BIOLOGICAL OVERVIEW

In the Drosophila embryo, the correct association of muscle cells with their specific ectodermally derived tendon cells, also known as epidermal muscle attachment or EMA cells, is achieved through reciprocal interactions between these two distinct cell types. Vein, a neuregulin-like factor secreted by the approaching myotube, activates the EGF-receptor signaling pathway within the tendon cells to initiate tendon cell differentiation. kakapo, renamed short stop because it is now realized that the gene was first discovered by van Vactor et al. (1993), is expressed in the tendons and is essential for muscle-dependent tendon cell differentiation. Short stop/Kakapo is a large intracellular protein and contains structural domains also found in cytoskeletal-related vertebrate proteins (including plakin, dystrophin, and Gas2 family members). kakapo mutant embryos exhibit abnormal muscle-dependent tendon cell differentiation. A major defect in the kakapo mutant tendon cells is the failure of Vein to localize at the muscle-tendon junctional site; instead, Vein is dispersed and its levels are reduced. This may lead to aberrant differentiation of tendon cells and consequently to the kakapo mutant's deranged somatic muscle phenotype (Strumpf, 1998). The flies display wing blisters because the mutant epidermal cells fail to adhere to the opposing layer of wild-type cells in the wing bilayer. This led to the significance of Kakapo’s Maori name, based upon an ineptly flying New Zealand parrot (Gregory, 1998).

To elucidate the function of kak in epidermal muscle attachment (EMA) cell differentiation, an examination was made of the expression of various markers characteristic of tendon cell terminal differentiation, including Stripe, Delilah, and beta1 tubulin mRNA. The expression of the regulatory protein Stripe, a transcription factor of the early growth response (EGR) family, determines the fate of the EMA competent cells at the first phase of tendon cell development. Stripe expression leads to the expression of an array of EMA-specific genes that contribute to the correct guidance of the myotubes. The second phase of tendon cell differentiation depends on inductive interactions between the myotube and the EMA cell. These interactions lead to terminal differentiation of the EMA competent cells into tendon cells, in which high protein levels of Stripe, Groovin (now known as Kakapo), and Alien are maintained, and the transcription of the genes delilah and beta1 tubulin is induced (Strumpf, 1998 and references).

In kak mutants, an excess of EMA cells, marked by the expression of Stripe and Delilah, is observed at a number of sites in the epidermis. This phenotype is particularly notable in domains in which a group of muscles extend together towards neighboring epidermal attachment cells, such as along the ventral segmental border cells, to which the four ventral longitudinal muscles bind. To further study the state of differentiation of the EMA cells in the kak mutant embryos, the expression of the beta1 tubulin gene was examined. In wild-type embryos, the expression of the beta1 tubulin gene is significantly elevated toward the end of tendon cell differentiation. In contrast to the expression of Stripe and Delilah, the mRNA expression of beta1 tubulin in kak mutant embryos is significantly reduced, suggesting that transcription of the latter gene requires different levels of signaling. It is suspected that Vein signaling from mesodermal cells, which is required for terminal differentiation of tendon cells (Yarnitzky, 1997), may be reduced in the mutant embryos; while there is enough signal to trigger Delilah and Stripe expression, the signal is not capable of inducing beta1 tubulin transcription (Strumpf, 1998).

The expression of delilah, stripe, and beta1 tubulin is induced in the epidermal attachment cells as a result of the EGF-receptor pathway activation by the neuregulin-like growth factor, Vein (Yarnitzky, 1997). Vein is secreted by mesodermal cells underlying the EMA cells. Vein protein localization is restricted to the muscle-tendon junctional site in wild-type embryos. However, in kak mutant embryos, Vein protein is not localized and appears rather diffuse. This altered pattern of Vein may explain the multiple number of cells expressing delilah and stripe: since Vein is not strictly localized at a given muscle-tendon junction site, it apparently weakly activates the EGF-receptor pathway in neighboring cells as well. It is presumed that the only cells that can respond to the ectopic Vein protein are the competent population of EMA cells, defined by the early expression of stripe. These cells express stripe during early developmental stages in a muscle-independent manner and normally lose their stripe expression by stage 16 of embryonic development. When these competent EMA cells receive the muscle-derived Vein signal, the expression of stripe and delilah is reactivated. It appears that only this population of cells is capable of responding to Vein, since the pattern of the ectopic Stripe- or Delilah-expressing cells in the kak mutant embryos resembles that of the early population of Stripe-expressing cells. The reduced levels of beta1 tubulin mRNA in the mutant tendon cells may also result from the abnormal pattern of Vein localization, since lower levels of Vein may not be sufficient to induce maximal beta1 tubulin expression. It therefore appears that the primary defect in kak mutant embryos stems from the lack of Vein accumulation at the muscle-tendon junctional site (Strumpf, 1998).

