Rat oligodendroglia express c-met and focal adhesion kinase, protein tyrosine kinases implicated in regulating epithelial cell motility

The integrins have recently been implicated in signal transduction. A likely mediator of integrin signaling is focal adhesion kinase (pp125FAK or FAK), a structurally distinct protein tyrosine kinase that becomes enzymatically activated upon engagement of integrins with their ligands. A second candidate signaling molecule is paxillin, a focal adhesion associated, cytoskeletal protein that coordinately becomes phosphorylated on tyrosine upon activation of pp125FAK. Paxillin physically complexes with two protein tyrosine kinases, pp60src and Csk (COOH-terminal src kinase), and the oncoprotein p47gag-crk, each of which could function as part of a paxillin signaling complex. Using an in vitro assay we have established that the cytoplasmic domain of the beta 1 integrin can bind to paxillin and pp125FAK from chicken embryo cell lysates. The NH2-terminal, noncatalytic domain of pp125FAK can bind directly to the cytoplasmic tail of beta 1 and recognizes integrin sequences distinct from those involved in binding to alpha-actinin. Paxillin binding is independent of pp125FAK binding despite the fact that both bind to the same region of beta 1. These results demonstrate that the cytoplasmic domain of the beta subunits of integrins contain binding sites for both signaling molecules and structural proteins suggesting that integrins can coordinate the generation of cytoplasmic signals in addition to their role in anchoring components of the cytoskeleton.

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Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.147-155.1994 ◽

1994 ◽

Vol 14 (1) ◽

pp. 147-155

Author(s):

B S Cobb ◽

M D Schaller ◽

T H Leu ◽

J T Parsons

Keyword(s):

Focal Adhesion ◽

Tyrosine Kinases ◽

Protein Tyrosine Kinase ◽

Src Family Kinases ◽

Protein Tyrosine Kinases ◽

Protein Tyrosine ◽

Sh2 Domains ◽

Embryo Cells ◽

Stable Association

Changes in cellular growth and dramatic alterations in cell morphology and adhesion are common features of cells transformed by oncogenic protein tyrosine kinases, such as pp60src and other members of the Src family. In this report, we present evidence for the stable association of two Src family kinases (pp60src and pp59fyn) with tyrosine-phosphorylated forms of a focal adhesion-associated protein tyrosine kinase, pp125FAK. In Src-transformed chicken embryo cells, most of the pp125FAK was stably complexed with activated pp60src (e.g., pp60(527F). The stable association of pp125FAK with pp60(527F) in vivo required the structural integrity of the Src SH2 domain. The association of pp60(527F) and pp125FAK could be reconstituted in vitro by incubation of normal cell extracts with glutathione S-transferase fusion proteins containing SH2 or SH3/SH2 domains of pp60src. Furthermore, the association of isolated SH2 or SH3/SH2 domains with in vitro 32P-labeled pp125FAK protected the major site of pp125FAK autophosphorylation from digestion with a tyrosine phosphatase, indicating that the autophosphorylation site of pp125FAK participates in binding with Src. Immunoprecipitation of Src family kinases from extracts of normal chicken embryo cells revealed stable complexes of pp59fyn and tyrosine-phosphorylated pp125FAK. These data provide evidence for a direct interaction between two cytoplasmic nonreceptor protein tyrosine kinases and suggest that Src may contribute to changes in pp125FAK regulation in transformed cells. Furthermore, pp125FAK may directly participate in the targeting of pp59fyn or possibly other Src family kinases to focal adhesions in normal cells.

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Differential Expression of Protein Tyrosine Kinases in the Cortical and Medullary Thymic Epithelial Cell Lines

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1995.2196 ◽

1995 ◽

Vol 213 (3) ◽

pp. 768-773 ◽

Cited By ~ 5

Author(s):

T. Kuwata ◽

H. Takahashi ◽

M. Kasai ◽

C. Kurashima ◽

M. Utsuyama ◽

...

Keyword(s):

Epithelial Cell ◽

Differential Expression ◽

Cell Lines ◽

Tyrosine Kinases ◽

Protein Tyrosine Kinases ◽

Thymic Epithelial Cell ◽

Protein Tyrosine ◽

Epithelial Cell Lines ◽

Medullary Thymic Epithelial Cell

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Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK.

