Focal adhesions are specialized regions of cell membranes that are foci for the transmission of signals between the outside and the inside of the cell. Intracellular signaling events are important in the organization and stability of these structures. In previous work, we showed that the counter-adhesive extracellular matrix proteins, thrombospondin, tenascin, and SPARC, induce the disassembly of focal adhesion plaques and we identified the active regions of these proteins. In order to determine the mechanisms whereby the anti-adhesive matrix proteins modulate cytoskeletal organization and focal adhesion integrity, we examined the role of protein kinases in mediating the loss of focal adhesions by these proteins. Data from these studies show that cGMP-dependent protein kinase is necessary to mediate focal adhesion disassembly triggered by either thrombospondin or tenascin, but not by SPARC. In experiments using various protein kinase inhibitors, we observed that selective inhibitors of cyclic GMP-dependent protein kinase, KT5823 and Rp-8-Br-cGMPS, blocked the effects of both the active sequence of thrombospondin 1 (hep I) and the alternatively-spliced segment (TNfnA-D) of tenascin-C on focal adhesion disassembly. Moreover, early passage rat aortic smooth muscle cells which have high levels of cGMP-dependent protein kinase were sensitive to hep I treatment, in contrast to passaged cGMP-dependent protein kinase deficient cells which were refractory to hep I or TNfnA-D treatment, but were sensitive to SPARC. Transfection of passaged smooth muscle cells with the catalytic domain of PKG I alpha restored responsiveness to hep I and TNfnA-D. While these studies show that cGMP-dependent protein kinase activity is necessary for thrombospondin and tenascin-mediated focal adhesion disassembly, kinase activity alone is not sufficient to induce disassembly as transfection of the catalytic domain of the kinase in the absence of additional stimuli does not result in loss of focal adhesions.

REFERENCES

Adams
J.
,
Lawler
J.
(
1993
).
The thrombospondin family.
Curr. Biol
3
,
188
190
Adams
J. C.
,
Lawler
J.
(
1994
).
Cell-type specific adhesive interactions of skeletal myoblasts with thrombospondin-1.
Mol. Biol. Cell
5
,
423
437
Adams
J. C.
(
1995
).
Formation of stable microspikes containing actin and the 55 kDa actin bundling protein, fascin, is a consequence of cell adhesion to thrombospondin 1: implications for the anti-adhesive activities of thrombospondin-1.
J. Cell Sci
108
,
1977
1990
Boerth
N. J.
,
Lincoln
T. M.
(
1994
).
Expression of the catalytic domain of cyclic GMP-dependent protein kinase in a baculovirus system.
FEBS Lett
342
,
255
260
Bornstein
P.
,
Sage
E. H.
(
1994
).
Thrombospondins.
Meth. Enzymol
245
,
62
85
Burridge
K.
,
Turner
C. E.
,
Romer
L. H.
(
1992
).
Tyrosine phosphorylation of paxillin and pp 125FAKaccompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly.
J. Cell Biol
119
,
893
903
Butte
E.
,
Van Bemmelen
M.
,
Fischer
L.
,
Walter
U.
,
Jastorff
B.
(
1990
).
Inhibition of cGMP-dependent protein kinase by (Rp)-guanosine 3,5 -monophosphorothioates.
FEBS Lett
263
,
47
50
Chen
Q. M.
,
Kinch
M. S.
,
Lin
T. H.
,
Burridge
K.
,
Juliano
R. L.
(
1994
).
Integrin-mediated cell adhesion activates mitogen-activated protein kinases.
J. Biol. Chem
269
,
26602
26605
Chiquet-Ehrismann
R.
,
Kalla
P.
,
Pearson
C. A.
(
1989
).
Tenascin interferes with fibronectin action.
Cell
53
,
383
390
Chiquet-Ehrismann
R.
,
Kalla
R.
,
Pearson
C. A.
(
1989
).
Participation of tenascin and TGF-in reciprocal epithelial-mesenchymal interactions of MCF7 cells and fibroblasts.
Cancer Res
49
,
4322
4325
Chiquet-Ehrismann
R.
(
1991
).
Anti-adhesive molecules of the extracellular matrix.
