InteractiveFly: GeneBrief

shark: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - SH2 ankyrin repeat kinase

Synonyms - shark

Cytological map position - 52F7--8

Function - signaling

Keywords - ectoderm, dorsal closure, oogenesis, dorsal-appendage morphogenesis

Symbol - shark

FlyBase ID: FBgn0015295

Genetic map position - 2-

Classification - SH2 domains, ankyrin repeats, a proline-rich and basic region, and a tyrosine kinase domain

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

Shark (SH2 domain ankyrin repeat kinase, Ferrante, 1995) is a Drosophila nonreceptor tyrosine kinase that contains from amino to carboxyl terminus, a Src homology 2 (SH2) domain (N-SH2), five ankyrin repeats, a second SH2 domain (C-SH2), a proline-rich and basic region, and a tyrosine kinase domain. Analysis of the phenotypes associated with a shark loss-of-function mutation demonstrates that Shark activity is essential for the migration of the dorsolateral epidermis of the embryo during dorsal closure (DC). Shark kinase functions in DC upstream of Dpp expression by leading edge (LE) cells (Fernandez, 2000).

shark is also required for dorsal-appendage (DA) morphogenesis in Drosophila oogenesis. shark function is required in follicle cells for cell migration and chorion deposition. bullwinkle (bwk) regulates, through a novel germline-to-soma signal, morphogenesis of the eggshell dorsal appendages. A screen was carried out for dominant modifiers of the bullwinkle mooseantler eggshell phenotype and shark was idenfied as a dominant modifier of bullwinkle. At the onset of dorsal-appendage formation, shark is expressed in a punctate pattern in the squamous stretch cells overlying the nurse cells. Confocal microscopy with cell-type-specific markers demonstrates that the stretch cells act as a substrate for the migrating dorsal-appendage-forming cells and extend cellular projections towards them. Mosaic analyses reveal that shark is required in follicle cells for cell migration and chorion deposition. Proper shark RNA expression in the stretch cells requires bwk activity, while restoration of shark expression in the stretch cells suppresses the bwk dorsal-appendage phenotype. These results suggest that shark plays an important downstream role in the bwk-signaling pathway. Candidate testing implicates Src42A in a similar role, suggesting conservation with a vertebrate signaling pathway involving non-receptor tyrosine kinases (Tran, 2003).

The folding and remodeling of epithelia into more complex structures is a recurrent phenomenon in metazoan development. Intercellular interactions are important regulatory components of these processes. Adjacent cells typically provide cues that direct morphogenesis or establish an extracellular milieu permissive for cell movements (Tran, 2003).

In Drosophila melanogaster, remodeling epithelia can interact with an adjacent epithelium. Two well-studied examples include the migration of the embryonic dorsal epithelium over the amnioserosa, and eversion of leg and wing primordia relative to the peripodial tissue that bounds the imaginal discs. These cell layers actively regulate the patterning and movements of neighboring epithelia. Ablation of the peripodial membrane results in growth and patterning defects in the eye and wing discs. In the embryo, the amnioserosa contributes signals and mechanical force to dorsal closure. During germband retraction, the amnioserosa also signals to and extends lamellipodia-like structures towards the retracting germband cells. This study elaborates on a novel extracellular pathway defined by bullwinkle (bwk) (Rittenhouse, 1995) that is essential for proper tubulogenesis of the follicular epithelium during synthesis of the dorsal appendages (DAs), specialized respiratory structures of the eggshell. Additionally, it is demonstrated that an adjacent squamous cell layer acts as a substrate for the migrating epithelium and expresses factors required for this morphogenetic process (Tran, 2003).

DA formation occurs within the context of the Drosophila egg chamber, which consists of ~650 somatically derived follicle cells surrounding a germline cyst composed of one oocyte and 15 nurse cells. The germ cells are interconnected via cytoplasmic bridges called ring canals, which provide access for the transfer of nurse-cell material into the developing oocyte. At stage 11, the nurse cells transport most of their cytoplasm into the oocyte, and then undergo programmed cell-death. DA morphogenesis begins at stage 11, coincident with nurse-cell apoptosis (Tran, 2003).

During DA formation, the somatic layer consists of two major populations with distinctive morphologies, the stretch cells and columnar cells. At the anterior, ~50 squamous stretch cells cover the nurse cells. These cells provide signals that pattern the anterior eggshell-forming cells and ensure proper nurse-cell cytoplasmic dumping. The columnar cells overlie the oocyte at the posterior and secrete the layers and specialized structures of the eggshell. The anterior-most columnar cells (the centripetal cells)migrate inwards, closing off the anterior end of the oocyte while synthesizing the operculum and micropyle. In addition, two subpopulations of ~65 dorsoanterior follicle cells form the two dorsal appendages through a complex reshaping and reorganization of a flat epithelium into three-dimensional tubes (Tran, 2003 and references therein).

These DA-forming cells apically constrict and evert outwards, changing from a flat layer into tubular structures that extend anteriorly. Secretion of chorion proteins into the tube lumens creates the appendages. This process occurs during the final stages of oogenesis, downstream of the events that pattern the eggshell and embryonic axes (Tran, 2003).

Although much is known about the induction and refinement of follicle-cell patterning, little is known about the factors that govern the cellular movements. One pathway that contributes to the morphogenesis is the Jun-kinase (JNK) pathway. The Drosophila Jun and Fos transcription factors are expressed highly in the stretch cells and in an anterior subset of the two DA-forming cell populations. Loss of JNK-pathway function results in two short paddleless DAs and defective nurse-cell cytoplasmic transport (Tran, 2003 and references therein).

The DA-forming cells require additional extracellular cues for normal tubulogenesis. Mosaic analyses demonstrate that bwk is required in the germline to regulate formation of the dorsal appendages (Rittenhouse, 1995). bwk encodes several SOX/TCF transcription factors with pleiotropic functions, regulating dorsal follicle-cell migration, anteroposterior (AP) patterning in the embryo, and transport of nurse-cell cytoplasm into the oocyte. In bwk mutants, the DA-forming cells not only fail to migrate anteriorly, but instead extend much more laterally (Dorman, 2004), as indicated by the wide DA paddle (Tran, 2003 and references therein).

To elucidate the role of bwk in DA formation, other components of this germline-to-soma signaling pathway were sought. Second-chromosome deficiencies were screened for regions that genetically interact with bwk. Tests of candidate mutations identified shark as a strong Enhancer of bwk. shark encodes an SH2-ankyrin-repeat, tyrosine-kinase protein (Ferrante, 1995) that functions upstream of the JNK pathway (Fernandez, 2000) during dorsal closure of the embryo (Tran, 2003 and references therein).

shark is shown to act downstream of bwk in the squamous stretch cells, and shark mediates the regulation of DA formation by bwk. Furthermore, detailed cellular analyses with stretch-cell markers show that the stretch cells provide a substrate for the DA-forming cells and appear morphogenetically active (Tran, 2003).

