c-Src (for
review, see Ref. 28). Our studies with the clone 28 antibody show the
association of significant amounts of Tyr-527 unphosphorylated kinase
in the cytoskeletal fraction of pressure-overloaded myocardium. In
addition, at least a part of this form of kinase shows tyrosine
phosphorylation distinct from Tyr-527, indicating that the kinase
undergoes active phosphorylation in vivo, possibly at
Tyr-416. Although we have not eliminated the possibility that some of
the tyrosine phosphorylation of c-Src in the pressure-overloaded
myocardium is at Tyr-527, it is important to note that negative
regulation of c-Src by the Tyr-527 phosphorylated C-terminal residue
requires intact SH2 and SH3 domains of c-Src (42). Furthermore, any
alterations in these domains have been shown to result in the failure
of negative regulation, and the kinase thus becomes activated even
while Tyr-527 is phosphorylated by Csk (28, 42). Interestingly, the SH2
domain of c-Src has also been shown to be responsible for the
cytoskeletal association of c-Src (43). Taken together, these studies
indicate that interaction of the SH2 and/or SH3 domain of c-Src with
the cytoskeletal protein could result in the loss of negative
regulation mediated by the Tyr-527 phosphorylated residue. More
important, our studies show that a significant proportion of the
cytoskeleton-associated c-Src is phosphorylated at a residue other than
Tyr-527. Thus, it is likely that this in vivo
phosphorylation corresponds to Tyr-416 autophosphorylation. In this
context, it is important to note that phosphorylation of any tyrosine
residue other than Tyr-527 has not been implicated in negative
regulation of c-Src (for review, see Ref. 28). Unfortunately, isolation
of cytoskeleton-bound c-Src in pressure-overloaded myocardium required
harsh denaturing conditions, such that we were unable to
immunoprecipitate and show the kinase activity. Nevertheless, our
studies on the in vitro kinase reaction using the
cytoskeletal fraction of pressure-overloaded myocardium show strong
phosphorylation of a 60-kDa protein band corresponding to the size of
c-Src. In addition, studies using a synthetic peptide, employed as a
specific substrate for Src family kinases (21), also suggest enhanced
tyrosine kinase activity in the cytoskeletal fraction of
pressure-overloaded myocardium. Finally, our in vivo studies
reveal several newly tyrosine-phosphorylated proteins in the
cytoskeletal fraction of pressure-overloaded myocardium, where this
kinase is associated in substantial amounts. Taken together, these data
further support our contention that the c-Src that is present in the
cytoskeletal fraction of pressure-overloaded myocardium is functioning
as an activated kinase.
The time course of the cytoskeletal association of c-Src and the
appearance of a 60-kDa tyrosine-phosphorylated protein band containing
c-Src suggests that both events occur concurrently. Two conclusions can
thus be drawn from these observations. (i) In the context of
hypertrophy, our earlier studies (3) have shown a similar time course
for the pressure overload-induced increase in total RNA content. There
is substantial evidence that mRNA and polysomes are associated with
the cytoskeleton (44). As increased protein synthesis is the essential
feature of hypertrophic cardiac growth (1), it is possible that
cytoskeletal association of c-Src and phosphorylation of associated
proteins might result in changes in the internal cellular architecture
that could increase the size of the cytoskeleton-associated polysome
pool. (ii) From a mechanistic point of view, we suggest that tyrosine
phosphorylation of c-Src might occur subsequent to its cytoskeletal
association. This is different from the view of Fox et al.
(16), who, based on their platelet studies, suggest that tyrosine
phosphorylation and activation of c-Src are necessary for its
cytoskeletal association. Our interpretation is based on our
observations that both c-Src phosphorylation and its cytoskeletal
association occur concurrently in pressure-overloaded myocardium and
that under these conditions, tyrosine-phosphorylated c-Src is
exclusively present in the cytoskeletal fraction. This view is further
supported by our unpublished observation2 that a non-ionic
detergent extract obtained from adult cardiocytes exhibits strong c-Src
suppressor activity, suggesting that kinase phosphorylation and
activation may be prevented in the Triton X-100-soluble
compartment.
