Focal adhesion kinase-like: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Focal adhesion kinase-like

Synonyms -

Cytological map position - 56D5--7

Function - signal transduction

Keywords - integrin signaling, growth response, insulin signaling pathway

Symbol - Fak

FlyBase ID: FBgn0020440

Genetic map position -

Classification - protein tyrosine kinase

Cellular location - cytoplasmic

NCBI links: Precomputed BLAST | Entrez Gene

Cloning of Drosophila Focal adhesion kinase-like (Fak56D) was accomplished using a polymerase chain reaction (PCR) strategy, and was reported almost simultaneously from three laboratories (Fujimoto, 1999; Palmer, 1999 and Fox, 1999). Focal adhesion kinase (FAK) was one of the first molecules identified as playing a role in integrin signaling. Integrins are a family of cell surface molecules that link the extracellular matrix with the actin cytoskeleton. As such, they are in a position to transmit information into and out of the cell, and it is now well established that integrin-mediated signaling influences many intracellular events, including rearrangement of the actin cytoskeleton, cell migration, cell survival, and gene expression. Much of the early work on FAK focused on identifying the molecules with which it interacts, including focal contact and adaptor proteins like talin, paxillin, and p130cas, and kinases like src and PI3K. More recently, it has been observed that increasing the expression of FAK in cells can stimulate both migration and cell survival, and further research into these phenomena has emphasized the importance of FAK's interactions with src, PI3K, and CAS. Ablation of FAK in mouse embryos produces early embryonic lethality, and FAK-null cells show reduced motility (Fox, 1999 and references therein).

FAK is the founder member of a structurally conserved family of cytoplasmic nonreceptor protein-tyrosine kinases implicated in controlling cellular responses to the engagement of cell surface receptors. This protein-tyrosine kinase (PTK) subfamily so far comprises two mammalian members: FAK and Pyk2 (also known as CAK, RAFTK, FAK2, and CADTK); these proteins have 45% overall sequence identity to one another, contain a central catalytic domain flanked by large N- and C-terminal domains, and possess a conserved Src SH2 binding autophosphorylation site. The FAK N-terminal domain can bind in vitro to the tails of beta-integrins (see Drosophila Myospheroid), and the C-terminal region contains a focal adhesion targeting sequence that localizes FAK to focal adhesions, and binds paxillin and talin. A number of cellular stimuli in addition to ECM proteins can induce tyrosine phosphorylation of FAK, including growth factors, and this, in combination with direct association of growth factor receptors with integrins, provides a linkage between growth control and adhesion. Like FAK, Pyk2 tyrosine phosphorylation can be stimulated by integrin activation, but this is generally a weak response, and stronger Pyk2 activation is elicited by elevation of intracellular Ca2+ levels, particularly in response to activation of G protein-coupled receptors (Palmer, 1999 and references therein).

Because no Fak56D mutants are yet available, the role of Fak56D in vivo was examined using the GAL4-UAS system to overexpress Fak56D in the wing. The Drosophila wing provides an excellent system for the study of morphogenesis in an intact animal. Because the wing is nonessential, manipulations affecting it need not affect viability. In addition, wing morphogenesis is a relatively simple process involving the conversion of a single layered columnar epithelium to a flattened bilayer in which the basal surfaces of the dorsal and ventral epithelia are in close contact. Previous data suggest that integrins function in early signaling processes as well as in adhesion of the dorsal and ventral surfaces. Homozygous myospheroid (beta integrin) mutant cell clones induced in the wing disc during larval stages result in wing blisters in which the dorsal and ventral wing epithelia in and around the clone fail to adhere. Ectopic expression of Fak56D under the control of the Actin5C promoter driving GAL4 (Actin5C-GAL4) results in 100% pupal lethality. When Engrailed:GAL4 (which targets expression to the posterior compartment of the wing) is used to drive Fak56D expression, the formation of wing blisters is observed in the posterior region of the wing at 22° or 25°C. At higher temperatures an increased level of severity and penetrance is observed (Palmer, 1999).