Shark non-receptor kinase is conserved, with homologs in Hydra (Chan, 1994) and sponge (Suga, 1999). The mammalian counterparts contain homologous SH2 and tyrosine-kinase domains but lack the ankyrin repeats (Chan, 1991; Taniguchi, 1991). These mammalian proteins, Zap70 and Syk, are recruited to immunoreceptor complexes upon ligand binding and regulate immune-cell activation and differentiation, functioning alongside Src kinases (reviewed by Chu, 1998). In T-cells, Zap70 also mediates signaling downstream of integrin-receptor complexes that feature in T-cell motility (Bearz, 1999; Soede, 1998: Tran, 2003 and references therein).

Mosaic analyses with loss-of-function shark alleles have established two somatic functions in DA formation. First, shark is required in the DA cells for proper DA-chorion deposition, a complex process regulated at many levels. Mutations that disrupt chorion-gene amplification or chorion-protein synthesis result in thin, collapsed DAs and main-body eggshell. Unlike those mutations, loss of shark in the main-body follicle cells does not cause defects in follicular imprints, alter the appearance of the eggshell under darkfield optics, or produce thin chorion and collapsed eggs. Although the methods used in this study may miss subtle defects in main-body chorion, shark may play a DA-cell-specific role in the production/formation of chorion. Although regulatory sequences and a putative binding protein drive specific spatial expression of chorion-reporter constructs, no reported mutants disrupt DA-specific chorion expression (Tran, 2003).

The second function of shark lies in the stretch cells and affects the migration of the DA cells. Large stretch-cell clones resulted in shortened DAs that vary in their morphology and penetrance. This variability could result from residual activity of these mutant alleles, non-cell autonomy, or functional redundancy. Although no Shark paralogs are encoded in the genome, several non-receptor tyrosine kinases share homology in the SH2 and kinase domains, including Src42A (Tran, 2003).

In addition, stretch-cell expression of shark strongly suppresses the bwk-mutant DA phenotype, in concurrence with a direct role for bwk in regulating shark expression in this tissue. These results indicate that shark is key in regulating DA migration downstream of bwk. Full rescue was not likely achieved because of insufficient expression levels, the need to localize shark RNA, or the existence of shark-independent branches downstream of bwk (Tran, 2003).

These data suggest a model in which Bwk regulates factors in the germline that are required for proper shark expression in the stretch cells. Shark then regulates the activity of targets required for DA-cell movement across the stretch-cell layer. Another factor that could be regulated by bwk is the Src42A kinase, which behaves similarly to shark. Loss of Src42A enhances bwk mutants, while stretch-cell expression of activated Src42A suppresses bwk. Mammalian homologs of Shark function together with Src kinases, suggesting a conserved signaling cascade (Tran, 2003).

Two other stretch-cell signaling pathways, JNK and DPP, regulate DA morphogenesis. Tests with bwk and shark, however, fail to reveal strong or definitive interactions. Loss of JNK activity in oogenesis results in shortened and paddleless DAs, yet expression of UAS-basket+ and reduction of bsk dose do not alter the morphology of bwk eggshells. Furthermore, expression of the AP-1 components is unaffected in bwk mutants and shark clones. These data support the hypothesis that the bwk/shark pathway does not primarily act through JNK signaling (Tran, 2003).

Moderate overexpression of dpp and loss of the type I receptors, tkv and sax, can lead to shortened and somewhat broadened DAs, resembling bwk mutants. The expression of dpp RNA and a dpp enhancer trap, however, are unaffected in bwk mutants. Both hypomorphic dpp alleles and loss of type I receptors fail to interact with a strong bwk mutant. These data suggest that bwk does not directly regulate dpp4 expression or activity but rather may modulate downstream targets (Tran, 2003).

DA-cell movement over the stretch cells may require expression of stretch-cell factors that guide or facilitate migration. Mammalian proteins that share homology with Shark can bind to and regulate integrin complexes. Shark may bind these and/or other adhesion receptors to regulate cell migration either through signaling cues or by modulating the extracellular matrix (Tran, 2003).

Shark could also regulate stretch cell behaviors, controlling the small cellular projections that extend toward the DA cells during their anterior movement. These extensions may guide or signal the DA-forming cells, as occurs in imaginal discs (Tran, 2003).

Extracellular signals and interactions are key components of morphogenetic processes. Two downstream components of the bwk pathway have been identified that act in the stretch-cell layer to relay a novel germline signal required for the movement of a third tissue, the remodeling epithelium of the dorsal appendage cells (Tran, 2003).


REGULATION

Protein Interactions

The cellular machinery promoting phagocytosis of corpses of apoptotic cells is well conserved from worms to mammals. An important component is the Caenorhabditis elegans engulfment receptor CED-1 and its Drosophila orthologue, Draper. The CED-1/Draper signalling pathway is also essential for the phagocytosis of other types of 'modified self' including necrotic cells, developmentally pruned axons and dendrites, and axons undergoing Wallerian degeneration. This study shows that Drosophila Shark, a non-receptor tyrosine kinase similar to mammalian Syk and Zap-70 (Ferrante, 1995), binds Draper through an immunoreceptor tyrosine-based activation motif (ITAM) in the Draper intracellular domain. Shark activity is essential for Draper-mediated signalling events in vivo, including the recruitment of glial membranes to severed axons and the phagocytosis of axonal debris and neuronal cell corpses by glia. The Src family kinase (SFK) Src42A can markedly increase Draper phosphorylation and is essential for glial phagocytic activity. It is proposed that ligand-dependent Draper receptor activation initiates the Src42A-dependent tyrosine phosphorylation of Draper, the association of Shark and the activation of the Draper pathway. These Draper-Src42A-Shark interactions are strikingly similar to mammalian immunoreceptor-SFK-Syk signalling events in mammalian myeloid and lymphoid cells. Thus, Draper seems to be an ancient immunoreceptor with an extracellular domain tuned to modified self, and an intracellular domain promoting phagocytosis through an ITAM-domain-SFK-Syk-mediated signalling cascade (Ziegenfuss, 2008).

Developing tissues produce excessive numbers of cells and selectively destroy a subpopulation through programmed cell death to regulate growth. Rapid clearance of cell corpses is essential for maintaining tissue homeostasis and preventing the release of potentially cytotoxic or antigenic molecules from dying cells, and defects in cell corpse clearance are closely associated with autoimmune and inflammatory diseases. In C. elegans the CED-1 receptor is expressed in engulfing cells, where it acts to recognize cell corpses and drive their phagocytosis. CED-1 promotes engulfment through an intracellular NPXY motif, a binding site for proteins containing a phosphotyrosine-binding (PTB) domain, and a YXXL motif, a potential interaction site for proteins containing SH2 domains. The PTB domain adaptor protein CED-6 can bind the NPXY motif of CED-1, is required for cell corpse engulfment and acts in the same genetic pathway as CED-1. CED-1 ultimately mediates actin-dependent cytoskeletal reorganization through the Rac1 GTPase, and Dynamin modulates vesicle dynamics downstream of CED-1 during engulfment, but the molecular signalling cascade that allows CED-1 to execute phagocytic events remains poorly defined (Ziegenfuss, 2008).

