BIOLOGICAL OVERVIEW

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 p130^cas, 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).

Novel functions for integrin-associated proteins revealed by analysis of myofibril attachment in Drosophila

This study used the myotendinous junction of Drosophila flight muscles to explore why many integrin associated proteins (IAPs) are needed and how their function is coordinated. These muscles revealed new functions for IAPs not required for viability: Focal Adhesion Kinase (FAK), RSU1, tensin and vinculin. Genetic interactions demonstrated a balance between positive and negative activities, with vinculin and tensin positively regulating adhesion, while FAK inhibits elevation of integrin activity by tensin, and RSU1 keeps PINCH activity in check. The molecular composition of myofibril termini resolves into 4 distinct layers, one of which is built by a mechanotransduction cascade: vinculin facilitates mechanical opening of filamin, which works with the Arp2/3 activator WASH to build an actin-rich layer positioned between integrins and the first sarcomere. Thus, integration of IAP activity is needed to build the complex architecture of the myotendinous junction, linking the membrane anchor to the sarcomere (Green, 2018).

The adult indirect flight muscles of Drosophila have proved to be an excellent system to identify functions for integrin-associated proteins (IAPs) that are not essential for viability. The mechanical linkage between the last Z-line of each myofibril and the plasma membrane is a well ordered and multi-layered structure, ideal for elucidating the mechanisms by which actin can be organized into different structures at subcellular resolution. In the layer closest to the membrane, the integrin signaling layer, an important counterbalancing is found between IAPs, with FAK inhibiting the activation of integrin by tensin, and RSU1 inhibiting excess PINCH activity. It was discovered that the muscle actin regulatory layer (MARL) has a different composition to the fibroblast ARL, containing a mechanotransduction cascade of vinculin and filamin, which, together with WASH and the Arp2/3 complex, builds an actin-rich zone linking the adhesion machinery at the membrane to the first Z-line (Green, 2018).

The modified terminal Z-lines [MTZ - composed of 4 zones: (1) an integrin signalling layer at the membrane; and then zones containing different actin structures-(2) a force transduction layer (FTL); (3) a muscle actin regulatory layer (MARL); and (4) the first Z-line followed by the first sarcomere] revealed both positive and inhibitory actions of FAK, with the latter consistent with the role of FAK in adhesion disassembly. Both loss of FAK and activated integrin suppressed the phenotypes caused by loss of RSU1 or vinculin, but only activated integrin alleviated the defects caused by the absence of tensin, suggesting that FAK inhibition requires tensin activity, and in turn, tensin elevates integrin activity. This fits with the recent discovery that tensin contributes to the inside-out activation of integrins via talin (Georgiadou, 2017). FAK and tensin thus form a balanced cassette that is thought to respond to upstream signals to regulate integrin activity. Further work is needed to discover how tensin increases integrin activity, how this is inhibited by FAK, and what signals control this regulatory cassette. One model would have tensin activating integrin by direct binding to the β subunit cytoplasmic tail, and FAK inhibition by phosphorylation of tensin, but an alternative is that they have antagonistic roles in integrin recycling (Green, 2018).

RSU1 is part of the complex containing ILK, PINCH and Parvin (IPP complex), and binds the 5th LIM domain of PINCH. Loss of RSU1 causes milder phenotypes than loss of ILK, PINCH or parvin, and these phenotypes have previously been interpreted as a partial loss of IPP activity. The current findings indicate that the phenotypes observed in the absence of RSU1 are due to too much PINCH activity, and therefore the role of RSU1 is to keep PINCH activity in check. This suggests that PINCH is perhaps the key player of the IPP complex, and is recruited to adhesions by integrin via ILK, and kept in check by integrin and RSU1. The importance of regulating active PINCH levels is consistent with the dosage sensitivity of PINCH: reducing PINCH partially rescues the dorsal closure defect in embryos lacking the MAPK Misshapen, and elevating PINCH rescues hypercontraction caused by loss of Myosin II phosphatase. Reducing the interaction of PINCH with ILK had unexpectedly no phenotype, but in combination with the loss of RSU1 becomes lethal; the lethality can now be interpreted as being caused by too much PINCH activity, rather than too little. Excess 'free' PINCH results in elongated membrane interdigitations and elevated paxillin levels. This suggests that PINCH has an important role at the cell cortex, consistent with cortical proteins in the PINCH interactome. Too much parvin activity also causes lethality, which is suppressed by elevating ILK levels. Thus, it is increasingly clear that the functions of IPP components need to be tightly controlled. This study gained some insight into how RSU1 inhibits PINCH activity by demonstrating that ΔLIM4, 5 PINCH still caused longer interdigitations. This rules out RSU1 blocking the binding of another protein from binding LIM5, and suggests instead that RSU1 bound to LIM5 must be inhibiting the activity of LIM1-3 (Green, 2018).

