Cloning and Characterization of Dfak56, a Homolog of Focal Adhesion Kinase, in Drosophila melanogaster

Jiro Fujimoto,Kazunobu Sawamoto,Masataka Okabe,Yasumitsu Takagi,Tohru Tezuka,Shingo Yoshikawa,Haruko Ryo,Hideyuki Okano,Tadashi Yamamoto

doi:10.1074/jbc.274.41.29196

Jiro Fujimoto, Kazunobu Sawamoto + Show 7 more

Open Access

https://doi.org/10.1074/jbc.274.41.29196

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Abstract

The focal adhesion kinase (FAK) protein-tyrosine kinase plays important roles in cell adhesion in vertebrates. Using polymerase chain reaction-based cloning strategy, we cloned a Drosophila gene that is homologous to the vertebrate FAK family of protein-tyrosine kinases. We designated this gene Dfak56 and characterized its gene product. The overall protein structure and deduced amino acid sequence of Dfak56 show significant similarity to those of FAK and PYK2. Dfak56 has in vitro autophosphorylation activity at tyrosine residues. Expression of the Dfak56 mRNA and the protein was observed in the central nervous system and the muscle-epidermis attachment site in the embryo, where Drosophila position-specific integrins are localized. The results suggest that like FAK in vertebrates, Dfak56 functions downstream of integrins. Dfak56 was tyrosine-phosphorylated upon integrin-dependent attachment of the cell to the extracellular matrix. We conclude that the Dfak56 tyrosine kinase is involved in integrin-mediated cell adhesion signaling and thus is a functional homolog of vertebrate FAK.

Highlights

Focal adhesion kinase (FAK)1 is a member of a growing family of non-receptor protein-tyrosine kinases [1] and was originally identified as a putative substrate for the oncogenic protein-tyrosine kinase pp60v-src [2]
Accumulating data, show that FAK tyrosine phosphorylation is induced upon adhesion of cells to the extracellular matrix through the surface integrin [3] and upon stimulation of cells by a variety of other extracellular factors including those for receptor tyrosine kinases and for G-protein-coupled receptors (4 – 6)
We have identified a Drosophila homolog of vertebrate focal adhesion kinase and termed it Dfak56

Summary

EXPERIMENTAL PROCEDURES

Isolation of cDNA and Genomic DNA Clones—Polymerase chain reaction was performed using fully degenerate oligonucleotides corresponding to the amino acid sequences HRDIAAR (for the forward primer) and DVWAFG (for the reverse primer) as primers and 108 plaque-forming units of Drosophila embryo cDNA library To generate rabbit anti-Dfak polyclonal antibodies, glutathione S-transferase (GST)-Dfak was constructed by inserting the cDNA fragment encoding amino acid residues 1–280 of the Dfak protein into the pGEX-2T expression vector (Amersham Pharmacia Biotech). Expression of the GST fusion proteins in Escherichia coli BL21 was induced with isopropyl-␤-D-thiogalactopyranoside, and the proteins were purified with glutathione-Sepharose (Amersham Pharmacia Biotech) as described previously [27]. Whole-mount immunohistochemistry was performed as described [29] using rabbit anti-Dfak antibodies at 1:100 dilution. The cell lysates were precleared with protein A-Sepharose 4B (Amersham Pharmacia Biotech) for 1 h at 4 °C. The lysates were incubated for 2 h at 4 °C with anti-Dfak antibodies and protein-A Sepharose. Phosphoamino acid analysis of phosphorylated proteins and two-dimensional analysis of tryptic phosphopeptides were performed as described previously [31]

RESULTS

Focal Adhesion Kinase in Drosophila

DISCUSSION