Glia are the primary phagocytic cell type in the developing and mature brain. Glia rapidly engulf neuronal cell corpses produced during development, as well as neuronal debris generated during axon pruning or during Wallerian degeneration in the adult brain. In Drosophila, glial phagocytosis of these engulfment targets requires Draper, the fly orthologue of CED-1. Draper, like CED-1, contains 15 extracellular atypical epidermal growth factor (EGF) repeats, a single transmembrane domain, and NPXY and YXXL motifs in its intracellular domain. Drosophila Ced-6 is also required for the clearance of pruned axons, indicating possible conservation of the interaction between CED-1 and CED-6 in flies, but additional signalling molecules acting downstream of Draper have not been identified (Ziegenfuss, 2008).

Draper was identified in a yeast two-hybrid screen for molecules interacting with the regulatory region of Shark. When LexA-Shark, constitutively active Src kinase and AD-Draper are present, Shark and Draper interact physically. In the absence of Src kinase, Shark and Draper fail to interact, indicating that phosphorylation of Draper by Src may be essential for Shark-Draper interactions. The Draper intracellular domain contains an ITAM (YXXI/L-X6-12-YXXL), a key domain found in many mammalian immunoreceptors including Fc, T-cell and B-cell receptors. SFKs phosphorylate the tyrosines in ITAM domains, thereby allowing ITAM association with SH2-domain-containing signal transduction proteins including Syk and Zap-70. Y-->F substitutions of the tyrosine residues were generated within or near the Draper ITAM, and it was found that Tyr 949 and Tyr 934 are critical for robust Draper-Shark binding. These correspond to the consensus tyrosine residues in the predicted Draper ITAM. Plasmids were transfected with carboxy-terminally haemagglutinin-tagged Draper (Draper-HA) or with Draper-HA and Shark with an amino-terminal Myc tag (Myc-Shark) into Drosophila S2 cells, immunoprecipitated with anti-HA antibodies, and western blots were performed with anti-phosphotyrosine, anti-Myc and anti-HA antibodies. Myc-Shark was found to co-immunoprecipitate with Draper-HA, and that anti-phosphotyrosine antibodies label a band corresponding to the position of Draper-HA that is absent in empty vector controls. Further, it was found that a Y949F substitution markedly reduces Draper-Shark association. Taken together, these data indicate Draper and Shark can associate physically through the Draper ITAM domain (Ziegenfuss, 2008).

Attempts were made to determine whether Shark is required for glial phagocytic activity in vivo. Severing adult Drosophila olfactory receptor neurons (ORNs) initiates Wallerian degeneration of ORN axons. Antennal lobe glia surrounding these severed axons respond to this injury by extending membranes towards severed axons and engulfing degenerating axonal debris. These glia express high levels of Draper, and in draperδ5 null mutants, glia fail to respond morphologically to axon injury, and severed axons are not cleared from the central nervous system (CNS). Thus, both the extension of glial membranes to severed axons and the phagocytosis of degenerating axonal debris require Draper signalling (Ziegenfuss, 2008).

Whether Shark function in glia is essential for glial responses to axon injury was explored by driving a UAS-regulated double-stranded RNAi construct designed to target shark (sharkRNAi) with the glial-specific repo-Gal4 driver, severing ORN axons, and assaying the recruitment of Draper and green fluorescent protein (GFP)-labelled glial membranes to severed axons. Maxillary palp-derived ORN axons project to 6 of the roughly 50 glomeruli in the antennal lobe. Within hours after maxillary palps have been ablated in control animals, Draper immunoreactivity decorates severed axons projecting to and within maxillary palp ORN-innervated glomeruli, and GFP-labelled glial membranes are recruited to these severed axons. Strikingly, knocking down Shark in glia completely suppresses these events. Next, antennal ORN axons were severed; these axons project to about 44 of the 50 antennal lobe glomeruli. Antennal ablation therefore injures nearly all glomeruli in the antennal lobe and results in the majority of antennal lobe glia in control animals upregulating Draper and undergoing hypertrophy. It was found that knocking down Shark in glia also blocks this glial response to axon injury. Thus, Shark is essential for all axon-injury-induced changes in glial morphology and Draper expression (Ziegenfuss, 2008).

To determine whether Shark is required for glial phagocytosis of severed axons, a subset of maxillary palp ORN axons were labelled with mCD8::GFP, Shark function was knocked down in glia, and the clearance of severed axons was assayed. In control animals severed GFP-labelled ORN axonal debris was cleared from the CNS within 5 days. In contrast, glial-specific sharkRNAi potently suppresses the clearance of degenerating axons, with severed axons lingering in the CNS for at least 5 days. Then whether mutations in the shark gene affected the glial clearance of degenerating axons was examined. The null allele of shark, shark1, is pupal lethal (Fernandez, 2000). Therefore glial responses to axon injury were assayed in shark1 heterozygous mutants, and dominant genetic interactions between draperδ5 and shark1 were tested. It was found that both draperδ5/+ and shark1/+ animals showed defects in glial phagocytic function: 5 days after injury, significant amounts of axonal debris remained within OR85e-innervated glomeruli. Moreover, shark1/+; draperδ5/+ animals showed a striking suppression of glial clearance of severed axons almost equivalent to that of draperδ5 mutants. Thus, shark mutations dominantly suppress the glial clearance of degenerating ORN axons, and this phenotype is strongly enhanced by removing one copy of draper. These data, taken together with sharkRNAi data, show that Shark is essential for the clearance of degenerating axons by glia (Ziegenfuss, 2008).

Is Shark required for the glial clearance of neuronal cell corpses? In embryonic stage 14-15 control animals, 24.4 cell corpses were found per hemisegment. In contrast, it was found that shark1 null mutants showed a marked increase in CNS cell corpses, with null mutants containing almost twice as many corpses per hemisegment. shark1/Df(2R)6063 mutants accumulate cell corpses at levels similar to those in shark1, indicating that this phenotype maps to shark. These cell corpse engulfment phenotypes are indistinguishable from that of draperδ5 mutants. It is concluded that Shark, like Draper, is also essential for the efficient clearance of embryonic neuronal cell corpses by glia (Ziegenfuss, 2008).

Because it was found that Shark binds Draper only in the presence of an active Src kinase in two-hybrid assays, Drosophila Src kinases were screened for roles in glial phagocytic activity. Interestingly, it was found that glia-specific knockdown of Src42A (src42ARNAi) potently suppress glial phagocytic activity: in src42ARNAi animals, Draper is not recruited to severed maxillary palp axons; glial hypertrophy and upregulation of Draper after antennal ablation was blocked; and GFP-labelled severed axons lingered in the CNS for 5 days. Knockdown of two other Drosophila Src kinases, Btk29A and Src64B, had no effect on the glial phagocytosis of severed axons. Thus, Src42A seems to be essential for all morphological responses of glia to axon injury and for the efficient clearance of degenerating axonal debris from the CNS (Ziegenfuss, 2008).