Vinculin has a dual function in the MTZ: its head domain promotes force transduction layer (FTL; containing actin, the C-terminus of talin and vinculin) stability via binding talin, and its tail promotes muscle actin regulatory layer (MARL) formation. This analysis of the vinculin mutant by electron microscopy showed a phenotype within the electron dense layer close to the membrane that is presumed to corresponds to the integrin signalling layer. It suggests that vinculin may mediate interactions between IAPs that aid in keeping this as an even layer. The fact that the disruption to this layer is only evident on the muscle side of the interaction raises the question of how similar the integrin junctions are on the two sides of this cell-cell interaction via an intervening ECM. Many other sites of integrin-mediated adhesion in Drosophila involve integrins on both sides of the interaction and by electron microscopy the electron dense material looks similar on the two sides, and it would be expected that both sides need to resist the same forces. Even with structured illumination microscopy the two sides of the membrane cannot be resolved, but the results show that the C-terminus of talin and vinculin are not pulled away from the membrane in the adult tendon cells. This suggests either that vinculin has a different role in the tendon cell, with a different configuration, as was observed for talin in the pupal wing, or it is absent (Green, 2018).

The vinculin tail function in MARL formation does not require that vinculin is bound to talin, but it is suspected that in the wild type it is talin-binding that converts vinculin into an open conformation, permitting the tail to trigger MARL formation with filamin, as outlined in a working model (see Model of IAP function in the IFM MTZ). A key function of vinculin tail in the MARL is to aid the mechanical opening of the filamin mechanosensitive region. This study presents evidence suggesting this is achieved by the vinculin tail anchoring the C-terminus to actin, but further work is required to determine if there is direct binding between the two proteins. Similarly, the results indicate that the Arp2/3 nucleation promoting factor WASH is part of the same pathway as filamin and acts downstream of it, but the connection between the two has yet to be resolved. This new function for WASH is distinct from its best characterized role regulating actin on intracellular vesicles during endosomal sorting and recycling, but WASH also has additional roles in the nucleus and the oocyte cortex, showing that it is a versatile protein (Green, 2018).

Given the myofibril defects seen with loss of RSU1, tensin, vinculin and filamin it might be expected that mutations in genes encoding these IAPs might be implicated in muscle disease. Indeed, mutations in integrin α7, talin and ILK are associated with muscular myopathies in humans and mice. Mutations in the genes encoding RSU1, tensin and vinculin have not been linked to muscle myopathies, but mutations in filamin are linked to myofibrillar myopathies. However, given the subtlety of these defects in Drosophila, one might predict that mutations in genes encoding these IAPs are associated with subtle defects in humans such as reduced sporting performance or susceptibility to muscle injury. The authors were unaware of any mutations in genes encoding these IAPs being related to athletic performance or injury susceptibility, but these IAPs would be good candidates for further study in this area (Green, 2018).

One way that these IAPs may contribute to athletic performance is by building a muscle shock absorber, the MARL, which protects the myofibrils from contraction-induced damage. The concept of muscle shock absorbers is well established since tendons perform this function. The presence of filamin, Arp3, vinculin and α-actinin in the MARL suggests that the MARL contains branched and bundled actin filaments. Branched actin networks have been shown to be viscoelastic and actin crosslinkers such as filamin have been shown to reduce viscosity and increase elasticity of actin networks. Further study into the functional nature of the MARL should increase understanding of athletic performance and injury susceptibility (Green, 2018).

GENE STRUCTURE

Bases in 3' UTR - 445

PROTEIN STRUCTURE

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).

date revised: 18 February 2024

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