Curr. Opin. Cell Biol
3
,
800
804
Chung
C. Y.
,
Erickson
H. P.
(
1994
).
Cell surface annexin II is a high affinity receptor for the alternatively spliced segment of tenascin-C.
J. Cell Biol
126
,
539
548
Chung
C. Y.
,
Murphy-Ullrich
J. E.
,
Erickson
H. P.
(
1996
).
Mitogenesis, cell migration and loss of focal adhesions induced by tenascin-C interacting with its cell surface receptor, annexin II.
Mol. Biol. Cell
7
,
883
892
Cornwell
T. L.
,
Lincoln
T. M.
(
1989
).
Regulation of intracellular Ca2+levels in cultured vascular smooth muscle cells: reduction of Ca2+by atriopeptin and 8-bromo-cyclic GMP is mediated by cyclic GMP-dependent protein kinase.
J. Biol. Chem
264
,
1146
1155
Cornwell
T. L.
,
Soff
G. A.
,
Traynor
A. E.
,
Lincoln
T. M.
(
1994
).
Regulation of the expression of cyclic GMP-dependent protein kinase by cell density in vascular smooth muscle cells.
J. Vasc. Res
31
,
330
337
Couchman
J. R.
,
Rees
D. A.
(
1979
).
The behaviour of fibroblasts migrating from chick heart explants: changes in adhesion, locomotion, and growth, and in the distribution of actomyosin and fibronectin.
J. Cell Sci
39
,
149
165
Damsky
C.
,
Werb
Z.
(
1992
).
Signal transduction from the extracellular matrix: cooperative processing of extracellular information.
Curr. Opin. Cell Biol
4
,
772
781
Diwan
A. H.
,
Thompson
W. J.
,
Lee
A. K.
,
Strada
S. S.
(
1994
).
Cyclic GMP-dependent protein kinase activity in rat pulmonary microvascular endothelial cells.
Biochem. Biophys. Res. Commun
202
,
728
735
Dunlevy
J. R.
,
Couchman
J. R.
(
1995
).
Interleukin-8 induces motile behavior and loss of focal adhesions in primary fibroblasts.
J. Cell Sci
108
,
311
321
Erickson
H. P.
(
1993
).
Tenascin-C, tenascin-R, and teanscin-X: a family of talented proteins in search of functions.
Curr. Opin. Cell Biol
5
,
869
876
Frazier
W.
(
1991
).
Thrombospondins.
Curr. Opin. Cell Biol
3
,
800
804
Glass
W. F.
,
Kreisberg
J. I.
(
1993
).
Regulation of integrin-mediated adhesion at focal contacts by cyclic AMP.
J. Cell Physiol
157
,
296
306
Hahn
A. W. A.
,
Kern
F.
,
Jonas
U.
,
John
M.
,
Buhler
F. R.
,
Resnik
T. J.
(
1995
).
Functional aspects of vascular tenascin-C expression.
J. Vasc. Res
32
,
162
174
Harper
J. F.
,
Brooker
G.
(
1975
).
Femtomole sensitivity radioimmunoassay for cyclic AMP and cyclic GMP after 20 acetylation by acetic anhydride in aqueous solution.
J. Cyclic Nucl. Res
1
,
207
218
Herman
B.
,
Pledger
W. J.
(
1985
).
Platelet-derived growth factor-induced alterations in vinculin and actin distribution in BALB/c-3T3 cells.
J. Cell Biol
100
,
1031
1040
Herman
B.
,
Harrington
M. A.
,
Olashaw
N. E.
,
Pledger
W. J.
(
1986
).
Identification of the cellular mechanisms responsible for platelet-derived growth factor induced alterations in cytoplasmic vinculin distribution.
J. Cell Physiol
126
,
115
125
Herman
B.
,
Roe
M. W.
,
Harris
C.
,
Wray
B.
,
Clemmons
D.
(
1987
).
Platelet-derived growth factor-induced alterations in vinculin distribution in porcine vascular smooth muscle cells.
Cell Motil. Cytoskel
8
,
91
105
Hofmann
F.
,
Gensheimer
H. P.
,
Landgraf
W.
,
Hullin
R.
,
Jastorff
B.
(
1985
).
