Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, Indiana 46202
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The tyrosine
phosphorylation of paxillin increases in association with force
development during tracheal smooth muscle contraction, suggesting that
paxillin plays a role in the contractile activation of smooth muscle
[Z. L. Wang, F. M. Pavalko, and S. J. Gunst. Am.
J. Physiol. 271 (Cell
Physiol. 40): C1594-C1602, 1996]. We compared the Ca2+ sensitivity of
the tyrosine phosphorylation of paxillin and myosin light chain (MLC)
phosphorylation in tracheal muscle and evaluated whether MLC
phosphorylation is necessary to induce paxillin phosphorylation. Ca2+-depleted muscle strips were
stimulated with
107-10
4
M acetylcholine (ACh) in 0, 0.05, 0.1, or 0.5 mM extracellular Ca2+. In the absence of
extracellular Ca2+,
10
4 M ACh induced a maximal
increase in paxillin phosphorylation without increasing MLC
phosphorylation or force. Increases in extracellular
Ca2+ concentration did not further
increase paxillin phosphorylation. However, during stimulation with
10
6 M ACh, paxillin
phosphorylation increased with increases in extracellular Ca2+ concentration. We conclude
that the tyrosine phosphorylation of paxillin can be stimulated by
signaling pathways that do not depend on
Ca2+ mobilization and that the
activation of contractile proteins is not required to elicit paxillin
phosphorylation.
myosin light chain phosphorylation; cytoskeleton; focal adhesion proteins; smooth muscle contraction
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE CYTOSKELETAL PROTEINS paxillin and talin have been localized to the focal adhesion sites of cultured cells (5, 27) as well as to the membrane-associated dense plaque (MADP) sites of smooth muscle cells (9, 28). These proteins are thought to play a role in linking actin filaments to transmembrane integrins to enable force transmission across the membrane (4, 31). In cultured cells, talin and paxillin undergo phosphorylation during integrin-mediated cell adhesion and during stimulation by a variety of mitogens and growth factors (6, 16, 26, 29, 33). The phosphorylation of these proteins has been correlated with the assembly of actin stress fibers and with focal adhesion formation (6,7, 23, 29). It has been proposed that the phosphorylation of actin-membrane linker proteins may regulate the interaction of actin filaments with the cytoplasmic domains of transmembrane integrins in focal adhesions and smooth muscle dense plaques (5, 7, 18, 29).
In previous studies we reported that the contraction of tracheal smooth muscle with muscarinic agonists results in a three- to fourfold increase in the phosphorylation of paxillin and talin (17, 32). The increase in paxillin phosphorylation occurs on tyrosine residues, whereas talin is phosphorylated on serine-threonine residues. The changes in the phosphorylation of these MADP proteins occur with a time course similar to force development, suggesting that they may play a role in the contractile activation of smooth muscle. We have postulated that the stimulation of smooth muscle cells with contractile agonists may initiate active processes that regulate the organization of the actin cytoskeleton and the attachment of actin filaments to the membrane at MADP sites and that these events may occur in parallel to the activation of contractile proteins (17). The remodeling of the organization of actin filaments could serve to optimize force development to the physical conformation of the smooth muscle cell at the time of contractile activation.
If the tyrosine phosphorylation of paxillin plays a role in smooth muscle contraction, regulation of the phosphorylation of paxillin and of myosin light chains (MLCs) may be interdependent and may occur through the stimulation of a common signaling pathway. This is suggested by studies of cultured fibroblasts, in which the activation of contractile proteins was found to be prerequisite to the tyrosine phosphorylation of paxillin, the assembly of actin stress fibers, and focal adhesion formation (4, 8). Alternatively, the activation of contractile and cytoskeletal proteins may be parallel events elicited through distinct signaling pathways.
Agonists that activate smooth muscle tissues via receptor-coupled pathways can also stimulate pathways that increase the Ca2+ sensitivity of the serine-threonine phosphorylation of the regulatory MLC (10, 13, 20). It has been proposed that the mechanisms that regulate the Ca2+ sensitivity of contractile activation in smooth muscle may involve protein tyrosine phosphorylation (25).