The blistering phenotype associated with the overexpression of Fak56D under the control of the Engrailed promoter is of interest in light of the known phenotype of integrin mutant flies. However, because wing blistering is associated with loss of integrin function in integrin mutants, the blistering observed upon overexpression of Fak56D, a putative downstream effector of integrins, is a somewhat unexpected result. Interestingly, however, overexpression of various alpha PS subunits under the control of the UAS-GAL4 system in the developing wing disc can also lead to blistering. Although it is not currently understood why overexpression of integrin subunits causes blisters, this effect has been postulated to be due to increased signaling rather than a loss of mechanical adhesion (Palmer, 1999).

In the case of alpha PS integrin subunit overexpression, a period during early pupal development has been defined as being particularly sensitive to integrin alpha PS2 overexpression. If overexpression of Fak56D generates wing blisters for the same reason that overexpression of alpha PS integrin does, it would be expected that a similar critical period of Fak56D expression occurs. In testing this hypothesis, advantage was taken of the fact that expression of transgenes using the UAS-GAL4 system is increased at higher temperature. For these experiments a UAS:Fak56D(wild type) transgenic line was chosen that has a 50% penetrance of wing vein defects but only a 2-5% penetrance of wing blistering at 22°C. Flies carrying this UAS:Fak56D insertion and Engrailed:Gal4 were raised at 22°C and subjected to a single 24-h period at 29°C at specific developmental times, from embryo through late pupation. Consistent with the reported effect of alpha PS integrin subunit overexpression, an increase in wing blistering was clearly seen in Engrailed:GAL4-UAS:Fak56D(wild type) animals emerging 4 days after the 29 °C heat pulse, with the fraction of animals emerging with blistered wings reaching a peak at 5 days after the 29 °C pulse, corresponding to a 29°C pulse received during early pupation. Thereafter, the percentage of wing blisters returned to lower levels. Constant exposure to 29°C results in a sustained level of wing blisters (Palmer, 1999).

Because the engrailed promoter expresses broadly in the posterior wing, the blistering caused by Fak56D overexpression was thought to be an indirect effect due to the global expression of Fak56D. To determine whether Fak56D-induced blistering is a property associated with the regions where Fak56D is overexpressed, a combination of the flippase-out system and the GAL4-UAS system was used. In this system a fragment of DNA bracketed by FRT sites and containing transcription stop signals is inserted between the Actin5C promoter and GAL4. Heat shock induction of flippase activity induces recombination in which the transcription stop segment is flipped out, thereby allowing the Actin5C promoter to drive GAL4 expression. This system allows the creation of clones of cells expressing Fak56D, which are marked by green fluorescent protein (GFP) expression. Expression of Fak56D, as judged by immunostaining, and GFP are coincident, demonstrating that the system works for Fak56D and also establishing the specificity of the anti-Fak56D antibodies. Although endogenous Fak56D protein is expressed in the third instar wing disc during normal development, higher levels of Fak56D within overexpressing clones are clearly evident compared with endogenous levels. Fak56D overexpressing clones also display increased levels of Tyr(P), consistent with the overexpressed Fak56D being active and phosphorylating proteins in these clones. Upon eclosion a number of animals with heat shock-induced Fak56D-overexpressing clones also display wing blisters, consistent with previous results. Further, the observed wing blisters have been found to be GFP-positive, thus confirming that the site of Fak56D expression is coincident with the wing blistering phenotype observed and therefore that Fak56D is responsible for the blistering (Palmer, 1999).