Diastereomers of adenosine 3,5 -monothionophosphate (cAMP{S}) antagonize the activation of cGMP-dependent protein kinase.
Eur. J. Biochem
150
,
85
88
Huttenlocher
A.
,
Sandborg
R. R.
,
Horwitz
A. F.
(
1995
).
Adhesion in cell migration.
Curr. Opin. Cell Biol
7
,
697
706
Hynes
R. O.
(
1992
).
Integrins: versatility, modulation and signaling in cell adhesion.
Cell
69
,
11
25
Ingber
D. E.
(
1993
).
Cellular tensegrity: defining new rules of biological design that govern the cytoskeleton.
J. Cell Sci
104
,
613
627
Jiang
H.
,
Colbran
J. L.
,
Francis
S. H.
,
Corbin
J. D.
(
1992
).
Direct evidence for cross-activation of cGMP-dependent protein kinase by cAMP in pig coronary arteries.
J. Biol. Chem
267
,
1015
1019
Juliano
R. L.
,
Haskill
S.
(
1993
).
Signal transduction from the extracellular matrix.
J. Cell Biol
120
,
577
585
Kase
H.
,
Iwahashi
K.
,
Nakanishi
S.
,
Matsuda
Y.
,
Yamada
K.
,
Takahasi
M.
,
Murakata
C.
,
Sato
A.
,
Kaneko
M.
(
1987
).
K-252 compounds, novel and potent inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases.
Biochem. Biophys. Res. Commun
142
,
436
440
Lahav
J.
(
1988
).
Thrombospondin inhibits adhesion of endothelial cells.
Exp. Cell Res
177
,
3290
3299
Lahav
J.
(
1993
).
The functions of thrombospondin and its involvement in physiology and pathophysiology.
Biochim. Biophys. Acta
1182
,
1
14
Lamb
N. J. C.
,
Fernandez
A.
,
Conti
M. A.
,
Adelstein
R.
,
Glass
D. B.
,
Welch
W. J.
,
Fermaisco
J. R.
(
1988
).
Regulation of actin microfilament integrity in living nonmuscle cells by the cAMP-dependentprotein kinase and the myosin light chain kinase.
J. Cell Biol
106
,
1955
1971
Lane
T. F.
,
Sage
E. H.
(
1990
).
Functional mapping of SPARC: peptides from two distinct Ca2+-binding sites modulate cell shape.
J. Cell Biol
111
,
3065
3076
Lane
T. F.
,
Sage
E. H.
(
1994
).
The biology of SPARC, a protein that modulates cell-matrix interactions.
FASEB J
8
,
163
173
Lightner
V. A.
,
Erickson
H. P.
(
1990
).
Binding of hexabrachion (tenascin) to the extracellular matrix and substratum and its effect on cell adhesion.
J. Cell Sci
95
,
263
277
Lincoln
T. M.
,
Cornwell
T. L.
,
Taylor
A. E.
(
1990
).
cGMP-dependent protein kinase mediates the reduction of Ca2+by cAMP in vascular smooth muscle cells.
Am. J. Physiol
258
,
399
–.
Lincoln
T. M.
,
Cornwell
T. L.
(
1993
).
Intracellular cyclic GMP receptor proteins.
FASEB J
7
,
328
338
Lo
S. H.
,
Chen
L. B.
(
1994
).
Focal adhesions as a signal transduction organelle.
Cancer Metast. Rev.
13
,
9
24
Lotz
M. M.
,
Burdsal
C. A.
,
Erickson
H. P.
,
McClay
D. R.
(
1989
).
Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding and subsequent strengthening response.
J. Cell Biol
109
,
1795
1805
MacMillan-Crow
L.
,
Murphy-Ullrich
J. E.
,
Lincoln
T. M.
(
1994
).
Identification and possible localization of cGMP-dependent protein kinase in bovine aortic endothelial cells.
Biochem. Biophys. Res. Commun
201
,
531
537
Majack
R. A.
,
Milbrandt
J.
,
Dixit
V. M.
(
1987
).
Induction of thrombospondin messenger RNA levels occurs as an immediate primary response to platelet-derived growth factor.
J. Biol. Chem
262
,
8821
8825
Moretto
M.
,
Lopez
F. J.
,
Negro-Vilar
A.