It was predicted that Draper phosphorylation status should be sensitive to the SFK inhibitor PP2. Indeed, addition of PP2 to S2 cultures led to a decrease in the phosphorylation of Draper and Draper-Shark association. Strikingly, co-transfection of Draper and Src42A led to a marked increase in Draper phosphorylation, which was PP2-sensitive and Draper-specific. Draper-Shark interactions are not dependent on Shark kinase activity because kinase-dead Shark (Shark K698R) associates with Draper. These data indicate that Src42A may phosphorylate the Draper intracellular domain, thereby increasing the association of Shark with Draper and the activation of downstream glial phagocytic signalling (Ziegenfuss, 2008).

This study has identified Shark and Src42A as novel components of the Draper pathway. One potential model for Draper-Shark-Src42A interactions is that Shark and Src42A drive the recruitment of Draper to engulfment targets. However, CED-1 has been shown to cluster around cell corpses even in the absence of its intracellular domain. Moreover, Zap-70 and Syk bind phosphorylated ITAM domains in mammalian immunoreceptors when ITAM domains are phosphorylated by Src after ligand-dependent receptor. Therefore a model is favoured in which the engagement of Draper with its ligand (presumably presented by engulfment targets) promotes receptor clustering, tyrosine phosphorylation of Draper by Src42A, association of Shark, and activation of downstream phagocytic signalling events. This work suggests that Draper is an ancient immunoreceptor in which the extracellular domain is tuned to recognize modified self and the intracellular domain signals through ITAM-Src-Syk-mediated mechanisms. This is the first identification of ITAM-Src-Syk signalling in invertebrates, and it suggests that a pathway similar to Draper-Ced-1 may ultimately have given rise to ITAM-based signalling cascades in mammalian myeloid and lymphoid cells, including those regulated by Fc, B-cell and T-cell receptors (Ziegenfuss, 2008).


DEVELOPMENTAL BIOLOGY

Tyrosine kinases, ankyrin repeats, and Src homology 2 domains play central roles in developmental processes. The cloning of a cDNA for Shark, a single protein that possesses all three domains, is described. During Drosophila embryogenesis, Shark is expressed exclusively by ectodermally derived epithelia and is localized preferentially to the apical surface of these cells. Initiating expression at the end of gastrulation, the most prominantly Shark positive structure is the cephalic furrow. Ectodermal structures associated with the ventral midline and the anterior and posterior gut ectoderm express Shark. Tracheal placodes and tracheal lumen express Shark, and expression in the trachea persists throughout its development. The stomatogastric nervous system precursors express Shark in a pattern that overlaps that of Crumbs. Gnathal structures including the labium, mandible and maxilla express Shark from the time they are visible as distinct protruberances. Apical localization of Shark persists, even as tissues undergo complex invaginations, moving from the external surface of embryos to form internal structures, but expression is lost when cells lose their polarity. This pattern closely mimics the expression of Crumbs, a protein necessary for proper organization of ectodermal epithelia. Shark's structure and localization pattern suggest that it functions in a signaling pathway for epithelial cell polarity, possibly transducing the Crumbs signal (Ferrante, 1995).

To localize the distribution of shark transcripts, whole-mount in situ hybridization was performed in wild-type embryos. Large amounts of shark mRNA are observed in ovarian nurse cells, unfertilized eggs, and syncitial blastoderm embryos. Owing to the abundant maternal contribution, shark message expression appears ubiquitous during early embryogenesis up until about stages 6-7. By the end of germ-band extension, shark mRNA is expressed primarily in the ectoderm. Following germ-band retraction, stages 12-15, the strongest shark mRNA expression occurs uniformly in the epidermis and other ectodermally derived structures such as the anterior foregut, hindgut, and Malphigian tubules. Most of the shark message is down-regulated by the end of embryogenesis. Protein expression was analyzed using an antiserum raised to the Shark N-SH2 domain. In general, staining with this antiserum paralleled the expression of shark mRNA. Shark protein is expressed abundantly from the onset of cellularization, resulting in ubiquitous expression until the commencement of gastrulation. From this stage on, its expression is most prominent in the ectoderm and some of the ectodermally derived tissues. At stage 10 (full germ-band extension) and thereafter, Shark protein expression increases again but is restricted to the ectoderm, foregut, hindgut, and Malphigian tubules. During germ-band extension (stages 9-10) and in migrating epidermal cells during DC, Shark protein expression is particularly strong at the LE of the epidermis. By the end of embryogenesis, epidermal expression decreases. Embryos homozygous for deficiency Df(2R)Jp7, which removes the shark locus, as well as shark1 homozygote embryos, show a reduction in immunoreactivity only after stages 13-14, consistent with a substantial contribution of maternal mRNA. In contrast, staining is reduced in embryos derived from shark1 germ-line clones (GLCs), consistent with the specificity of the antiserum for Shark, which could detect residual mutant protein. Also, in agreement with genetic evidence for a predominant role of Shark in the epidermis, late shark1 GLC-derived embryos consistently lack all immunoreactivity in epidermal cells. This pattern of Shark protein expression differed significantly from the previously reported immunolocalization of Shark protein with antibodies directed to the kinase domain (Ferrante, 1995), where poor pregastrulation and epidermal staining was seen that was contributed to by the dominance of apical staining of epithelia and staining of the tracheal system by one of three antibodies in the mixture of antipeptide antibodies used. This antibody was subsequently shown to detect an unrelated epitope (Fernandez, 2000).


EFFECTS OF MUTATION

Shark is required for embryonic dorsal closure

shark maps to polytene segment 53A1 (Ferrante, 1995) on the right arm of the second chromosome. Further molecular and genetic mapping has localized the shark locus to the 52F interval. The shark gene is found to be contained in a P1 clone (DS06638), cytologically mapped by the Berkeley Genome Project, to 52F2-52F8. In addition, independently mapped YAC clones were used to further confirm the chromosomal location. Clones DY506 (52F-53C) and DY664 (52E1-53A2), but not DYR15-76 (52D7-52E9), were shown by PCR and Southern blot analysis to contain the entire shark gene. In situ hybridization to polytene chromosomes with the P1 clone DS06638 indicates that shark is removed by deficiencies Df(2R)Jp7, Jp8, and Jp4 but not by Jp1, defining the position for the shark locus to a genetic interval between the proximal breakpoint of Df(2R)Jp8 and the distal breakpoint of DfJp(2R)P4 (Fernandez, 2000).

A search for recessive lethal mutations in the region uncovered by Df(2R)Jp8 identified 20 lethal complementation groups. Of these, 12 complementation groups were removed by Df(2R)Jp4 as well and represented candidate mutations in the shark locus. To determine if shark was represented by one of these lethal complementation groups and would therefore encode an essential function, transgenes expressing shark cDNA were introduced (under the control of a heat shock promoter) into flies mutant for each complementation group in trans to the deficiency. The lethality of the single mutant allele of complementation group W-4 was the only phenotype rescued when shark expression was induced by heat shock treatment. This was true for either of the two hs-shark transgenic lines, and therefore the rescued mutation was renamed shark1). Moreover, a hs-shark transgene encoding a catalytically inactive form (K698R) of Shark failed to rescue shark1 mutants, indicating that the Shark tyrosine kinase activity is required to rescue the mutant phenotype (Fernandez, 2000).