We have shown here for the first time cytoskeletal association and
activation of c-Src in response to cardiac pressure overloading. The
role that c-Src plays during this very important physiological response
is far from understood. But since many of the cytoskeletal changes in
pressure-overloaded myocardium occur at the level of individual
cardiocytes, one possible role for this kinase might lie in promoting
the assembly and stabilization of cardiocyte cytoskeletal structures.
The further, critical question is what role these events may have in
transducing cardiac load into a hypertrophic response.
We thank Mary Barnes for excellent technical
assistance and Dr. M. Koji Owada and Dr. Hisaaki Kawakatsu for the
clone 28 anti-c-Src monoclonal antibody.
Cell Biology,
Biochemistry,
and ** Physiology, Gazes Cardiac Research Institute and Veterans
Administration Hospital, Medical University of South Carolina,
Charleston, South Carolina 29425-2221
3-integrin, but no Fyn, in the pressure-overloaded
right ventricle. This association was seen as early as 4 h after
right ventricular pressure overloading, increased through 48 h,
and reverted to normal in 1 week. Cytoskeletal binding of non-receptor tyrosine kinases was synchronous with tyrosine phosphorylation of
several cytoskeletal proteins, including c-Src. Examination of
cytoskeleton-bound c-Src revealed that a significant portion of the
tyrosine phosphorylation was not at the Tyr-527 site and therefore
presumably was at the Tyr-416 site. Thus, these studies strongly
suggest that non-receptor tyrosine kinases, in particular c-Src, may
play a critical role in hypertrophic growth regulation by their
association with cytoskeletal structures, possibly via load activation
of integrin-mediated signaling.
ocal
dhesion
inase) has been proposed as a prime candidate for transmitting integrin-mediated signaling, and ligation of
integrins with their extracellular ligands increases tyrosine phosphorylation of several proteins, including FAK (4-12). FAK has
been shown to physically associate with at least two non-receptor tyrosine kinases, c-Src and Fyn, after autophosphorylation and activation (13). Interestingly, it has been shown in platelets that
thrombin-induced platelet aggregation mediated by fibrinogen binding to
integrins (glycoprotein IIb-IIIa) results in a redistribution of c-Src
and Yes to cytoskeletal actin filaments, and this redistribution is
associated with tyrosine phosphorylation of several
cytoskeleton-associated proteins (14-16). Such phosphorylation has
been suggested as a mechanism for the cytoskeletal rearrangement seen
during platelet activation. In addition, cytoskeleton-membrane
junctions are believed to contain binding domains for Src family
kinases, and phosphorylation mediated by these kinases could be
important for signal transmission between the extracellular matrix and
the intracellular cytoskeleton (16). Thus, at least in terminally
differentiated cells such as platelets, integrin-ligand interactions
could cause cytoskeletal reorganization of several proteins, and Src
family kinases could be a critical factor in such events.
3-integrin is seen as early as 4 h after pressure overloading and is maximal at 48 h. (ii) There is
tyrosine phosphorylation of several cytoskeleton-associated proteins
including c-Src. (iii) At least part of the cytoskeleton-associated c-Src exists in the active form. (iv) There is reversion of these early
cytoskeletal changes to their base-line state after long-term pressure
overloading when the hypertrophic growth response is complete. (v) The
movement of c-Src to the cytoskeleton that was observed in cardiac
tissue was also found in cardiocytes isolated from that tissue. (vi)
Several other Src family members do not participate in this
cytoskeletal association. Interestingly, c-Src, as a
tyrosine-phosphorylated kinase, was exclusively present in the
cytoskeletal fraction of pressure-overloaded myocardium and was
completely absent in the normal heart.
Experimental Animal Models
1-integrin (CD29);
Transduction Laboratory for Lyn (C42), FAK (C77),
3-integrin (C26),
and annexin II (C5); and Chemicon International, Inc. for actin (1501).