Is the abnormal differentiation of the epidermal muscle attachment (EMA) cells in kak mutant embryos reflected by the pattern of the somatic musculature? kak mutant embryos at stage 16 of embryonic development were labeled with anti-myosin heavy chain antibody to visualize the somatic muscles, and the muscle pattern was compared with that of wild-type embryos. A significant disruption of the somatic muscle pattern is observed in kak mutant embryos. In many cases, individual myotubes are not oriented correctly, and in some cases the myotube rounds up. Since Kak cannot be detected in myotubes using the available antibodies, it is assumed that the somatic muscle derangement is secondary to the abnormal differentiation of the EMA cells. A similar phenotype is also observed in stripe mutant embryos, in which the EMA cells do not differentiate correctly (Frommer, 1996). The similarity between the stripe and kak muscle phenotype and the reduced beta1 tubulin mRNA expression are consistent with the conclusion that EMA cell differentiation is defective in kak mutants. The correct recognition between the muscle and the tendon cell is essential for arresting the extension of the myotube and establishment of the final pattern of somatic musculature (Yarnitzky, 1997). It appears that the muscle development in kak partial loss of function embryos does not represent a complete loss of function phenotype since a more severe muscle defect is observed in kakV104/DfMK1 embryos (Strumpf, 1998).

How could this intracellular protein affect the localization of Vein at the extracellular matrix surrounding the EMA cell? At least two possibilities, which are not mutually exclusive, are considered. The first is the association of Kak with the unique cytoskeletal network of the EMA cell, which is critical for the cell's polarized organization. Tendon cell polarity may be essential for maintaining the characteristic junctional complexes formed between the basal surfaces of the EMA cell and the muscle cells. The space between these junctional complexes contains many extracellular matrix proteins, some of which may possess a Vein binding function. Impaired tendon cell polarity may lead to the loss of the putative Vein-binding component(s). Alternatively, Kak may be associated with a transmembrane protein(s) responsible for Vein localization either by direct binding or by association with additional extracellular matrix components that may directly bind Vein. Immunoprecipitation experiments with anti-Kak antibody indicated that Kakapo forms protein complexes containing the extracellular protein Tiggrin. These results favor the latter possibility that Kak is directly associated with protein complexes that may be important for Vein binding. The reduced amount of electron-dense material observed at the muscle-tendon junction site in the kak mutant embryos described in Prokop, et al. (1998) is in agreement with both mechanisms mentioned above (Strumpf, 1998).

The excess number of Stripe- and Delilah-expressing cells in the kak mutant embryos may be attributed to the dispersed levels of Vein, which could induce partial activation of the EGF-receptor signaling pathway in neighboring cells. An alternative explanation is that muscle-dependent differentiation of tendon cells may be accompanied by lateral inhibition of neighboring cells. The differentiated tendon cell may activate the Notch-signaling pathway in the surrounding cells. Aberrant contacts between tendon cells and their neighboring EMA competent cells in the kak mutant embryos may prevent efficient lateral inhibition, resulting in an excess of Stripe- and Delilah-expressing cells. An observation that supports this possibility is that an excess in beta1 tubulin-expressing cells is detected in Delta mutant embryos. Delta, a well-characterized Notch ligand, mediates lateral inhibition in a large array of tissues during embryonic and adult development. The lack of Delta may prevent lateral inhibition of the competent EMA cells, leading to their differentiation into beta1 tubulin-expressing cells. The impaired integrity of the epidermis described by Gregory (1998) is consistent with this explanation (Strumpf, 1998).

