To determine whether DFak56 has protein tyrosine kinase (PTK) activity, Fak56D and
Fak56D(K513R), in which the conserved Lys in the kinase subdomain II (K513) was mutated to Arg to reduce catalytic activity, were
transiently expressed in 293 cells. Anti-Fak56D antibodies were used to immunoprecipitate Fak56D and Fak56D(K513R)
from cell lysates. Fak56D
antibodies specifically recognize a 140-kDa protein, which is present when cells were transfected with either Fak56D or Fak56D(K513R) but not with vector alone. Immunoprecipitates were also analyzed by anti-Tyr(P) immunoblotting. Fak56D is detected as a 140-kDa tyrosine-phosphorylated protein; mutation of Lys513 abrogates tyrosine
phosphorylation of Fak56D. These results imply that Fak56D is indeed a PTK (Palmer, 1999).
Fak56D can also be detected as a tyrosine-phosphorylated protein in vivo. Anti-Fak56D antibodies recognize a doublet of endogenous Fak56D at
140 kDa from Schneider 2 (S2) tissue culture cells. This doublet is observed both by immunoblotting of whole cell lysates and by
immunoprecipitation. The upper band comigrates with Fak56D overexpressed in 293 cells and may represent a more highly posttranslationally modified
Fak56D species. Similar results were obtained for whole Drosophila embryo and third instar extracts as well as for S2 cell extracts (Palmer, 1999).
Mammalian FAK has been shown to be tyrosine-phosphorylated in response to plating on fibronectin (FN). Therefore, it was asked whether Fak56D is
tyrosine-phosphorylated under similar circumstances. For this purpose, primary cell cultures prepared from 4-6-h-old Drosophila embryos were plated on
the Drosophila ECM components tiggrin or laminin. After 24 h multiple cell types differentiated, including muscles and neurites. These differentiated primary cell cultures were lysed, and the endogenous Fak56D was immunoprecipitated and resolved on
SDS-PAGE. Anti-Tyr(P) immunoblotting shows that after 24 h on tiggrin endogenous Fak56D has an increased level of Tyr(P). Very low levels of Tyr(P) in Fak56D are observed before plating. Interestingly, differentiation on
the ECM protein laminin causes a smaller increase in phosphorylation. It is concluded that Fak56D encodes a 140-kDa PTK, which exists in vivo as a
tyrosine-phosphorylated protein. Additionally, the increase in Tyr(P) content of Fak56D when cells are plated on the Drosophila ECM protein, tiggrin, is
consistent with activation of the Fak56 PTK (Palmer, 1999).
A test was performed for protein kinase activity and phosphorylation of Fak56D with mutational analysis. By site-directed mutagenesis,
an amino acid substitution at residue 430 (tyrosine to phenylalanine) was introduced (Y430F mutant). This residue corresponds to the major
autophosphorylation site in vertebrate FAK (tyrosine 397). Methionine was substituted for lysine at residue 513, a possible ATP-binding site essential
for tyrosine kinase activity (K513M mutant). The cDNAs for the wild-type Fak56D protein and the mutant proteins constructed in expression plasmids
were then introduced in simian COS cells. Cell lysates were immunoprecipitated with anti-Fak56D antibodies, and the precipitates were subjected to in
vitro kinase assay (Fujimoto, 1999).
Autophosphorylation of the Fak56D protein is observed in the wild-type protein and the Y430F mutant, but not in the K513M mutant. The
Fak56D kinase activity observed is not attributable to co-immunoprecipitated kinases because the lysine at residue 513 of Fak56D is essential for the
kinase activity. Phosphoamino acid analysis of in vitro phosphorylated Fak56D detected phosphorylated tyrosine only, confirming that Fak56D is
a protein-tyrosine kinase. Furthermore, the incorporated radioactivity of the phosphorylated Y430F mutant is approximately half that of wild-type
Fak56D, suggesting that the tyrosine at residue 430 is a major autophosphorylation site. This was confirmed by two-dimensional phosphopeptide
mapping of the wild-type and Y430F mutant proteins. A major spot in the peptide map of wild-type Fak56D is not observed in that of
the Y430F mutant (Fujimoto, 1999).
Drosophila S2 cells and stable transfectants expressing
Drosophila PS integrin chains of alphaPS1 and betaPS (called PS1 cells) or alphaPS2 and betaPS (called PS2 cells) were
cultured on plastic dishes. Among them, only PS2 cells could attach to a plastic dish in serum-containing medium because of the cross-reactivity of alphaPS2betaPS integrin to vertebrate vitronectin and fibronectin. Using these cell lines, the levels of phosphotyrosine in the Fak56D
protein were examined following integrin-dependent cell attachment to the extracellular matrix. The Fak56D protein is tyrosine-phosphorylated in PS2 cells that attached to the surface of the dish. This indicates that Fak56D is tyrosine-phosphorylated upon integrin-dependent cell attachment to the extracellular matrix. Tyrosine-phosphorylated proteins of 50-60 kDa were associated with the Fak56D
protein. These phosphoproteins are also present in the anti-Fak56D precipitates of PS1 cells or suspended PS2 cells, but not in precipitates of parental S2 cells. This suggests that the interaction between Fak56D and these phosphoproteins requires the presence of integrins and is enhanced by cell attachment to the extracellular matrix (Fujimoto, 1999).
FAK acts as a suppressor of RTK-MAP kinase signalling in Drosophila melanogaster epithelia and human cancer cells
Receptor Tyrosine Kinases (RTKs) and Focal Adhesion Kinase (FAK) regulate multiple signalling pathways, including mitogen-activated protein (MAP) kinase pathway. FAK interacts with several RTKs but little is known about how FAK regulates their downstream signalling. This study investigated how FAK regulates signalling resulting from the overexpression of the RTKs RET and EGFR. FAK was found to suppress RTKs signalling in Drosophila epithelia by impairing MAPK pathway. This regulation was also observed in MDA-MB-231 human breast cancer cells, suggesting it is a conserved phenomenon in humans. Mechanistically, FAK reduces receptor recycling into the plasma membrane, which results in lower MAPK activation. Conversely, increasing the membrane pool of the receptor increases MAPK pathway signalling. FAK is widely considered as a therapeutic target in cancer biology; however, it also has tumour suppressor properties in some contexts. Therefore, the FAK-mediated negative regulation of RTK/MAPK signalling described in this study may have potential implications in the designing of therapy strategies for RTK-driven tumours (Macagno, 2014).