Polyclonal antibodies were from Santa Cruz for Fyn (FYN3) and Lyn and
from Transduction Laboratory for Yes. The monoclonal antibody against
c-Src that specifically recognizes Tyr-527 unphosphorylated kinase was
obtained as a generous gift from Dr. Koji Owada. Incubations with
primary antibodies were done for either 2 h at room temperature or
overnight at 4 °C. Blots were washed five times with TBST buffer for
5 min and incubated for 1 h with horseradish peroxidase-conjugated
secondary antibodies (Amersham Corp.). After incubation, the blots were
washed with TBST buffer once again, and the proteins were detected by
enhanced chemiluminescence (ECL, Amersham Corp.). For detecting
tyrosine-phosphorylated proteins, the blots after the protein transfer
were blocked with 1% bovine serum albumin for 30 min at 37 °C and
probed with horseradish peroxidase-labeled anti-phosphotyrosine
antibody (PY20, Transduction Laboratory) for 20 min at 37 °C. The
blots were washed, and the tyrosine-phosphorylated proteins were
detected as described above.
-glycerophosphate, 1 mM dithiothreitol, and 1 mM EGTA); phosphorylation was performed as described by Kuppuswamy et al. (20) in the presence or absence of 2 mM synthetic peptide corresponding to amino acids 6-20 of
p34cdc2 kinase (21).
Cytoskeletal Redistribution of Non-receptor Tyrosine Kinases and
Integrins in Pressure-overloaded Myocardium
3-integrin for the
sham-operated control (A), 4-h RVPO (B), 48-h
RVPO (C), and 1-week RVPO (D) are shown in Fig.
1. The levels of these proteins in each fraction were compared between the normally loaded same-animal control LV and pressure-overloaded RV samples as well as fractions from sham-operated controls (Fig. 1A).
Fig. 1.
Western blot analysis for the detection of
actin, c-Src, FAK, and 3-integrin in Triton X-100-lysed ventricular
subfractions. Ventricular tissue samples were processed to obtain
Triton X-100-lysed subfractions, namely soluble, low-spin
(cytoskeleton), and high-spin (membrane skeleton) fractions as
described under "Materials and Methods." The proteins were resolved
by SDS-PAGE and Western-blotted with specific antibodies. In the case
of c-Src, monoclonal antibody GD11, which recognizes all forms of
c-Src, was employed. For RVPO, the pulmonary artery was partially
occluded for various time periods either by inflating a balloon
catheter within it for 4 h or by pulmonary artery-banding for
48 h or longer. A represents the sham-operated control,
and B-D represent 4 h, 48 h, and 1 week of RVPO,
respectively. Lanes 1, 3, and 5 represent normally loaded same-animal control LV, and lanes
2, 4, and 6 represent either normally loaded
RV or pressure-overloaded RV, as indicated.
[View Larger Version of this Image (50K GIF file)]
3-integrin was present in the Triton
X-100-soluble fractions, just as has been reported by Fox et
al. (16) for platelet preparations. They were either absent or
present at very low levels in the Triton X-100-insoluble low- and
high-spin fractions of all normally loaded ventricles. It should be
noted that in sham-operated control cats, where both ventricles were
normally loaded, we found no major difference between these two
ventricles when the levels of any of these three proteins were compared
for each set of Triton X-100-lysed subfractions.
3-integrin when compared
with the normally loaded same-animal control LV. However, the Triton X-100-insoluble low-spin fraction (cytoskeleton) prepared from the
pressure-overloaded RV (lane 4) showed a significant amount of cytoskeleton-associated c-Src, which is completely absent in the
similarly prepared and normally loaded control LV (lane 3). In addition, FAK and
3-integrin also showed increased association with the Triton X-100-insoluble fraction (cytoskeleton) in the pressure-overloaded ventricle.
3-integrin, only a moderate increase in
its association with the cytoskeletal fraction of the
pressure-overloaded RV was noticed. On the other hand, a significant
increase in the association of
3-integrin with the membrane skeleton
due to pressure overloading was observed. All of these changes in the
cytoskeletal redistribution of c-Src, FAK, and
3-integrin returned
to their normal levels when RVPO was prolonged for 1 week (Fig.
1D), and the same finding was obtained for RVPO durations as
long as 5 weeks (data not shown).
3-integrins) redistribute to
the Triton X-100-insoluble cytoskeletal pellet and that these changes
are seen as early as 4 h, are maximal by 48 h, and return to
their normal levels by 1 week of RVPO. These results were confirmed in
at least three cats for each time point.