short stop (shot) is required for sensory and motor axons to reach their targets in the Drosophila embryo. Growth cones in shot mutants initiate at the normal times, and they appear normal with respect to overall morphology and their abilities to orient and fasciculate. However, sensory axons are unable to extend beyond a short distance from the cell body, and motor axons are unable to reach target muscles. The shot gene encodes novel actin binding proteins that are related to plakins and dystrophin and expressed in axons during development. The longer isoforms identified are predicted to contain an N-terminal actin binding domain, a long central triple helical coiled-coil domain, and a C-terminal domain that contains two EF-hand Ca2+ binding motifs and a short stretch of homology to the growth arrest-specific 2 protein. Other isoforms lack all or part of the actin binding domains or are truncated and contain a different C-terminal domain. Only the isoforms containing full-length actin binding domains are detectably expressed in the nervous system. shot is allelic to kakapo, a gene that may function in integrin-mediated adhesion in the wing and embryo. It is proposed that Shot's interactions with the actin cytoskeleton allow sensory and motor axons to extend (Lee, 1999).

kakapo mutation affects terminal arborization and central dendritic sprouting of Drosophila motoneurons. Four mutant alleles of kak are described that are embryonic lethals, that fail to complement one another, and that show a paralytic phenotype when homozygous, transheterozygous or hemizygous over deficiencies. Paralysis might be caused by dysfunction or developmental defects in either the nervous system or the musculature. In kakapo mutant embryos, defects are found in both tissues: muscles detach from the epidermis in all alleles, and there is a reduction in the size of motoneuronal terminals on muscles and of neuronal branches in the CNS at late stage 17 (Prokop, 1998).

In wild-type embryos at stage 17, motoneuronal terminals have branches on their target muscles with varicosities (boutons) of up to 1 micro meters in diameter. In kak mutant embryos NMJs in all locations occupy far less surface on their respective muscles; their branches are reduced in length, and boutons appear reduced in number and size. Whereas some allelic combinations exhibit an almost complete absence of NMJs, other combinations show less severe phenotypes, but their phenotype is nevertheless significant. Although NMJs are severely reduced in kak mutant embryos, presynaptic marker expression is mainly restricted to neuromuscular sites and can hardly be found in ectopic locations. This reduced and restricted appearance of synaptic markers in kak mutant embryos hints at a requirement for kak within the presynaptic terminal (Prokop, 1998).

Ultrastructural analyses of kak mutant embryos reveal that presynaptic boutons can form normal cell junctions with the muscle, interspersed by morphologically normal synapses. However, examples are found where synapses are indicated by structured material in the neuromuscular cleft, but typical presynaptic specializations (T-bars) are missing. If T-bars are found, they are restricted to neuromuscular sites, corroborating light microscopic findings. Furthermore, neuromuscular contacts and synapses are found less frequently when compared with controls, which is in agreement with the reduction of NMJs observed at the light microscopic level. To test whether transmission occurs at kak mutant NMJs, patch recordings were carried out on kak mutant muscles. These recordings reveal excitatory junctional currents, clearly indicating that neuromuscular transmission occurs. In four cases the NMJs were stained with antibodies raised against cysteine string protein subsequent to recording and it was confirmed that in all cases the NMJ is clearly misshapen and reduced in size. Occurrence of neuromuscular transmission is furthermore demonstrated by the presence of strong muscle contractions in kak mutant embryos observed under polarized light in vivo. Taken together, ultrastructural, electrophysiological and in vivo observations suggest that NMJs, although abnormal in shape, are functional in kak mutant embryos. This suggests that kak might be required specifically for growth and shaping of branches at motoneuronal terminals. In kak mutant embryos motoneurons appear capable of navigating along correct paths to their target muscles and maintaining these contacts thereafter. This suggests that the kak mutant alleles affect NMJ formation during the differentiation phase, when muscle-attached growth cones reshape into the branches and boutons of mature NMJs (Prokop, 1998).