(
1993
).
Nitric oxide regulates luteinizing hormone-releasing hormone secretion.
Endocrinology
133
,
2399
2402
Murphy-Ullrich
J. E.
,
Mosher
D. F.
(
1987
).
Interactions of thrombospondin with cells culture: rapid degradation of both soluble and matrix thrombospondin.
Semin. Thromb. Hemost
13
,
343
351
Murphy-Ullrich
J. E.
,
Höök
M.
(
1989
).
Thrombospondin modulates focal adhesions in endothelial cells.
J. Cell Biol
109
,
1309
1919
Murphy-Ullrich
J. E.
,
Lightner
V. A.
,
Aukhil
I.
,
Yan
Y. Z.
,
Erickson
H. P.
,
Höök
M.
(
1991
).
Focal adhesion integrity is downregulated by the alternatively spliced domain of tenascin.
J. Cell Biol
115
,
1127
1136
Murphy-Ullrich
J. E.
,
Gurusiddappa
S.
,
Frazier
W. A.
,
Höök
M.
(
1993
).
Heparin-binding peptides from thrombospondins 1 and 2 contain focal adhesion-labilizing activity.
J. Biol. Chem
268
,
26784
26789
Murphy-Ullrich
J. E.
,
Lane
T. F.
,
Pallero
M. P.
,
Sage
E. H.
(
1995
).
SPARC mediates focal adhesion disassembly in endothelial cells through a follistatin-like region and the Ca2+-binding EF-hand.
J. Cell. Biochem
57
,
341
350
Nobes
C. D.
,
Hall
A.
(
1995
).
Rho, Rac, and Cdc42 GTPases regulates the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia.
Cell
81
,
53
62
Penttinen
R. O.
,
Kobayashi
S.
,
Bornstein
P.
(
1988
).
Transforming growth factorincreases mRNA for matrix proteins both in the presence and in the absence of changes in mRNA stability.
Proc. Nat. Acad. Sci. USA
85
,
1105
1108
Prieto
A. L.
,
Andersson-Fisone
C.
,
Crossin
K. L.
(
1992
).
Characterization of multiple adhesive and counteradhesive domains in the extracellular matrix protein cytotactin.
J. Cell Biol
119
,
663
678
Pryzwansky
K.B.
,
Wyatt
J.A.
,
Lincoln
T.M.
(
1995
).
Cyclic guanosine monophosphate-dependent protein kinase is targeted to intermediate filaments and phosphorylates vimentin in A23187-stimulated human neutrophils.
Blood
85
,
222
230
Qwarnström
E. E.
,
MacFarlane
S. A.
,
Page
R. C.
,
Dower
S. K.
(
1991
).
Interleukin 1induces rapid phosphorylation and redistribution of talin: a possible mechanism for modulation of fibroblast focal adhesions.
Proc. Nat. Acad. Sci USA
88
,
1232
1236
Reinhard
M.
,
Halbrugge
M.
,
Sheer
U.
,
Wiegand
C.
,
Jockbusch
B. M.
,
Walter
U.
(
1992
).
The 46/50 kDa VASP purified from human platelets is a novel protein associated with actin filaments and focal contacts.
EMBO J
11
,
2063
2070
Reinhard
M.
,
Giehl
K.
,
Abel
K.
,
Haffner
C.
,
Jarchau
T.
,
Hoppe
V.
,
Jockusch
B. M.
,
Walter
U.
(
1995
).
The proline-rich focal adhesion and microfilament protein VASP is a ligand for profilins.
EMBO J
14
,
1583
1589
Reinhard
M.
,
Jouvenal
K.
,
Tripier
D.
,
Walter
U.
(
1995
).
Identification, purification, and characterization of a zyxin-related protein that binds the focal adhesion and microfilament protein VASP (vasodilator-stimulated phosphoprotein).
Proc. Nat. Acad. Sci. USA
92
,
7956
7960
Ridley
A. J.
,
Hall
A.
(
1994
).
Signal transduction pathways regulating rho-mediated stress fiber formation: requirement for a tyrosine kinase.
EMBO J
13
,
2600
2610
Riou
J.-F.
,
Alfandari
D.
,
Eppe
M.
,
Tachetti
C.