In the present study we have compared the Ca2+ sensitivity of the tyrosine phosphorylation of paxillin with the Ca2+ sensitivity of MLC phosphorylation during the muscarinic stimulation of tracheal smooth muscle strips. We evaluated whether the activation of contractile proteins is required to induce the tyrosine phosphorylation of paxillin in this tissue.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue preparation.
Mongrel dogs weighing 20-25 kg were anesthetized with
pentobarbital sodium and quickly exsanguinated. A 10- to 15-cm segment of extrathoracic trachea was immediately removed and immersed in
physiological saline solution (PSS) composed of (in mM) 110 NaCl, 3.4 KCl, 2.4 CaCl2, 0.8 MgSO4, 25.8 NaHCO3, 1.2 KH2PO4, and 5.6 glucose. The solution was aerated with 95%
O2-5%
CO2 to maintain a pH of 7.4. Rectangular strips of trachealis muscle 12-15 mm long and 2-3
mm wide were dissected from the trachea after removal of the epithelium
and connective tissue layer. Muscle strips were mounted in PSS at
37°C in a 25-ml glass tissue bath and attached to a Grass force
transducer at a resting tension of 2-4 g and then equilibrated for
~90 min. Each muscle was then stimulated repeatedly with
105 M acetylcholine (ACh;
Sigma Chemical). The optimal length for maximal active force
(Lo) was determined by increasing the muscle length progressively after each stimulation until the force of active
contraction reached a maximum
(Fmax).
Ca2+
depletion of muscle strips.
After the determination of Lo, muscle strips
were depleted of Ca2+ as described
previously (10). Briefly, strips were incubated in
Ca2+-free PSS containing 0.1 mM
ethylene glycol-bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA) for 10 min for the removal of extracellular
Ca2+. No change in resting tension
occurred when the bath was changed from PSS to
Ca2+-free PSS containing 0.1 mM
EGTA. Muscle strips were then stimulated for 10 min by addition of
10
5 M ACh to the
Ca2+-free PSS. This step was
repeated three to four times with
10
5 M ACh. Between each
stimulation the strips were incubated in Ca2+-free PSS containing 0.1 mM
EGTA for 10 min. Stimulation with 10
5 M ACh initially
produced a force of ~70% Fmax,
but subsequent stimulations resulted in progressively smaller
contractions. At the end of the depletion protocol, force in response
to 10
5 M ACh was <10% of
Fmax. The strips did not contract
in response to 60 mM KCl after the
Ca2+-depletion protocol.
Experimental design.
Up to 14 muscle strips from a single trachea were studied concurrently.
Duplicate muscle strips were used for each measurement. Ca2+-depleted strips were
stimulated with ACh in the absence of extracellular Ca2+ for 5 min and then rapidly
freeze clamped with liquid
N2-cooled tongs for measurement of
the tyrosine phosphorylation of paxillin or MLC phosphorylation. The
effects of Ca2+ on paxillin and
MLC phosphorylation were also assessed by increasing the extracellular
Ca2+ concentration
([Ca2+]o)
during stimulation with ACh. Muscle strips not subjected to the
Ca2+-depletion protocol were also
frozen at rest or after stimulation with
104 or
10
6 M ACh. In most cases,
separate experiments were performed for the analysis of MLC
phosphorylation and paxillin phosphorylation for any given
protocol.