To investigate whether kak function might be required directly within the nerve terminal, an anti-kak antiserum was used. The staining procedure used fails to detect strong staining at neuro-muscular junctions in wild type or hemizygous embryos, however, the NMJs of kak overexpressing embryos are labeled more reliably and strongly than wild type. Local neuronal growth is not restricted to branch formation at the NMJ but also occurs within the CNS during the development of dendritic branches at stage 16/17. A test was carried out to see if this growth might also be affected in kak mutant embryos. Dendrites were labeled in a retrograde manner by applying DiI to the NMJ of RP3 motoneurons on muscles VL3/4. In wild-type embryos, RP3 sends an axon contralaterally through the dorsally located anterior root of the intersegmental nerve. On the ipsilateral side, a second projection leaves the soma of RP3, projecting along a similar path as the contralateral process, but remaining confined to the neuropile. Both projections have numerous local arborizations. In kak mutant embryos, the ipsilateral local arborizations are almost normal, but the contralateral arborizations are severely reduced and often form swellings or blobs. The failure of RP3 to elaborate its contralateral dendrites is apparent from late stage 16, suggesting kak function is required for the process of outgrowth rather than maintenance of dendrites. Consistent with the findings for the RP3 dendrites, the whole neuropile is reduced in size in kak mutant embryos, as compared with wild type, but appears normal in its organization. Taken together, these findings suggest that neurons project correctly, but fail to elaborate part of their local branches, leading to smaller NMJs in the periphery and smaller dendrites and thus reduced neuropile volume in the central nervous system (Prokop, 1998).

The phenotypes shown so far strongly suggest a specific requirement for kak function in specific local growth events. Below, two further kak mutant phenotypes are described: mislocalization of axonal proteins and disorganization of the cytoskeleton, both of which are potential causes underlying the specific defects in neuronal branch formation. A mislocalization of proteins along neuronal processes has been observed. For example, Fas II, which encodes a transmembrane protein of the immunoglobulin superfamily, is expressed at low levels in the nerve roots and stops at the entry point into the neuropile of stage 16 and 17 control embryos. By stage 17, Fas II expression in all nerve roots is strongly upregulated and the anterior root of the intersegmental nerve extends to the dorsal part of the neuropile. Thus, it appears as if Fas II fails to localize properly along neuronal processes. In contrast, 22C10 immunoreactivity (see Futsch) appears to be distributed normally in kak mutant nerve roots, but a mislocalization phenotype is found in another type of neuron, the dorsal bipolar neuron of the peripheral nervous system. The dorsal bipolar neurons have longitudinal projections that span the entire length of the segment, but only the proximal regions of these processes are labeled by 22C10 antibodies in the wild type. However, in kak mutant embryos the entire length of these lateral bipolar projections is 22C10-positive. Thus, kak function is required for the correct localization of (membrane) proteins within neuronal processes, and the mislocalization of such proteins is a potential cause for defects in local branching in the neuropile or at the NMJ (Prokop, 1998).

Both defects, the localization of axonal proteins and defects in the microtubule organization may be the underlying cause for the observed reduction in local growth of dendrites and at NMJs. Branching of motoneuronal terminals and axonal defasciculation require a reduction of neuronal cell adhesion molecule (N-CAM)-mediated interaxonal adhesion in vertebrates and, in agreement with this, the overexpression of Fas II, the Drosophila homolog of N-CAM, antagonizes nerve branching. Hence, it can be reasoned that the inhibition of dendrite and branch formation might be due to the observed mislocalization of Fas II to axonal areas where dendrites and terminal branches are usually forming. However, combining kak0 with a Fas II null allele does not show any obvious suppression of the neuromuscular phenotype. Thus, mislocalization of Fas II alone does not explain the growth defects, but its involvement might be obscured by mislocalization of other redundant CAMs of similar function. Mislocalization of membrane proteins might be the consequence of their lack of a Kak-mediated linkage to the membrane-associated cytoskeleton. Conversely, loss of such a physical link could cause disruption of growth regulation, since transmembrane proteins have been shown to instruct the assembly of the actin cytoskeleton in neuronal growth cones (Prokop, 1998).