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.147 ◽

1994 ◽

Vol 14 (1) ◽

pp. 147-155 ◽

Cited By ~ 343

Author(s):

B S Cobb ◽

M D Schaller ◽

T H Leu ◽

J T Parsons

Keyword(s):

Focal Adhesion ◽

Tyrosine Kinases ◽

Protein Tyrosine Kinase ◽

Src Family Kinases ◽

Protein Tyrosine Kinases ◽

Protein Tyrosine ◽

Sh2 Domains ◽

Embryo Cells ◽

Stable Association

Changes in cellular growth and dramatic alterations in cell morphology and adhesion are common features of cells transformed by oncogenic protein tyrosine kinases, such as pp60src and other members of the Src family. In this report, we present evidence for the stable association of two Src family kinases (pp60src and pp59fyn) with tyrosine-phosphorylated forms of a focal adhesion-associated protein tyrosine kinase, pp125FAK. In Src-transformed chicken embryo cells, most of the pp125FAK was stably complexed with activated pp60src (e.g., pp60(527F). The stable association of pp125FAK with pp60(527F) in vivo required the structural integrity of the Src SH2 domain. The association of pp60(527F) and pp125FAK could be reconstituted in vitro by incubation of normal cell extracts with glutathione S-transferase fusion proteins containing SH2 or SH3/SH2 domains of pp60src. Furthermore, the association of isolated SH2 or SH3/SH2 domains with in vitro 32P-labeled pp125FAK protected the major site of pp125FAK autophosphorylation from digestion with a tyrosine phosphatase, indicating that the autophosphorylation site of pp125FAK participates in binding with Src. Immunoprecipitation of Src family kinases from extracts of normal chicken embryo cells revealed stable complexes of pp59fyn and tyrosine-phosphorylated pp125FAK. These data provide evidence for a direct interaction between two cytoplasmic nonreceptor protein tyrosine kinases and suggest that Src may contribute to changes in pp125FAK regulation in transformed cells. Furthermore, pp125FAK may directly participate in the targeting of pp59fyn or possibly other Src family kinases to focal adhesions in normal cells.

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Protein tyrosine kinases transcribed in a murine thymic medullary epithelial cell line

Gene ◽

10.1016/0378-1119(94)90106-6 ◽

1994 ◽

Vol 143 (2) ◽

pp. 257-260 ◽

Cited By ~ 7

Author(s):

Benoît Hébert ◽

Jean Bergeron ◽

Peter Tijssen ◽

Edouard F. Potworowski

Keyword(s):

Epithelial Cell ◽

Cell Line ◽

Tyrosine Kinases ◽

Protein Tyrosine Kinases ◽

Epithelial Cell Line ◽

Protein Tyrosine ◽

Medullary Epithelial Cell

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An SH3 domain-containing GTPase-activating protein for Rho and Cdc42 associates with focal adhesion kinase.

Molecular and Cellular Biology ◽

10.1128/mcb.16.6.3169 ◽

1996 ◽

Vol 16 (6) ◽

pp. 3169-3178 ◽

Cited By ~ 222

Author(s):

J D Hildebrand ◽

J M Taylor ◽

J T Parsons

Keyword(s):

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Tyrosine Kinases ◽

Binding Proteins ◽

Sh3 Domain ◽

Gtpase Activating Protein ◽

Protein Tyrosine ◽

Gtp Binding Proteins ◽

Gtp Binding ◽

Rho Family

The integrin family of cell surface receptors mediates cell adhesion to components of the extracellular matrix (ECM). Integrin engagement with the ECM initiates signaling cascades that regulate the organization of the actin-cytoskeleton and changes in gene expression. The Rho subfamily of Ras-related low-molecular-weight GTP-binding proteins and several protein tyrosine kinases have been implicated in mediating various aspects of integrin-dependent alterations in cell homeostasis. Focal adhesion kinase (FAK or pp125FAK) is one of the tyrosine kinases predicted to be a critical component of integrin signaling. To elucidate the mechanisms by which FAK participates in integrin-mediated signaling, we have used expression cloning to identify cDNAs that encode potential FAK-binding proteins. We report here the identification of a cDNA that encodes a new member of the GTPase-activating protein (GAP) family of GTPase regulators. This GAP, termed Graf (for GTPase regulator associated with FAK), binds to the C-terminal domain of FAK in an SH3 domain-dependent manner and preferentially stimulates the GTPase activity of the GTP-binding proteins RhoA and Cdc42. Subcellular localization studies using Graf-transfected chicken embryo cells indicates that Graf colocalizes with actin stress fibers, cortical actin structures, and focal adhesions. Graf mRNA is expressed in a variety of avian tissues and is particularly abundant in embryonic brain and liver. Graf represents the first example of a regulator of the Rho family of small GTP-binding proteins that exhibits binding to a protein tyrosine kinase. We suggest that Graf may function to mediate cross talk between the tyrosine kinases such as FAK and the Rho family GTPase that control steps in integrin-initiated signaling events.

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