Analysis of the lethal phase of the single shark mutant recovered indicated that either shark1 homozygotes, or shark1/Df(2R)Jp7 or shark1/Df(2R)Jp4 trans-heterozygotes, results in pupal lethality. Sequencing of shark1 revealed the introduction of a stop codon at position 210 that predicts a protein containing only the first 210 amino acids of the shark ORF, which is likely to be nonfunctional, because two ankyrin repeats and the C-SH2, the proline-rich, and the tyrosine kinase domains are deleted completely. The truncation of the shark ORF encoded by this mutant allele confirms the identity of the W-4 complementation group as the shark locus (Fernandez, 2000).

Wild-type hs-shark transgene rescue experiments without heat shock provided suboptimal rescue conditions (defined as those that yield progeny below the 33% of adults expected for a fully viable genotype), which often gave rise to flies with a split thorax and/or hep-like defects. This indicates that rescue of lethality can occur at levels of Shark that are insufficient to support normal thoracic closure. These phenotypes also point to a role for Shark in processes in which the Jun kinase kinase hep and the fos homolog kay have been shown to be involved. Both hep and kay have been shown to be components of the JNK signaling module, which regulates cell elongation in epithelial cell sheets undergoing morphogenetic stretching. In addition to the split thorax defects, rescued shark1/Df(2R)Jp4; hs-shark females fertilized with sperm carrying the shark1 allele gave rise to embryos that showed a DC-defective phenotype. Like the split thorax phenotype, defects in embryonic DC have also been associated with loss of function in components of the JNK pathway that regulates dpp expression at the LE of the embryonic epidermis (Fernandez, 2000).

To investigate the phenotypic effects of the loss of Shark function, maternal Shark activity was removed by generating shark1 homozygote GLCs. Zygotes derived from GLCs with the shark1 mutation show a marked reduction in reactivity in anti-Shark immunostaining. Because shark1 is predicted to encode a truncated polypeptide containing the entire N-SH2 domain, which was the immunogen used to raise the antiserum in these experiments, the reduced immunoreactivity with the anti-GST/N-SH2 serum suggests that this allele produces a mutant protein that is expressed at low levels. This result is consistent with Western blot analysis of balanced shark1 mutant embryos and adults, which also show low levels of the truncated protein predicted to be encoded by shark1 (Fernandez, 2000).

Analysis of cuticles derived from shark1 homozygote GLCs fertilized with males of the genotypes shark1/CyO or Df(2R)Jp4/CyO demonstrates that loss of maternal and zygotic Shark activity yields a strong DC-defective phenotype, similar to embryos produced by partially rescued shark1/Df(2R)Jp4; hs-shark flies. Moreover, shark1 GLCs fertilized with sperm carrying either the shark1 allele or Df(2R)Jp7 give identical phenotypes, providing genetic evidence that shark1 is a null mutation. Finally, zygotic rescue of all cuticular defects via the paternal allele, which leads to the eventual production of viable, fertile adults, indicates that Shark activity is not required for early embryonic events such as organization of the cellular blastoderm or gastrulation (Fernandez, 2000).

Cuticles of maternal and zygotic shark null embryos are identical to those of strong alleles or maternal and zygotic loss-of-function alleles in hep, msn, bsk, kay, and D-jun mutants, among others. Cellular analysis of the epidermal cells in this group of mutants has revealed that the defect in cuticle secretion on the dorsal-anterior side, together with the failure to enclose the embryonic gut, is due to a defect in the directional elongation of the cells of the LE and lateral epidermis. Immunostaining of epidermal cells with monoclonal antibodies to the band 4.1 homolog protein Coracle reveals that shark1 has the same effect on epidermal cell function and epithelial cell morphogenesis as all other members of the JNK pathway. Both LE and lateral epidermal cells fail to stretch toward the dorsal midline over the anterior three-quarters of the embryo, consistent with the deficient cuticle secretion in the anterior-dorsal region (Fernandez, 2000).

This defect, evident in all epidermal cells, demonstrates a requirement of Shark for the elongation of LE cells and suggests a role for Shark in the regulation of Dpp transcription, or the production of other signals activated in LE cells that subsequently target elongation of adjacent lateral cells. Because Shark is expressed throughout the epidermis, it is possible that it also has an active role in the lateral cells, operating downstream or in parallel to the Dpp/Tkv-Put pathway. To investigate these possibilities, Dpp expression was analyzed by in situ hybridization in shark1 null embryos and the effects of reconstitution of the JNK and the Dpp pathways, as well as Shark function, in a shark1 null background were examined by using a combination of upstream activating sequence (UAS) transgenes and Gal4 driver lines specific for the LE region and lateral cells of the embryonic epidermis (Fernandez, 2000).

A comparison of the embryonic dpp mRNA expression by whole-mount in situ hybridization, between wild-type and shark1 GLCs, showed that Shark is required for Dpp expression by cells of the LE following germ-band retraction. From stage 12 on, Dpp expression is absent in LE cells of shark1 GLCs, whereas expression in the ventral cord and midgut, which occurs in a JNK-independent manner, remains unaffected (Fernandez, 2000).

Consistent with the Shark expression pattern, the shark1 DC phenotype is completely rescued by expression of UAS-shark under control of the 69BGal4 driver, which expresses ubiquitously throughout the ectoderm. Interestingly, however, expression of the LE region driver Pnr-Gal4 in the same UAS-shark line fails to rescue the shark1 DC defect. This result, with a driver that expresses in three to five rows of the most dorsal ectodermal cells, suggests that either Shark expression is also required in the lateral epidermis or that Shark is required for Pnr-Gal4 expression in the LE region (Fernandez, 2000).

Because no obvious genetic interactions were obtained between Shark and mutations of the JNK pathway, tests were performed to see whether constitutive activation of the JNK or the Dpp pathway could rescue the shark1 DC phenotype. When shark1 GLCs were generated in the background of flies carrying a shark1 chromosome with an inserted transposon expressing an activated form of c-Jun (hs-SEjunAsp), shark1 DC defects were completely rescued, in some cases, as determined by the decreased penetrance of embryonic lethality (~10% lower than the fully penetrant 50% observed without the expression of hs-SEjunAsp) and by the complete or partial enclosure observed in unhatched embryos. These results are consistent with the action of Shark upstream of bsk (JNK) in the JNK pathway in LE cells (Fernandez, 2000).

If Shark was required solely for Dpp expression in LE cells, expression of a constitutively activated Tkv receptor (Tkv*) under the control of the 69B driver throughout the ectoderm should rescue the shark1 DC phenotype. Although embryos expressing the combinations of UAS-Tkv* and 69B-Gal4 can be readily identified by the partial dorsalization of their cuticles, two distinctive kinds of partially dorsalized cuticles were observed: (1) those that should be expressing the combination of transgenes in a paternally rescued background and that showed complete enclosure (~12%), and (2) an equivalent percentage of embryos, showing marginal or no rescue of the DC defect, in spite of being dorsalized, demonstrating expression of both Tkv* and 69B-Gal4. Because msn and bsk mutations are rescued by 69B-Gal4-driven UAS-Tkv*, these findings raise the possibility that Shark may also function in an additional signal generating pathway in LE cells, in the lateral epidermis, or both (Fernandez, 2000).