Neuronal growth defects in kak mutant embryos might be caused directly by defects in cytoskeleton assembly. Microtubules are essential for axonal growth and are regulated in a complex way. The assembly of microtubules during growth is preceded by formation of the actin cytoskeleton. The fine regulation of actin could require actin-associated proteins, and Kak might be one of them. This might explain why loss of kak function suppresses only a specific subset of neuronal growth events, i.e., local growth at NMJs and of contralateral RP3 dendrites but not long distance growth or ipsilateral RP3 arbors. The specific growth defects in kak mutant embryos might be due to subcellular-specific compartmentalization of Kak or local posttranslational modifications. Alternatively, unaffected branches may contain redundant cytoskeletal molecules that the affected branches lack. Possible molecular differences might reflect a general difference between affected and unaffected branches. For example, affected branches might represent preferentially presynaptic output branches (certainly true for NMJs) and unaffected branches may represent postsynaptic or input branches. Alternatively, the qualitative differences might consist in the origin of the branches: arborizations derived from an axon (NMJ, contralateral RP3 dendrites) may require Kak function, but not those derived from somatic extensions (ipsilateral RP3 dendrites) (Prokop, 1998).

GENE STRUCTURE

Northern blot analysis using a kak cDNA fragment reveals two transcripts of ~17.6 and ~15.4 kbp (Strumpf, 1998). Two cDNA sequences overlap to give mRNAs of 17,420 nucleotides encoding a 5,497-amino acid protein (form A) and 17,217 nt encoding a 5,385-amino acid protein (Gregory, 1999)

PROTEIN STRUCTURE

Amino Acids - 4,151 (Strumpf, 1998) and 5,497 and 5,338 (Gregory, 1998)

Structural Domains

The primary amino acid sequence of Kak reveals several domains and motifs that show high degrees of similarity to three distinct vertebrate cytoskeletal-related protein families: plakin (Ruhrberg, 1997a); dystrophin (Koenig, 1988), and Gas2/GAR22 (Schneider, 1988; Zucman-Rossi, 1996). The NH2-terminal region of Kak is homologous to the NH2-terminal domain of members of the plakin family of cytoskeletal cross-linker proteins, comprising plectin, BPAG1, and ACF7 (Ruhrberg, 1997). These large proteins link actin microfilaments and intermediate filaments to the plasma membrane at specialized attachment sites, called hemidesmosomes. Abnormal function of various plakin family members leads to skin (e.g., bullous pemphigous) as well as neurological (e.g., dystonia musculorum) disorders (for reviews see Ruhrberg, 1997a; Fuchs, 1998). The region of similarity between Kak and plakin family members includes the actin binding region but does not exhibit similarity to the intermediate filament-associated domain. The area of strongest similarity is with an actin-binding domain originally defined in alpha-actinin, but subsequently found in dystrophins and spectrins as well as the plakin family. Across this 240-amino acid region, Kakapo shares ~65% amino acid identity with plectin and BPAG1. The high level of conservation suggests that this domain in Kak does bind to actin. All proteins so far described in the plakin family have a carboxy-terminal domain that binds intermediate filaments, encoded by a single large final exon (Ruhrberg, 1997a). This does not appear to be the case for the Kak protein, which, after residue 1200, has no further sequence similarity with plakins, and instead becomes similar to dystrophin. The central region of Kak (amino acids 408-3574) consists of 22 repeats, 105-113 amino acids long. A computerized search has indicated that the central region of Kak shares sequence similarity (~20% identity) with spectrin-like repeats present in an array of cytoskeletal-associated proteins, including dystrophin, alpha-actinin, and spectrin. These repeats are predicted to adopt a triple-helical conformation. In dystrophin, the multiple repeat domain functions as a spacer between the NH2-terminal actin-binding domain and the COOH-terminal domain associated with a group of membrane proteins. The consensus sequence deduced from the alignment of the spectrin-like repeats in Kak shares 46% identity (51% similarity) with the human dystrophin repeat consensus, CS1 (Koenig, 1990). This similarity suggests the presence of a similar domain containing multiple spectrin-like, triple-helical repeats in the central region of Kak protein. This region also contains five Leucine-zipper motifs. A somewhat lower degree of similarity between the Kak COOH-terminal domain (sequence 3725-3793) and the region in dystrophin containing the two EF-hand motifs is also observed. The COOH-terminal domain of dystrophin-related proteins is highly conserved and includes a WW domain, implicated in mediating interactions with the transmembrane protein, beta-dystroglycan. This domain also includes two putative Ca2+-binding EF-hands (Kawasaki, 1995) and a region involved in the binding to members of the syntrophin family of PDZ domain-containing proteins. The similarity between Kak and dystrophin in the COOH-terminal domain is detected only along the EF-hand motifs (~30% identity along a sequence of 70 amino acids). This limited similarity may suggest that although Kak does not appear to be a dystrophin family member, these genes may share a common ancestor (Strumpf, 1998; Gregory, 1998, and their references).