Measurement of the tyrosine phosphorylation of paxillin. The tyrosine phosphorylation of paxillin was determined by Western blot as described previously (32). Frozen muscle strips were pulverized under liquid N2, and the powder was transferred to dry-ice-cooled centrifuge tubes. While on dry ice, 300 µl of extraction buffer were added to each of the tubes, and then they were quickly vortexed. The extraction buffer contained 20 mM tris(hydroxymethyl)aminomethane (pH 7.4), 1% Triton X-100, 0.2% sodium dodecyl sulfate, 2 mM EDTA, phosphatase inhibitors (2 mM sodium orthovanadate, 2 mM molybdate, and 2 mM sodium pyrophosphate), and protease inhibitors (2 mM benzamidine, 0.5 mM aprotinin, and 1 mM phenylmethylsulfonyl fluoride). Each sample was then boiled for 5 min to inactivate phosphatases and proteases, and then it was kept at 4°C for 1 h. The concentration of protein in each sample was determined using a standard bicinchoninic acid protein assay kit (Pierce).
Protein (40 µg) from each muscle extract was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were then transferred to nitrocellulose, blocked with 2% gelatin, and probed with mouse monoclonal antiphosphotyrosine antibody (ICN Pharmaceuticals) and then with horseradish peroxidase anti-mouse immunoglobulin (Amersham) for visualization by chemiluminescence. Nitrocellulose membranes were then stripped of bound antibodies and reprobed with mouse monoclonal antibody against paxillin (Transduction Laboratories) to confirm the location of paxillin and to normalize for minor differences in protein loading. Phosphotyrosine-containing proteins and paxillin were visualized by chemiluminescence and quantitated by scanning densitometry. In previous studies, similar increases in paxillin phosphorylation were obtained from phosphotyrosine blots of paxillin immunoprecipitated from muscle extracts and from blots of whole muscle extracts (17, 32).Measurement of MLC phosphorylation. MLC phosphorylation was measured in separate experiments. Frozen muscle strips were immersed in dry ice-cooled acetone containing 10% (wt/vol) trichloroacetic acid (TCA) and 10 mM dithiothreitol (DTT). Strips were thawed in acetone-TCA-DTT at room temperature and then washed with acetone-DTT. MLCs were extracted for 60 min in 8 M urea, 20 mM tris(hydroxymethyl)- aminomethane, 22 mM glycine, and 10 mM DTT. Proteins were separated by glycerol-urea polyacrylamide gel electrophoresis and blotted to nitrocellulose. MLCs were specifically labeled with polyclonal rabbit anti-MLC 20 antibody. The primary antibody was detected with 125I-labeled recombinant protein A (New England Nuclear). Unphosphorylated and phosphorylated bands of MLCs were localized on nitrocellulose membranes by autoradiography. Bands were cut out and counted in a gamma counter. Background counts were subtracted, and fractional phosphorylation was calculated as the ratio of phosphorylated MLCs to total MLCs.
Statistical analysis. Comparisons among different groups were performed by one-way analysis of variance or Kruskal-Wallis one-way analysis of variance. Differences between pairs of groups were analyzed by Student's t-test or Dunn's method. All statistical analyses were performed using SigmaStat software. Values of n represent the number of experiments used to obtain each value. P < 0.05 was considered to be significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of ACh concentration on MLC phosphorylation and the tyrosine
phosphorylation of paxillin in
Ca2+-depleted
muscle strips.
Duplicate muscle strips were depleted of
Ca2+ and then stimulated with
107-10
4
M ACh in the absence of extracellular
Ca2+ for 5 min. They were then
quickly frozen for the measurement of MLC phosphorylation and the
tyrosine phosphorylation of paxillin. MLC phosphorylation and tyrosine
phosphorylation of paxillin were also determined in muscle strips that
had not been depleted of Ca2+.
(Changes in the tyrosine phosphorylation of paxillin are illustrated in
the immunoblot shown in Fig.
1.)
|
|
Ca2+
sensitivity of paxillin phosphorylation and MLC phosphorylation in
response to ACh.
Ca2+-depleted muscle strips were
stimulated with 104 or
10
6 M ACh in
Ca2+-free buffer. After 1 min in
ACh, 0.05, 0.1, or 0.5 mM extracellular Ca2+ was added or strips were
maintained in 0 mM extracellular
Ca2+. Muscle strips were frozen 5 min after the addition of extracellular Ca2+, and MLC phosphorylation and
the tyrosine phosphorylation of paxillin were determined (Fig.