Fig. 2.
Western blot showing tyrosine-phosphorylated
proteins present in the Triton X-100-lysed subfractions of 48-h
pressure-overloaded RV and normally loaded same-animal control LV.
Triton X-100-lysed fractions were prepared from a 48-h pulmonary
artery-banded cat, and the proteins in each sample were resolved by
SDS-PAGE as described under "Materials and Methods."
Tyrosine-phosphorylated proteins were detected by Western blotting with
anti-phosphotyrosine antibody. Arrows indicate the positions
of tyrosine-phosphorylated protein bands corresponding to the size of
FAK (top), c-Src (middle), and annexin II
(bottom).
[View Larger Version of this Image (29K GIF file)]
60-kDa tyrosine-phosphorylated protein band (Fig. 2,
arrow) in the cytoskeletal fraction of the
pressure-overloaded RV matches the size of c-Src shown in the previous
experiment (Fig. 1). This would be consistent with the increase in
cytoskeletal association of c-Src in pressure-overloaded myocardium. It
is interesting to note that this band is present only in the
cytoskeletal fraction, while it is completely absent both in the
soluble and membrane skeleton fractions. Furthermore, in none of the
subfractions of the normally loaded control LV was a
60-kDa
tyrosine-phosphorylated protein band present in detectable amounts.
Such an observation raises two possibilities. First, phosphorylation of
the protein(s) in the
60-kDa band might be occurring only after its
association with the cytoskeleton; and second, the phosphorylation
might occur in other compartments prior to cytoskeletal binding in
pressure-overloaded myocardium and then completely translocate to the
cytoskeleton.
5% of the total) of this protein was associated with the
cytoskeletal fraction of the pressure-overloaded RV, but was almost
absent in the normally loaded LV (data not shown).
60-kDa Protein Band
60-kDa tyrosine-phosphorylated protein band
was also maximal during this time period. For this purpose, the
cytoskeletal fractions obtained from control and pressure-overloaded
ventricles for various time periods were examined for the presence of
tyrosine-phosphorylated proteins. The
60-kDa tyrosine-phosphorylated
protein could be detected as early as 4 h after RVPO and was
maximal 48 h after RVPO (Fig. 3). This change in
the level of the
60-kDa tyrosine-phosphorylated protein band
reverted to a normal low level following a 1 week or longer (5 weeks)
period of RVPO, when the hypertrophic response to a step increase in
load is complete. During the entire time course, the level of the
42-kDa tyrosine-phosphorylated protein band that corresponds to actin
did not vary appreciably between the two ventricular samples. More
important, the time course for tyrosine phosphorylation of the
60-kDa protein band parallels that of the cytoskeletal association
of c-Src shown in Fig. 1. All of these experiments suggest that the
cytoskeletal association of c-Src and the phosphorylation of the
60-kDa protein band occur concurrently, with both being maximal
after 48 h of pressure overloading.
Fig. 3.
Time course of RVPO versus the
appearance of a 60-kDa tyrosine-phosphorylated protein band in the
cytoskeletal fraction. LVs and RVs from either sham-operated
control cats or RVPO cats at 4 h, 48 h, 1 week, or 5 weeks
were lysed with Triton X-100 buffer; cytoskeletal pellets were prepared
as described under "Materials and Methods." The pellets were washed
once with Triton X-100 buffer, and the proteins were eluted with SDS
sample buffer. After resolving the proteins by SDS-PAGE, the
tyrosine-phosphorylated proteins were detected by Western blotting with
anti-phosphotyrosine antibody. The position of the
tyrosine-phosphorylated protein band corresponding in size to c-Src is
shown by the closed arrow; the position of
tyrosine-phosphorylated actin is shown by the open
arrow.