Thus, Shark is essential for activation of the JNK pathway in LE cells. shark loss of function results in phenotypes similar to a subset of defects associated with loss of Dpp signaling activity in migrating ectodermal epithelial layers. Temperature-dependent rescue of the lethality of shark mutants with heat shock-driven cDNA transgenes resulted in dramatic adult phenotypes that suggested a role for Shark in morphogenesis of ectodermal epithelia. Adult flies expressing insufficient levels of Shark exhibit a split thorax phenotype, indicating a role for Shark activity in the dorsal joining of the wing imaginal epithelia derived from the left and right wing discs during pupal development. Unilateral deletions of wings and a cleft along the middle of the dorsal thorax have been reported in homozygous escapers of weak hep alleles and partially rescued kay mutants. Since both hep and kay are components of the JNK/Dpp/Tkv-Put pathway, shown to be essential for epidermal cell sheet elongation and fusion during embryonic DC, it has been proposed that wing disc epithelial stretching may be regulated by a JNK/Dpp/Tkv-Pnt pathway similar to the one operating in DC. Zeitlinger (1999) has formally demonstrated that thorax closure is controlled by the same molecular events that regulate DC, in which the JNK pathway promotes LE expression of Dpp and Puckered. Consistent with this, mutations that affect Dpp signaling show a split thorax and/or hep-type defect(s) under a variety of hypomorphic conditions that yield adult flies. For example, this is the case in viable tkv, mad, and med heteroallelic combinations, as well as in tkv6/tkv6 adults, whose phenotypes have been enhanced by loss of function in one copy of the TGFbeta-like 60A locus (also known as Glass bottom boat), mad, med, or put (Fernandez, 2000 and references therein).

Adult shark1 rescued females also show reduced Shark accumulation in developing eggs as evidenced by the production of shark1 homozygous embryos with strong DC zygotic defects. Considering that homozygous shark1 mutants die as pupae and the early preblastoderm expression of shark mRNA is high, it seems plausible that the maternally derived Shark activity is sufficient to facilitate embryonic development. The requirement of Shark for DC was ultimately confirmed by removing Shark activity through the generation of shark mutant GLCs. Even though the shark mutant phenotypes that have been described are consistent with loss of Dpp expression and/or failure of signaling by Dpp receptors, unlike some other mutations in these pathways -- dpp, tkv, put, mad, and med -- that affect pattern formation and cell determination during eye, wing, and leg imaginal disc development, the shark1 mutation does not seem to affect the development of imaginal disc-derived structures, aside from dorsal fusion of the thorax. That is, typical rough eye phenotypes, abnormalities in wing vein patterning, lack of distal tarsal segments in the adult legs, as well as dorsoventral patterning defects in the embryo, were not observed in shark1 mutants. Thus, as with other loci controlling the epidermal LE-specific JNK pathway, shark1 phenotypes are clearly restricted to cell migration and the stretching of ectodermal cell sheets. However, it remains possible that further reduction in Shark expression, below levels in the developing shark1 pupae, would lead to effects in other structures (Fernandez, 2000).

To date, two functionally different cell types have been defined among the ectodermal cells of the embryo that eventually differentiate into cuticle-secreting epidermis: (1) LE cells, and (2) the cells of the lateral epidermis. In response to extracellular cues believed to emanate from the underlying amnioserosa, ectodermal LE cells are specifically induced to secrete Dpp, from mid- to late-embryogenesis. LE expression of Dpp is not only essential to coordinate developmental processes within the ectoderm itself but also to promote patterning of the juxtaposing dorsal mesoderm. In addition to their ability to transduce a signaling cascade mediated by a SAPK/JNK cascade that leads to Dpp expression, the morphology of the LE cells is dramatically altered following germ-band retraction, when the LE cells elongate along the dorso-ventral axis as they migrate in the direction of the dorsal midline. Dpp secretion by LE cells triggers a response in the immediately adjacent cells of the epidermis, which results in the concerted elongation of the entire epidermis along the dorso-ventral axis and enclosure of the dorsal side of the embryo. This response in lateral epidermal cells is transduced by the type I and II heterodimeric Dpp receptors Tkv and Put. Analysis of the effect of the combined loss of maternal and zygotic shark function, with markers that reveal changes in epidermal cell shape and function in the developing embryo, indicate that elongation of all cells of the epidermis is equally affected. This results in a strong DC-defective cuticular phenotype that is caused, at least in part, by loss of Dpp expression by LE cells, as revealed by Dpp in situ hybridization (Fernandez, 2000).

However, apart from its involvement upstream of the JNK pathway in LE cells, transgenic rescue experiments suggest that Shark is either required in another pathway in the LE, in a pathway other than the Dpp pathway in the lateral epidermis, or both. Shark is expressed throughout the ectoderm, and expression of Shark driven by the 69B driver in both the LE and the lateral epidermis completely rescues the shark1 DC phenotype. Consistent with Shark regulation of JNK or JNK-like pathways, the shark1 DC phenotype is rescued by ubiquitous expression of activated c-Jun. The failure of 69B-Gal4-driven activated Tkv receptor to rescue the shark1 DC phenotype is consistent with the possible involvement of Shark in novel pathways in the lateral epidermis, or for the generation of LE signals in addition to Dpp. Additional investigation of these possibilities is warranted (Fernandez, 2000).

The current findings clearly indicate that Shark regulates Dpp expression in LE cells. The discovery that Shark is a member of this pathway should assist in the identification of additional upstream components. Recent studies indicate that a Drosophila c-src homolog, Src42A, also functions upstream of the JNK module in DC. Consistent with both observations, during DC, tyrosine phosphorylation is prominent at the dorsal border of LE cells and is also found along the cell peripheries in the lateral epidermis. It is speculated that Shark is an upstream regulator of multiple Ser/Thr protein kinase cascades, which regulate different biological effects, and that Shark transduces a signal(s) that is diversified in a manner analogous to receptor tyrosine kinase-mediated signaling that promotes pleiotropic effects in a variety of cell types (Fernandez, 2000).


EVOLUTIONARY HOMOLOGS

Structure of Shark homologs

The protein kinase ZAP-70 is involved in T-cell activation, and interacts with tyrosine-phosphorylated peptide sequences known as immunoreceptor tyrosine activation motifs (ITAMs), which are present in three of the subunits of the T-cell receptor. The tandem SH2 (tSH2) domains of ZAP-70 have been studied by both X-ray and NMR. The crystal structure of the apoprotein is presented, i.e., the tSH2 domain in the absence of ITAM. Comparison with the previously reported complex structure reveals that binding to the ITAM peptide induces surprisingly large movements between the two SH2 domains and within the actual binding sites. The conformation of the ITAM-free protein is partly governed by a hydrophobic cluster between the linker region and the C-terminal SH2 domain. The data suggest that the two SH2 domains are able to undergo large interdomain movements. The proposed relative flexibility of the SH2 domains is further supported by the finding that no NMR signals could be detected for the two helices connecting the SH2 domains; these are likely to be broadened beyond detection due to conformational exchange. It is likely that this conformational reorientation induced by ITAM binding is the main signaling event activating the kinase domain in ZAP-70. Another NMR observation is that the N-terminal SH2 domain can bind tetrapeptides derived from the ITAM sequence, apparently without the need to interact with the C-terminal domain. In contrast, the C-terminal domain has little affinity for tetrapeptides. The opposite situation is true for binding to plain phosphotyrosine, where the C-terminal domain has a higher affinity. Distinct features in the crystal structure, showing the interdependence of both domains, explain these binding data (Folmer, 2002).