3).
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our results show that maximal muscarinic stimulation of Ca2+-depleted tracheal smooth muscle strips in the absence of extracellular Ca2+ elicits a maximal increase in the tyrosine phosphorylation of paxillin. Under the same conditions, muscarinic receptor stimulation does not cause contraction of the muscle, nor does it produce a significant increase in MLC phosphorylation. Thus the tyrosine phosphorylation of paxillin can occur in the absence of the activation of contractile proteins, indicating that the tyrosine phosphorylation of paxillin and MLC phosphorylation can be elicited by independent signaling pathways. These observations demonstrate that contractile protein activation is not prerequisite to the tyrosine phosphorylation of paxillin in tracheal smooth muscle.
Our results also suggest a
Ca2+-independent pathway for the
tyrosine phosphorylation of paxillin in tracheal smooth muscle in addition to a Ca2+-activated
pathway. When Ca2+-depleted
tracheal smooth muscle strips were maximally stimulated with ACh in the
absence of extracellular Ca2+, a
maximal increase in paxillin phosphorylation was observed that was
unaffected by increases in
[Ca2+]o.
This indicates that maximal paxillin phosphorylation can be elicited
through a pathway that does not require
Ca2+ mobilization. Tyrosine
phosphorylation of paxillin was also elevated during submaximal
stimulation with 106 M ACh
in the absence of extracellular
Ca2+. However, at this ACh
concentration, significant increases in paxillin phosphorylation were
elicited when
[Ca2+]o
was increased. Thus, although the muscarinic stimulation of tracheal
smooth muscle in the absence of extracellular
Ca2+ can stimulate a maximal
increase in the tyrosine phosphorylation of paxillin, a
Ca2+-activated mechanism for
stimulating paxillin tyrosine phosphorylation is also present. When the
muscarinic stimulation of tracheal smooth muscle is submaximal, the
Ca2+-activated increase in
paxillin phosphorylation adds to the paxillin phosphorylation that
occurs in the absence of extracellular
Ca2+. Therefore, although
independent pathways for the phosphorylation of MLC and paxillin appear
to be present, a common
Ca2+-activated pathway may also be
able to stimulate the phosphorylation of both proteins. These results
are consistent with previous observations in cultured fibroblasts in
which the tyrosine phosphorylation of paxillin can be stimulated in the
absence of Ca2+ mobilization (24,
33). In some cultured cells, paxillin phosphorylation has also been
shown to be sensitive to Ca2+
activation (14).
Our present results are similar to our previous observation that a significant increase in MLC phosphorylation occurs in Ca2+-depleted canine tracheal smooth muscle strips in response to muscarinic stimulation only after [Ca2+]o is increased to 0.05 mM (10). In this previous study we also measured intracellular Ca2+ in Ca2+-depleted tracheal muscle strips. We found that muscarinic stimulation elicited a very small increase in intracellular Ca2+, even in the absence of extracellular Ca2+. Thus in the present study the maximal increase in paxillin phosphorylation elicited by ACh in the absence of extracellular Ca2+ may require a small increase in intracellular Ca2+; however, this increase in intracellular Ca2+ is insufficient to induce MLC phosphorylation.
The results of studies in cultured fibroblasts contrast with ours, in that they suggest that MLC phosphorylation and contractile protein activation are required for the tyrosine phosphorylation of the cytoskeletal proteins paxillin and pp125 focal adhesion kinase (FAK) (4, 8). There is evidence that FAK is the tyrosine kinase that phosphorylates paxillin in cultured smooth muscle cells and fibroblasts (3, 30). The activation of fibroblasts by a number of agents, including vasopressin, endothelin, and lysophosphatidic acid, stimulates the formation of stress fibers and the assembly of focal adhesions. These events are associated with a marked increase in the tyrosine phosphorylation of paxillin and FAK and with MLC phosphorylation (2, 4, 8, 19, 21, 22). The small GTP-binding protein rho has been shown to mediate these events (2, 4, 8, 19, 21, 22). The inhibition of fibroblast contractility and MLC phosphorylation by a number of mechanisms prevents rho-induced paxillin phosphorylation and FAK activation, and it also prevents actin stress fiber formation and focal adhesion assembly (4, 8).