[View Larger Version of this Image (37K GIF file)]
60-kDa
Tyrosine-phosphorylated Protein Band
60-kDa tyrosine-phosphorylated
protein band. Total extracts were prepared from 48-h
pressure-overloaded RV and same-animal normal LV as well as control RV
and LV. As can be seen in Fig. 4A, c-Src was
present in substantial amounts in each extract. These total lysates
were then immunoprecipitated with anti-c-Src antibody (clone GD11) and
examined for tyrosine-phosphorylated c-Src by Western blotting with
anti-phosphotyrosine antibody. A significant amount of
tyrosine-phosphorylated c-Src was present only in the
pressure-overloaded RV (Fig. 4B, lane 4), while
phosphorylated c-Src was undetectable in normally loaded ventricles
(lanes 1-3). This observation was further confirmed by
performing a reciprocal immunoprecipitation in which the total lysate
was used to first immunoprecipitate all of the tyrosine-phosphorylated
proteins and then examined for the presence of c-Src in the immune
complex by Western blotting with anti-c-Src antibody. As anticipated, tyrosine-phosphorylated c-Src was present only in the
pressure-overloaded ventricles (Fig. 4C, lane 4),
while it was absent in all control ventricles (lanes 1-3).
These results clearly show that c-Src is tyrosine-phosphorylated during
pressure overloading. Furthermore, since in our previous experiment, a
60-kDa tyrosine-phosphorylated protein band was present exclusively
in the cytoskeletal fraction of pressure-overloaded myocardium (Fig. 2,
lane 4), the results shown in Fig. 4 (B and
C, lane 4) should represent cytoskeleton-bound tyrosine-phosphorylated c-Src. To confirm this, we isolated
cytoskeletal fractions from normally loaded and pressure-overloaded
ventricles and then extracted the associated proteins with SDS sample
buffer. From these samples, c-Src was immunoprecipitated and then
probed for tyrosine-phosphorylated c-Src. As shown in Fig.
4D, tyrosine-phosphorylated c-Src was present in the
cytoskeletal fraction of pressure-overloaded myocardium. All of these
experiments clearly indicate that in pressure-overloaded myocardium,
cytoskeleton-bound c-Src (Fig. 1C, lane 4) exists
as a tyrosine-phosphorylated protein and that a significant amount of
the
60-kDa tyrosine-phosphorylated protein band shown in the
previous experiment (Fig. 2, lane 4) represents tyrosine-phosphorylated c-Src. Since this antibody has been suggested to have weak affinity for SDS-denatured c-Src, immunodepletion experiments were not performed to find out whether the entirety of the
60-kDa band is c-Src. Nevertheless, these experiments clearly
suggest that c-Src is at least one of the major components present in
the
60-kDa tyrosine-phosphorylated protein band.
Fig. 4.
Immunoprecipitation combined with Western
blot analysis for the detection of tyrosine-phosphorylated c-Src.
Ventricular tissue or cytoskeletal samples obtained from normal or 48-h
RVPO cats were extracted with SDS sample buffer. Aliquots of these samples were used for immunoprecipitation with anti-c-Src monoclonal antibody GD11 (B and D) or anti-phosphotyrosine
antibody (C). The proteins present in the immune complex as
well as in aliquots of total tissue lysate (A) were resolved
by SDS-PAGE and Western-blotted either with anti-c-Src antibody GD11
(A and C) or with anti-phosphotyrosine antibody
(B and D). Lanes 1 and 3 represent normally loaded LV, while lanes 2 and 4 represent normally loaded RV and 48-h pressure-overloaded RV,
respectively. WB, Western blot; IP,
immunoprecipitation; -c-Src, anti-c-Src antibody (GD11);
-PY, anti-phosphotyrosine antibody.
[View Larger Version of this Image (22K GIF file)]
Fig. 5.
Immunoprecipitation combined with Western
blot analysis for the detection of Tyr-527 unphosphorylated c-Src in
Triton X-100-lysed ventricular subfractions of a 48-h RVPO cat.
Ventricular tissue samples were processed to obtain Triton X-100-lysed
subfractions, namely soluble, low-spin (cytoskeleton), and high-spin
(membrane skeleton) fractions as described under "Materials and
Methods." For the experiment shown in A, the proteins were
resolved by SDS-PAGE and Western-blotted with the clone 28 anti-c-Src
monoclonal antibody. In B, aliquots of Triton X-100-lysed
subfractions were used for immunoprecipitation with
anti-phosphotyrosine antibody. The phosphorylated proteins present in
the immune complex were resolved by SDS-PAGE and Western-blotted with
the clone 28 anti-c-Src antibody. WB, Western blot;
IP, immunoprecipitation; -c-Src, anti-c-Src
antibody (clone 28);
-PY, anti-phosphotyrosine
antibody.