The observation that Fak56D overexpression causes wing blistering provides a Fak56D gain-of-function phenotype potentially consistent with a role in cell-cell interaction. But why should overexpression of Fak56D result in an integrin loss-of-function phenotype? Recent data suggest a possible answer to this unexpected result. It has been proposed that there are two distinct phases of integrin function in the wing, divided into distinct prepupal and pupal phases (Brabant, 1996). In the early phase, integrins primarily serve a signaling function, triggering or directing subsequent morphogenesis. Later, PS integrins provide a mechanical link between the epithelia to resist hydrostatic pressure, especially during the wing expansion. Such a model may help to account for the seemingly paradoxical observation that overexpression of an 'adhesion protein' leads to a loss of adhesion; the critical function of PS integrins during the early period, which is most sensitive to overexpression, is now postulated to be regulatory rather than adhesive. This hypothesis is consistent with the data presented here on the overexpression of Fak56D, which also leads to the formation of wing blisters. If, indeed, the overexpression of alphaPS subunits leads to the activation of downstream signaling events, then the overexpression of Fak56D, a putative downstream effector, would be expected to have a similar effect. Furthermore, it is interesting that both Fak56D and the alphaPS2 subunit display a very similar critical early period of sensitivity to overexpression, leading to blister formation. This indicates important roles for Fak56D and integrin-mediated signaling pathways during the multiple morphogenic processes occurring as the Drosophila larva undergoes pupation (Palmer, 1999).


cDNA clone length - 4370

Bases in 3' UTR - 445


Amino Acids - 1200

Structural Domains

The deduced amino acid sequence of the product of the Fak56D gene shows the presence of a protein kinase domain. Like FAK and PYK2, Drosophila Fak56D has large N- and C-terminal sequences flanking the kinase domain. Comparison of the amino acid sequence of the Fak56D kinase domain with the protein sequence data bases reveals that the kinase domain of Fak56D is most similar to that of FAK (59% identity to human FAK) and PYK2 (52% identity to human PYK2). The sequences of the N- and C-terminal regions are relatively divergent. Unlike the vertebrate counterparts, Fak56D contains an additional 24-amino acid sequence near the ATP-binding site of the kinase. In the C-terminal regions, FAK and PYK2 have a conserved sequence of ~150 amino acid residues. The sequence is called the focal adhesion targeting sequence (FAT) because it is essential for localization of FAK to focal adhesions. Drosophila Fak56D also carries a focal adhesion targeting-like sequence (~40% identity to human FAK) in the C-terminal region, suggesting that the focal adhesion targeting sequence is conserved as a functional domain (Fujimoto, 1999). An interesting difference between Fak56D and other FAK family members is that DFak56 contains a 104-aa insertion close to the C-terminal end of its FAT domain. This insert is not homologous with known sequences (Fox, 1999).

There are tyrosine phosphorylation sites within FAK that are conserved in PYK2. A major autophosphorylation site (Y397AEI for human FAK and Y402AEI for human PYK2) that mediates binding to the SH2 domain of Src-like protein-tyrosine kinases is well conserved in Drosophila Fak56D (Y430AEI). Another tyrosine phosphorylation site (Y925ENV for FAK and Y881LNV for PYK2) serves to bind the GRB2 SH2 domain. Tyrosine 954 in Fak56D is likely to correspond to this phosphorylation site, although the sequence is slightly divergent (Y954CAT). Tyr956 lacks the Asn at position +2 needed for Grb2 SH2 domain binding. The C-terminal region of FAK has two PXXP motifs, P712PKPSRP and P874PKKPPRP, which can bind to the SH3 domain. These sequences are highly conserved in PYK2, and only one of them (P772PSKPSR, corresponding to P712PKPSRP of FAK) is conserved in Fak56D. It is concluded that Fak56D is a Drosophila homolog of vertebrate FAK family protein-tyrosine kinases. The sequence data did not reveal which kinase, FAK or PYK2, is the counterpart of Fak56D (Fujimoto, 1999 and Palmer, 1999)

The kinase domain of DFak56 contains several sequence motifs conserved among PTKs, including the tripeptide motif DFG that is found in most protein kinases, and a consensus ATP-binding motif GXGXXG followed by an AXK sequence downstream. The N-terminal domain of DFak56 contains a YAEI consensus Src SH2 domain-binding sequence starting at Tyr430, identical to the YAEI motifs in FAK and Pyk2, which bind to the SH2 domain of mammalian Src when phosphorylated. A proline-rich region analogous to the first one in FAK is present in the C-terminal domain. There is 35% amino acid identity between Fak56D and FAK in the focal adhesion targeting region (Palmer, 1999).

Focal adhesion kinase-like: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 23 January 2000

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