The receptor-mediated activation of tracheal and vascular smooth muscles by agonists such as histamine, phenylephrine, or carbachol has been shown to induce a Ca2+ sensitization of MLC phosphorylation and force (10, 13, 20). This enhanced sensitivity of contractile activation appears to be G protein mediated (13). The small G protein rho has been implicated in the Ca2+ sensitization of contractile protein activation (11) by mediating the inhibition of MLC phosphatase (12, 15) or by stimulating the phosphorylation of MLC on serine-threonine residues by rho kinase (1). Inasmuch as rho is also implicated in cellular processes associated with cytoskeletal organization in cultured fibroblasts (21), receptor activation by contractile agonists in smooth muscle may initiate processes leading to cytoskeletal reorganization as well as to the sensitization of contractile protein activation (25). Although our data indicate that MLC phosphorylation is not prerequisite to paxillin phosphorylation in tracheal smooth muscle, it remains possible that the tyrosine phosphorylation of cytoskeletal proteins plays a role in mediating the Ca2+ sensitization of contractile proteins in this tissue.
In conclusion, the results of this study demonstrate that the muscarinic stimulation of tracheal smooth muscle can activate signaling pathways for the tyrosine phosphorylation of paxillin that appear to be independent of Ca2+ mobilization. In the absence of extracellular Ca2+, muscarinic stimulation elicits high levels of tyrosine phosphorylation on paxillin without eliciting significant increases in MLC phosphorylation or contraction. This observation demonstrates that MLC phosphorylation is not required to stimulate paxillin phosphorylation in tracheal smooth muscle; thus the activation of paxillin can occur independently of contractile protein activation. This is consistent with our previous suggestion that the activation of MADP proteins and contractile proteins may be parallel events in the contractile activation of smooth muscle. Paxillin phosphorylation may play a role in regulating the organization of actin filaments in smooth muscle tissues in response to contractile stimulation. Cytoskeletal proteins may also be involved in regulating the sensitivity of contractile proteins to activation by Ca2+.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by National Heart, Lung, and Blood Institute Grant HL-29289 and by a postdoctoral fellowship from the American Heart Association, Indiana Affiliate, to D. Mehta.
![]() |
FOOTNOTES |
---|
Address for reprint requests: S. J. Gunst, Dept. of Physiology and Biophysics, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202-5126.
Received 5 May 1997; accepted in final form 13 November 1997.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Amano, M.,
M. Ito,
K. Kimura,
Y. Fukata,
K. Chihara,
T. Niacin,
Y. Matsuura,
and
K. Kaibuchi.
Phosphorylation and activation of myosin by rho-associated kinase (rho-kinase).
J. Biol. Chem.
271:
20246-20249,
1996
2.
Barry, S. T.,
and
D. R. Critchley.
The Rho-A dependent assembly of focal adhesions in Swiss 3T3 cells is associated with increased tyrosine phosphorylation and the recruitment of both pp125FAK and protein kinase C- to focal adhesions.
J. Cell Sci.
107:
2033-2045,
1994
3.
Bellis, S. L.,
J. T. Miller,
and
C. E. Turner.
Characterization of tyrosine phosphorylation of paxillin in vitro by focal adhesion kinase.
J. Biol. Chem.
270:
17437-17441,
1995
4.
Burridge, K.,
and
M. Chrzanowska-Wodnicka.
Focal adhesions, contractility, and signaling.
Ann. Rev. Cell Dev. Biol.
12:
463-519,
1996.[Medline]
5.