[View Larger Version of this Image (27K GIF file)]
60-kDa Tyrosine-phosphorylated
Protein Band in the Cytoskeletal Fraction of Cardiocytes Isolated from
Pressure-overloaded Cat Ventricles
60-kDa tyrosine-phosphorylated
protein, which we have shown to contain c-Src, was detected only in the
cardiocyte samples obtained from the pressure-overloaded RV (Fig.
6). Furthermore, Western blot analysis for the detection
of c-Src (Upstate Biotechnology, Inc.) showed comigration of c-Src with
this
60-kDa protein band (data not shown). These data demonstrate
that the changes seen with c-Src in pressure-overloaded myocardium
occur at the level of individual cardiocytes.
Fig. 6.
Western blot showing the presence of a
60-kDa tyrosine-phosphorylated protein band in the cardiocyte
cytoskeletal fraction. Cardiocytes were isolated from a 48-h
pressure-overloaded RV and a normally loaded same-animal control LV.
Cells were extracted with Triton X-100 buffer, and the low-spin
cytoskeletal fractions were obtained. After resolving the proteins by
SDS-PAGE, tyrosine-phosphorylated proteins were detected by Western
blot analysis with anti-phosphotyrosine antibody. The presence of a
60-kDa tyrosine-phosphorylated protein band corresponding in size to
c-Src in the pressure-overloaded RV is shown by the closed
arrow, and the position of actin is indicated by the open
arrow. WB, Western blot;
-PY,
anti-phosphotyrosine antibody.
[View Larger Version of this Image (22K GIF file)]
Fig. 7.
Western blot analysis of the distribution of
actin, c-Src, and Fyn in Triton X-100-lysed fractions of a 48-h
pressure-overloaded RV and a normally loaded same-animal control
LV. Triton X-100-soluble and -insoluble fractions were prepared
from a 48-h RVPO cat as described under "Materials and Methods."
The proteins were separated by SDS-PAGE and Western-blotted with
antibodies raised against actin, c-Src (GD11), or Fyn
(polyclonal).
[View Larger Version of this Image (27K GIF file)]
3-integrin and FAK with c-Src suggests that
integrin-mediated signaling events might be necessary for the
cytoskeletal association of c-Src. Although
3-integrin was detected,
1-integrin appeared to be absent in all of the Triton X-100-lysed
subfractions based on our studies using a specific monoclonal antibody
against
1-integrin. Recent reports (38, 39) show a skeletal and
cardiac muscle-specific isoform of the
-integrins,
1-D. It will
be interesting to determine whether the cardiac muscle-specific
1-D
integrin isoform is a major factor in coupling external mechanical
signals to hypertrophic growth signals by translocating one or more
potential non-receptor tyrosine kinases such as c-Src. In the second
view of integrin-mediated signaling (35), FAK plays a major role in
mediating mitogenic signaling pathways. In normal and
pressure-overloaded adult feline myocardia, we did not observe a
substantial level of a tyrosine-phosphorylated protein band
corresponding to the size of FAK, although a significant amount of this
kinase was found to be associated with the cytoskeleton between 4 and
48 h of RVPO. It is possible that an initial wave of FAK tyrosine
phosphorylation might have occurred during the early time period of
pressure overloading prior to FAK's cytoskeletal association. However,
experiments with shorter time periods of RVPO are difficult to perform
with in vivo animal models.
*
This work was supported by Program Project Grant HL-48788
from NHLBI and by research funds from the Department of Veterans Affairs. The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Cardiology Div.,
Medical University of South Carolina, Charleston, SC 29425-2221. Tel.:
803-792-3405; Fax: 803-792-7771; E-mail:
kuppusd{at}smtpgw.musc.edu.
1
The abbreviations used are: RVPO, right
ventricular pressure overload; RV, right ventricle; LV, left ventricle;
PAGE, polyacrylamide gel electrophoresis.
2
V. S. Kasi and D. Kuppuswamy, manuscript in
preparation.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.