The COOH-terminal region of Kak shows sequence conservation with yet another family of cytoskeletal-related proteins represented by the Gas2/GAR22 proteins (Schneider, 1988; Zucman-Rossi, 1996). Mouse Gas2, a member of this protein family, belongs to a set of proteins that have been shown to be selectively expressed in growth-arrested cells in culture (Schneider, 1988). It is a highly regulated protein (Brancolini, 1992; Manzow, 1996) that interacts with the microfilament system (Brancolini, 1992; Brancolini, 1994). Deletion analysis of the Gas2 protein suggests that the region in Gas2 that is homologous to Kak has a significant function in cytoskeletal organization (Brancolini, 1995). During apoptosis, Gas2 is cleaved by ICE proteases, presumably leading to microfilament derangement (Brancolini, 1995). An additional partially cloned cDNA species of unknown function from human brain exhibits a high level of identity to the COOH-terminal domain of Kak protein. This similarity extends beyond the Gas2 homology domain and exhibits 40% overall identity along the entire 1,658-amino acid sequence available in the data base. It is not clear whether this partially cloned cDNA represents a human Kak homolog. In addition, a putative protein from C. elegans shares domain contents with Kak, including plectin, dystrophin, and Gas2/ GAR22-like domains. The function of this putative protein is yet to be elucidated (Strumpf, 1998 and references).

Taken together, the deduced amino acid sequence of Kak predicts a novel large intracellular protein that carries two distinct cytoskeletal-associated domains separated by a spacer consisting of elongated triple-helical spectrin-like repeats. At the NH2-terminal domain, Kak may interact with actin microfilaments, while at its COOH terminus, it may be associated with membrane structures or with additional cytoskeletal components. The similarities between Kak and its C. elegans and putative human homologs suggest that Kak structure is conserved through evolution (Strumpf, 1998 and references).

Two more isoforms of Shot/Kak have been identified. Two shot cDNAs (form C) encode a third unique N-terminal sequence of 210 amino acids followed by half of the actin binding domain. This second half of the actin binding domain is less evolutionarily conserved than the first half of the actin binding domain. The mammalian BPAG1n3 protein also contains a similar half actin binding domain and associates poorly with the actin cytoskeleton in cultured cells. Finally, isoform D lacks an identifiable actin binding domain and contains no N-terminal globular domain. The likely initiator methionine codon for isoform D is located downstream of sequences encoding the actin binding domain, at the start of sequences encoding the central rod domain. Thus, shot encodes various rod-like proteins predicted to differ in their actin binding properties. The genes for other plakins also encode similarly spliced actin binding variants. Four different 5' isoforms are encoded by the BPAG1 gene. Two neuronal isoforms, BPAG1n1 (dystonin-1) and BPAG1n2 (dystonin-2), contain an actin binding domain; a third neuronal isoform, BPAG1n3, contains a half-actin binding domain analogous to that found in Shot isoform C, and the epidermal isoform BPAG1e contains no actin binding domain, as in Shot isoform D. Mouse ACF7 is 71% identical to BPAG1 within the predicted actin binding domain and encodes N-terminal isoforms similar to Shot isoforms A, B, and C. Both isoforms 1 and 2 of mACF7 are predicted to contain a complete actin binding domain; mACF7 isoform 3 lacks the most conserved portion of the actin binding domain and contains the less conserved portion exactly as in Shot isoform C (Lee, 1999 and references therein).