Burridge, K.,
K. Fath,
T. Kelly,
G. Nuckolls,
and
C. E. Turner.
Focal-adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton.
Annu. Rev. Cell Biol.
4:
487-525,
1988.
6.
Burridge, K.,
C. E. Turner,
and
L. H. Romer.
Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly.
J. Cell Biol.
119:
893-903,
1992[Abstract].
7.
Burn, P.,
A. Kupfer,
and
S. J. Singer.
Dynamic membrane-cytoskeletal interactions: specific association of integrin and talin arises in vivo after phorbol ester treatment of peripheral blood lymphocytes.
Proc. Natl. Acad. Sci. USA
85:
497-501,
1988[Abstract].
8.
Chrzanowska-Wodnicka, M.,
and
K. Burridge.
Rho-stimulated contractility drives the formation of stress fibers and focal adhesions.
J. Cell Biol.
133:
1403-1415,
1996[Abstract].
9.
Drenckhahn, D.,
M. Beckerle,
K. Burridge,
and
J. Otto.
Identification and subcellular location of talin in various cell types and tissues by means of [125I]vinculin overlay, immunoblotting and immunocytochemistry.
Eur. J. Cell Biol.
46:
513-522,
1988[Medline].
10.
Gerthoffer, W. T.,
K. A. Murphey,
and
S. J. Gunst.
Aequorin luminescence, myosin phosphorylation, and active stress in tracheal smooth muscle.
Am. J. Physiol.
257 (Cell Physiol. 26):
C1062-C1068,
1989
11.
Hirata, K.,
A. Kikuchi,
T. Sasaki,
S. Kuroda,
K. Kaibuchi,
Y. Matsuura,
H. Seki,
K. Saida,
and
Y. Takai.
Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction.
J. Biol. Chem.
267:
8719-8722,
1992
12.
Kimura, K.,
M. Ito,
M. Amano,
K. Chihara,
Y. Fukata,
Y. Matsuura,
M. Nakafuku,
B. Yamameri,
J. Feng,
T. Nakano,
K. Okawa,
A. Iwamatsu,
and
K. Kaibuichi.
Regulation of myosin phosphatase by rho and rho-associated kinase.
Science
273:
245-248,
1996[Abstract].
13.
Kitazawa, T.,
B. D. Gaylinn,
G. H. Denney,
A. V. Somlyo,
and
A. P. Somlyo.
G protein-mediated Ca2+ sensitization of smooth muscle contraction through myosin light chain phosphorylation.
J. Biol. Chem.
266:
1708-1715,
1991
14.
Leduc, I.,
and
S. Meloche.
Angiotensin II stimulates tyrosine phosphorylation of the focal adhesion-associated protein paxillin in aortic smooth muscle cells.
J. Biol. Chem.
270:
4401-4404,
1995
15.
Noda, M. C.,
Y. Fukazawa,
K. Moriishi,
T. Kato,
T. Okuda,
K. Kurokawa,
and
Y. Takuwa.
Involvement of rho in GTPS-induced enhancement of phosphorylation of 20 kDa myosin light chain in vascular smooth muscle cells: inhibition of phosphatase activity.
FEBS Lett.
367:
246-250,
1995[Medline].
16.
Pasquale, E. B.,
P. A. Maher,
and
S. J. Singer.
Talin is phosphorylated on tyrosine in chicken embryo fibroblasts transformed by Rous sarcoma virus.
Proc. Natl. Acad. Sci. USA
83:
5507-5511,
1986[Abstract].
17.
Pavalko, F. M.,
L. P. Adam,
M.-F. Wu,
T. L. Walker,
and
S. J. Gunst.
Phosphorylation of dense plaque proteins talin and paxillin during tracheal smooth muscle contraction.
Am. J. Physiol.
268 (Cell Physiol. 37):
C563-C571,
1995
18.
Pavalko, F. M.,
and
C. A. Otey.
Role of adhesion molecule cytoplasmic domains in mediating interactions with the cytoskeleton.