,
Chiquet
M.
,
Boucaut
J. C.
,
Thiery
J. P.
,
Levi
G.
(
1991
).
Purification and partial characterization of Xenopus laevis tenascin from the XTC cell line.
FEBS Lett
279
,
346
350
Rosales
C.
,
O'Brien
V.
,
Kornberg
L.
,
Juliano
R.
(
1995
).
Signal transduction by cell adhesion receptors.
Biochim. Biophys. Acta
1242
,
77
98
Sage
E. H.
,
Vernon
R. B.
,
Decker
J.
,
Funk
S.
,
Iruela-Arispe
M. L.
(
1989
).
Distribution of the calcium-binding protein SPARC in tissues of embryonic and adult mice.
J. Histochem. Cytochem
37
,
819
829
Sage
E. H.
,
Bornstein
P.
(
1991
).
Extracellular proteins that modulate cell-matrix interactions: SPARC, tenascin, and thrombospondin.
J. Biol. Chem
266
,
14831
14834
Schaller
M. D.
,
Borgman
C. A.
,
Cobb
B. S.
,
Vines
R. R.
,
Reynolds
A. B.
,
Parson
J. T.
(
1992
).
pp125FAK, a structurally distinctive protein-tyrosine kinase associated with focal adhesions.
Proc. Nat. Acad. Sci USA
89
,
5192
5196
Schliwa
M.
,
Nakamura
T.
,
Porter
K. R.
,
Eutneneuer
U.
(
1984
).
A tumor promotor induces rapid and coordinated reorganization of actin and vinculin in cultured cells.
J. Cell Biol
99
,
1045
1059
Sims
J. R.
,
Karp
S.
,
Ingber
D. E.
(
1992
).
Altering the cellular mechanical force results in integrated changes in cell, cytoskeletal and nuclear shape.
J. Cell Science
103
,
1215
1222
Spring
J.
,
Beck
K.
,
Chiquet-Ehrismann
R.
(
1989
).
Two contrary functions of tenascin: dissection of the active sites by recombinant tenascin fragments.
Cell
59
,
325
334
Sriramarao
P.
,
Mendler
M.
,
Bourdon
M. A.
(
1993
).
Endothelial cell attachment and spreading on human tenascin is mediated by2 1 andv 3 integrins.
J. Cell Sci
105
,
1001
1012
Tang
D. G.
,
Diglio
C. A.
,
Honn
K. V.
(
1993
).
12(S)-HETE-induced microvascular endothelial cell retraction results from PKC-dependent rearrangement of cytoskeletal elements andv 3integrins.
Prostaglandins
45
,
249
268
Tidball
J. G.
,
Spencer
M. J.
(
1993
).
PDGF stimulation induces phosphorylation of talin and cytoskeletal reorganization in skeletal muscle.
J. Cell Biol
123
,
627
635
Tucker
R. P.
(
1993
).
The in situ localization of tenascin splice variants and thrombospondin 2 mRNA in the avian embryo.
Development
117
,
347
358
Turner
C. E.
,
Pavalko
F. M.
,
Burridge
K.
(
1989
).
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
Turner
C. E.
(
1991
).
Paxillin is a major phosphotyrosine-containing protein during embryonic development.
J. Cell Biol
115
,
201
207
Woods
A.
,
Couchman
J. R.
(
1992
).
Protein kinase C involvement in focal adhesion formation.
J. Cell Sci
101
,
277
290
Woods
A.
,
Couchman
J. R.
(
1994
).
Syndecan 4 heparan sulfate proteoglycan is a selectively enriched and widespread focal adhesion component.
Mol. Biol. Cell
5
,
183
192
Wyatt
T. A.
,
Lincoln
T. M.
,
Pryzwansky
K. B.
(
1991
).
Vimentin is transiently co-localized with and phosphorylated by cyclic GMP-dependent protein kinase in formyl-peptide stimulated neutrophils.
J. Biol. Chem
266
,
21274
21280
Yost
J.C.
,
Sage
E.H.
(
1993
).
Specific interaction of SPARC with endothelial cells is mediated through a carboxy-terminal sequence containing a Ca2+-binding EF hand.
J.Biol. Chem
268
,
25790
25796
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