Thus the shot gene may contain as many as four different promoters. The authors investigated the relationship between the P-element insertions in shot and the shot promoter and transcription start sites. A combination of PCR, Southern analysis, and sequencing was used to map the shotP1, shotP2, kakP1, and kakP2 insertion sites and the cDNA sequences onto a 15 kb genomic DNA contig. The kakP1 and kakP2 P-elements are inserted at the same site, in an intron 1917 bp before the first exon common to mRNAs encoding isoforms A and B. The shotP2 insertion is 49 bp upstream of the start of alternative transcript C. The shotP1 insertion is located in an exon common to both isoforms C and D, 131 bp downstream of the alternative splice site that generates isoform C. Although they are inserted at different sites with respect to the shot transcripts, all of the P-element insertions disrupt the protein expression of the long isoforms of Shot and appear to be similar in their axon growth phenotypes (Lee, 1999 and references therein).

Additional isoforms have been identified of Shot/Kak, the longest of which encodes a 5501 amino acid protein that is almost completely identical to the previously reported 5497 amino acid protein (Gregory, 1998; Strumpf, 1998). The central region of this protein is likely to be rod-like and contains 22 triple helical coiled-coil repeats similar to those found in spectrin and dystrophin. The C-terminal globular domain contains two EF-hand motifs. The C-terminal globular domain also contains a short stretch of sequence homology to the mammalian growth arrest-specific 2 (Gas2) protein. Gas2 is a cytoskeletal protein of unknown function that appears to be associated with microfilaments in cultured cells and is highly induced in cultured fibroblasts during serum starvation. The cDNA sequences isolated for the 3' end of the shot gene reveal that the central rod region is alternatively spliced. Seven of eight cDNA clones predict an isoform that shares the same central rod sequence reported previously (Strumpf, 1998). The other clone encodes a 300 amino acid sequence within the central rod region, as previously reported for Shot/Kak isoforms A and B (Gregory, 1998). A cDNA was also isolated in which the sequence for the 300 amino acid region is spliced to a novel sequence that encodes a globular domain of 436 amino acids. This 436 amino acid domain shows low homology to the six tandem repeat domains in the C-terminal globular region of plectin and is unlikely to form an extensive coiled-coil structure. Although it still contains considerable coiled-coil forming sequence, this truncated isoform lacks the 22 triple helical repeats found in the longer isoforms. By comparing the known lengths of plectin and dystrophin proteins, and the relative lengths of coiled-coil forming sequence in Shot, plectin and dystrophin, it is inferred that the truncated isoforms of Shot are ~75 nm long and the long isoforms of Shot are ~200-220 nm long. Thus, shot encodes rod-like proteins of varying length with different C-terminal domains, as well as different predicted actin binding properties (Lee, 1999 and references therein).

The sequence of the C-terminal domain in the longer Shot isoforms matches the C-terminal sequences of several vertebrate proteins, including the full-length sequence of mouse ACF7 recently reported in GenBank. The sequence of the EF-hand and GAS2 domains are particularly well conserved. Sequencing of the shot cDNAs also reveals diversity in this C-terminal domain. The C-terminal sequence after the Gas2 homology domain is alternatively spliced, with the variant reported here being a closer match to the vertebrate proteins. Taken together, overlapping cDNAs have been cloned that predict multiple isoforms of Shot/Kak and greatly expand the potential functional diversity of this gene. The gene encodes rod-like proteins of varying lengths, only some of which contain complete N-terminal actin binding domains. These proteins contain two different classes of globular C-terminal domains, which in plakins mediate protein-protein interactions. By analogy, the different Shot proteins may therefore interact with diverse cytoskeletal targets (Lee, 1999).

date revised: 20 February 2000

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

Please e-mail comments/corrections to brodyt@codon.nih.gov