Differential Signaling by the Focal Adhesion Kinase and Cell Adhesion Kinase β

Michael D Schaller,Terukatsu Sasaki

doi:10.1074/jbc.272.40.25319

Michael D Schaller, Terukatsu Sasaki

Open Access

https://doi.org/10.1074/jbc.272.40.25319

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Abstract

pp125(FAK) and CAKbeta/Pyk2/CadTK/RAFTK are related protein-tyrosine kinases. It is therefore of interest whether CAKbeta shares some of the properties of pp125(FAK). Using recombinant glutathione S-transferase fusion proteins, we show that the C-terminal domains of both proteins bind paxillin in vitro. The C-terminal domain of CAKbeta was engineered to be autonomously expressed in chicken embryo cells and, like pp125(FAK) and p41/43(FRNK) (the C-terminal noncatalytic domain of pp125(FAK)), was found to localize to cellular focal adhesions. In contrast, full-length CAKbeta was generally found diffusely distributed throughout the cell, although a fraction of the cells exhibited focal adhesion localization. Vanadate treatment of pp125(FAK)- and CAKbeta-overexpressing CE cells induced a dramatic increase in the phosphotyrosine content of a common set of proteins including tensin, paxillin, and p130(Cas), but some of these substrates, particularly p130(Cas), appeared to be differentially phosphorylated by pp125(FAK) and CAKbeta. Levels of tyrosine phosphorylation were higher in CAKbeta-overexpressing cells, and additional phosphotyrosine-containing species were specifically immunoprecipitated. In addition, vanadate treatment of CE cells overexpressing CAKbeta, but not pp125(FAK) overexpressors, induced a profound morphological change, which could be a consequence of the observed differences in substrate phosphorylation.

Highlights

Pp125FAK,1 the focal adhesion kinase, is a 125-kDa proteintyrosine kinase (PTK) that is discretely localized to cellular focal adhesions [1, 2]
Microinjection of a similar fragment of pp125FAK into human umbilical vein endothelial cells had little effect upon the structure of focal adhesions but abolished focal adhesion-associated phosphotyrosine and inhibited cell migration [12]. pp125FAK expression has been obliterated by gene knockout, which results in embryonic lethality [13]
A, chicken embryo (CE) cell lysates were precleared with GST immobilized on glutathione beads and incubated with immobilized GST or GST fusion proteins encoding the C-terminal noncatalytic domain of pp125FAK or CAK␤, and bound proteins were Western blotted for paxillin

Summary

Introduction

Pp125FAK, the focal adhesion kinase, is a 125-kDa proteintyrosine kinase (PTK) that is discretely localized to cellular focal adhesions [1, 2]. The integrins function in cell adhesion cytoskeleton anchorage and in the transduction of extracellular stimuli into cytoplasmic signals, including the phosphorylation of proteins on tyrosine [5, 6]. Overexpression of the C-terminal noncatalytic domain of pp125FAK, which contains the focal adhesion targeting sequence, blocks pp125FAK-induced tyrosine phosphorylation of substrates and impairs the spreading of chicken embryo cells on fibronectin [11]. In a megakaryocytic cell line, CAK␤ colocalizes with vinculin and exhibits cell adhesion-dependent tyrosine phosphorylation [18]. The C-terminal 150 residues of pp125FAK represent the focal adhesion targeting (FAT) sequence, which directs pp125FAK to its location in the cell and contains binding sites for two focal adhesion-associated proteins, paxillin and talin [21,22,23].

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