Mutation of Shark homologs

Rheumatoid arthritis (RA), which afflicts about 1% of the world population, is a chronic systemic inflammatory disease of unknown aetiology that primarily affects the synovial membranes of multiple joints. Although CD4(+) T cells seem to be the prime mediators of RA, it remains unclear how arthritogenic CD4(+) T cells are generated and activated. Given that highly self-reactive T-cell clones are deleted during normal T-cell development in the thymus, abnormality in T-cell selection has been suspected as one cause of autoimmune disease. A spontaneous point mutation of the gene encoding an SH2 domain of ZAP-70, a key signal transduction molecule in T cells, causes chronic autoimmune arthritis in mice that resembles human RA in many aspects. Altered signal transduction from T-cell antigen receptor through the aberrant ZAP-70 changes the thresholds of T cells to thymic selection, leading to the positive selection of otherwise negatively selected autoimmune T cells. Thymic production of arthritogenic T cells due to a genetically determined selection shift of the T-cell repertoire towards high self-reactivity might also be crucial to the development of disease in a subset of patients with RA (Sakaguchi, 2003).

One of the earliest functional responses of T lymphocytes to extracellular signals that activate the Ag-specific CD3/TCR complex is a rapid, but reversible, increase in the functional activity of integrin adhesion receptors. Previous studies have implicated the tyrosine kinase zeta-associated protein of 70 kDa (ZAP-70) and the lipid kinase phosphatidylinositol 3-kinase, in the activation of beta(1) integrins by the CD3/TCR complex. Human ZAP-70-deficient Jurkat T cells have been used to demonstrate that the kinase activity of ZAP-70 is required for CD3/TCR-mediated increases in beta(1) integrin-mediated adhesion and activation of phosphatidylinositol 3-kinase. A tyrosine to phenylalanine substitution at position 315 in the interdomain B of ZAP-70 inhibits these responses, whereas a similar substitution at position 292 enhances these downstream signals. These mutations in the ZAP-70 interdomain B region also specifically affect CD3/TCR-mediated tyrosine phosphorylation of residues 171 and 191 in the cytoplasmic domain of the linker for activation of T cells (LAT) adapter protein. CD3/TCR signaling to beta(1) integrins is defective in LAT-deficient Jurkat T cells, and can be restored with expression of wild-type LAT. Mutant LAT constructs with tyrosine to phenylalanine substitutions at position 171 and/or position 191 do not restore CD3/TCR-mediated activation of beta(1) integrins in LAT-deficient T cells. Thus, these studies demonstrate that the interdomain B region of ZAP-70 regulates beta(1) integrin activation by the CD3/TCR via control of tyrosine phosphorylation of tyrosine residues 171 and 191 in the LAT cytoplasmic domain (Goda, 2004).

Shark homologs in invertebrates

Segments of several protein-tyrosine kinase genes from Hydra vulgaris, a member of the ancient metazoan phylum Cnidaria, were amplified using the polymerase chain reaction with primers corresponding to conserved regions in the kinase domain of protein-tyrosine kinases. Characterization of cDNA clones for one of these genes, HTK16, revealed that it encodes a non-receptor protein-tyrosine kinase with two SH2 domains but no SH3 domain. In this regard the predicted HTK16 protein resembles two mammalian non-receptor protein-tyrosine kinases, the products of the ZAP-70 and syk genes. However, the HTK16 protein contains five ankyrin-like repeats, a structural motif that had not previously been found in protein-tyrosine kinases. The HTK16 protein also contains a potential tyrosine phosphorylation site in its carboxyl-terminal tail which resembles the phosphorylation site in members of the src family. RNA hybridization analysis indicates that the HTK16 gene is expressed in epithelial cells, cells which also express the Hydra homolog of the src protein. This finding of the HTK16 gene in Hydra indicates that diversification of genes encoding non-receptor protein-tyrosine kinases was a very early event in metazoan evolution (Chan, 1994).

Integrin signaling and Shark homologs

The beta1 integrins are a family of heterodimeric adhesion receptors involved in cell-to-cell contacts and cell-to-extracellular matrix interactions. Through their adhesive role, integrins participate in transduction of outside/inside signals and contribute to trigger a multitude of cellular events such as differentiation, cell activation, and motility. The fibronectin integrin receptors, alpha4beta1 and alpha5beta1, can function as costimulatory molecules in T-cell receptor (TCR)-dependent T-cell activation. The Jurkat T-cell line was used as a model system to investigate the TCR-independent role of cell adhesion to fibronectin in the activation of Zap-70, a central molecule in the signalling events in T cells. Upon adhesion to plastic immobilized fibronectin but not to bovine serum albumin (BSA) the phosphorylation of p125FAK, a protein kinase that localizes to focal adhesion sites, is induced. Moreover, clustering of fibronectin receptors leads to the detection of a p125FAK/Zap-70 complex. Finally, while the complex between fak-B, another protein kinase localized to focal adhesion sites, and Zap-70 is detected in cells plated either on BSA or on fibronectin, the formation of the p125FAK/Zap-70 complex appears specifically induced following fibronectin-mediated integrin clustering. These data suggest the existence of a high degree of specificity when the members of the beta1 integrin family mediate signalling pathways in T cells (Bearz, 1999).

Syk protein tyrosine kinase is essential for immune system development and function and for the maintenance of vascular integrity. In leukocytes, Syk is activated by binding to diphosphorylated immune receptor tyrosine-based activation motifs (pITAMs). Syk can also be activated by integrin adhesion receptors, but the mechanism of its activation is unknown. A novel mechanism is reported for Syk's recruitment and activation, which requires that Syk bind to the integrin beta3 cytoplasmic tail. Both Syk and the related kinase ZAP-70 bind the beta3 cytoplasmic tail through their tandem SH2 domains. However, unlike Syk binding to pITAMs, this interaction is independent of tyrosine phosphorylation and of the phosphotyrosine binding function of Syk's tandem SH2 domains. Deletion of the four C-terminal residues of the beta3 cytoplasmic tail [beta3(759X)] decreases Syk binding and disruptes its physical association with integrin alphaIIbbeta3. Furthermore, cells expressing alphaIIbbeta3(759X) fail to exhibit Syk activation or lamellipodia formation upon cell adhesion to the alphaIIbbeta3 ligand, fibrinogen. In contrast, FAK phosphorylation and focal adhesion formation are unimpaired by this mutation. Thus, the direct binding of Syk kinase to the integrin beta3 cytoplasmic tail is a novel and functionally significant mechanism for the regulation of this important non-receptor tyrosine kinase (Woodside, 2001).