Proc. Soc. Exp. Biol. Med.
205:
282-293,
1994[Abstract].
19.
Rankin, S.,
N. Morii,
S. Narumiya,
and
E. Rozengurt.
Botulinum C3 exoenzyme blocks the tyrosine phosphorylation of p125FAK and paxillin induced by bombesin and endothelin.
FEBS Lett.
354:
315-319,
1994[Medline].
20.
Rembold, C. M.,
and
R. A. Murphy.
Myoplasmic [Ca2+] determines myosin phosphorylation in agonist-stimulated swine arterial smooth muscle.
Circ. Res.
63:
593-603,
1988[Abstract].
21.
Ridley, A. J.,
and
A. Hall.
The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors.
Cell
70:
389-399,
1992[Medline].
22.
Ridley, A. J.,
and
A. Hall.
Signal transduction pathways regulating Rho-mediated stress fiber formation: requirement for a tyrosine kinase.
EMBO J.
13:
2600-2610,
1994[Abstract].
23.
Romer, L. H.,
N. McLean,
C. E. Turner,
and
K. Burridge.
Tyrosine kinase activity, cytoskeletal organization and motility in human vascular endothelial cells.
Mol. Biol. Cell
5:
349-361,
1994[Abstract].
24.
Seufferlein, T.,
and
E. Rozengurt.
Lysophosphatidic acid stimulates tyrosine phosphorylation of focal adhesion kinase, paxillin and p130. Signaling pathways and crosstalk with platelet derived growth factor.
J. Biol. Chem.
269:
9345-9351,
1994
25.
Steusloff, A.,
E. Paul,
L. A. Semenchuk,
J. Di-Salvo,
and
G. Pfitzer.
Modulation of Ca2+ sensitivity in smooth muscle by genistein and protein tyrosine phosphorylation.
Arch. Biochem. Biophys.
320:
236-242,
1995[Medline].
26.
Tidball, J. G.,
and
M. J. Spencer.
PDGF stimulation induces phosphorylation of talin and cytoskeletal reorganization in skeletal muscle.
J. Cell Biol.
123:
627-635,
1993[Abstract].
27.
Turner, C. E.,
J. R. Glenny, Jr.,
and
K. Burridge.
Paxillin: a new vinculin-binding protein present in focal adhesions.
J. Cell Biol.
111:
1059-1068,
1990[Abstract].
28.
Turner, C. E.,
N. Kramarcy,
R. Sealock,
and
K. Burridge.
Localization of paxillin, a focal adhesion protein, to smooth muscle dense plaques, and the myotendinous and neuromuscular junction of skeletal muscle.
Exp. Cell Res.
192:
651-655,
1991[Medline].
29.
Turner, C. E.,
F. M. Pavalko,
and
K. Burridge.
The role of phosphorylation and limited proteolytic cleavage of talin and vinculin in the disruption of focal adhesion integrity.
J. Biol. Chem.
264:
11938-11944,
1989
30.
Turner, C. E.,
M. D. Schaller,
and
J. T. Parsons.
Tyrosine phosphorylation of the focal adhesion kinase pp125FAK during development: relation to paxillin.
J. Cell Sci.
105:
637-645,
1993
31.
Wang, N.,
J. P. Butler,
and
D. E. Ingber.
Mechanotransduction across the cell surface and through the cytoskeleton.
Science
260:
1124-1127,
1993[Medline].
32.
Wang, Z. L.,
F. M. Pavalko,
and
S. J. Gunst.
Tyrosine phosphorylation of the dense plaque protein paxillin is regulated during smooth muscle contraction.
Am. J. Physiol.
271 (Cell Physiol. 40):
C1594-C1602,
1996
33.
Zachary, I.,
J. Sinnett-Smith,
C. E. Turner,
and
E. Rozengurt.
Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation of focal adhesion-associated protein paxillin in Swiss 3T3 cells.
J. Biol. Chem.
268:
22060-22065,
1993