Syk and ZAP-70 form a subfamily of nonreceptor tyrosine kinases that contain tandem SH2 domains at their N termini. Engagement of these SH2 domains by tyrosine-phosphorylated immunoreceptor tyrosine-based activation motifs leads to kinase activation and downstream signaling. These kinases are also regulated by beta3 integrin-dependent cell adhesion via a phosphorylation-independent interaction with the beta3 integrin cytoplasmic domain. The interaction of integrins with Syk and ZAP-70 depends on the N-terminal SH2 domain and the interdomain A region of the kinase. The N-terminal SH2 domain alone is sufficient for weak binding, and this interaction is independent of tyrosine phosphorylation of the integrin tail. Indeed, phosphorylation of tyrosines within the two conserved NXXY motifs in the integrin beta3 cytoplasmic domain blocks Syk binding. The tandem SH2 domains of these kinases bind to multiple integrin beta cytoplasmic domains with varying affinities [beta3 (Kd = 24 nm) > beta2 (Kd = 38 nm) > beta1 (Kd = 71 nm)] as judged by both affinity chromatography and surface plasmon resonance. Thus, the binding of Syk and ZAP-70 to integrin beta cytoplasmic domains represents a novel phosphotyrosine-independent interaction mediated by their N-terminal SH2 domains (Woodside, 2002).

The T-cell receptor and Shark homologs

Stimulation of the T-cell antigen receptor (TCR) leads to tyrosine phosphorylation of a number of cellular proteins, including phospholipase C (PLC) gamma 1 and the TCR zeta chain. A 70-kDa tyrosine phosphoprotein (ZAP-70) is describes that associates with zeta within 15 sec following TCR stimulation. The phosphorylation of ZAP-70 and its association with zeta is independent of the other TCR chains since stimulation of a functional CD8/zeta chimeric receptor in a TCR-negative T cell leads to coprecipitation of ZAP-70 with the chimeric protein. In a Jurkat cell expressing the TCR and the CD8/zeta chimeric protein, tyrosine phosphorylation and association of ZAP-70 occurs exclusively with the stimulated receptor complex. In addition, a tyrosine kinase that does not appear to be fyn associates with the cytoplasmic domain of zeta and phosphorylates zeta and ZAP-70 in vitro (Chan, 1991).

SAP-1 is a transmembrane-type protein-tyrosine phosphatase that is expressed in most tissues but whose physiological functions remain unknown. The cytoplasmic region of SAP-1 has now been shown to bind directly the tyrosine kinase Lck. Overexpression of wild-type SAP-1, but not that of a catalytically inactive mutant of SAP-1, inhibited both the basal and the T cell antigen receptor (TCR)-stimulated activity of Lck in human Jurkat T cell lines. Lck serves as a direct substrate for dephosphorylation by SAP-1 in vitro. Overexpression of wild-type SAP-1 in Jurkat cells also: (1) inhibits both the activation of mitogen-activated protein kinase and the increase in cell surface expression of CD69 induced by TCR stimulation; (2) reduces the extent of the TCR-induced increase in the tyrosine phosphorylation of ZAP-70 or that of LAT; (3) reduces both the basal level of tyrosine phosphorylation of p62dok, as well as the increase in the phosphorylation of this protein induced by CD2 stimulation, and (4) inhibits cell migration. These results thus suggest that the direct interaction of SAP-1 with Lck results in inhibition of the kinase activity of the latter and a consequent negative regulation of T cell function (Ito, 2003).

Regulation of protein tyrosine kinases (PTKs) by tyrosine phosphorylation is well recognized; in fact, nearly all PTKs require phosphorylation of tyrosine residues in their 'activation loop' for catalytic activity. In contrast, the phosphorylation of PTKs on serine and threonine residues has not been studied nearly as much. The ZAP-70 PTK contains predominately phosphoserine in normal T lymphocytes as well as in Jurkat T leukemia cells. One site of phosphorylation has been identified as Ser-520; this site is important for the recruitment and activation of ZAP-70 in T cells. Mutant ZAP-70-S520A has reduced ability to autophosphorylate and to mediate antigen receptor-induced interleukin 2 gene activation and is not enriched at the plasma membrane. These defects are rescued by addition of a myristylation signal to the N terminus of ZAP-70-S520A to force its plasma membrane and lipid raft localization. It is concluded that phosphorylation of ZAP-70 at Ser-520 plays an important role in the correct localization of ZAP-70 and in priming ZAP-70 for its acute recruitment and activation upon antigen receptor ligation (Yang, 2003).


REFERENCES

Search PubMed for articles about Drosophila shark

Bearz, A., Tell, G., Formisano, S., Merluzzi, S., Colombatti, A. and Pucillo, C. (1999). Adhesion to fibronectin promotes the activation of the p125(FAK)/Zap-70 complex in human T cells. Immunology 98: 564-568. 10594689

Chan, A. C., Irving, B. A., Fraser, J. D. and Weiss, A. (1991). The zeta chain is associated with a tyrosine kinase and upon T-cell antigen receptor stimulation associates with ZAP-70, a 70-kDa tyrosine phosphoprotein. Proc. Natl. Acad. Sci. 88: 9166-9170. 1717999

Chan, T. A., Chu, C. A., Rauen, K. A., Kroiher, M., Tatarewicz, S. M. and Steele, R. E. (1994). Identification of a gene encoding a novel protein-tyrosine kinase containing SH2 domains and ankyrin-like repeats. Oncogene 9: 1253-1259. 8134129

Chu, D. H., Morita, C. T. and Weiss, A. (1998). The Syk family of protein tyrosine kinases in T-cell activation and development. Immunol. Rev. 165: 167-180. 9850860

Dorman, J. B., James, K. E., Fraser, S. E., Kiehart, D. P. and Berg, C. A. (2004). bullwinkle is required for epithelial morphogenesis during Drosophila oogenesis. Dev. Biol. 267(2): 320-41. 1501379

Fernandez, R., Takahashi, F., Liu, Z., Steward, R., Stein, D. and Stanley, E. R. (2000). The Drosophila Shark tyrosine kinase is required for embryonic dorsal closure. Genes Dev. 14: 604-614. 10716948

Ferrante, Jr., A. W., Reinke, R. and Stanley, E. R. (1995). Shark, a Src homology 2, ankyrin repeat, tyrosine kinase, is expressed on the apical surfaces of ectodermal epithelia. Proc. Natl. Acad. Sci. 92: 1911-1915. 7892198

Folmer, R. H., Geschwindner, S. and Xue, Y. (2002). Crystal structure and NMR studies of the apo SH2 domains of ZAP-70: two bikes rather than a tandem. Biochemistry 41(48): 14176-84. 12450381

Goda, S., Quale, A. C., Woods, M. L., Felthauser, A. and Shimizu, Y. (2004). Control of TCR-mediated activation of beta 1 integrins by the ZAP-70 tyrosine kinase interdomain B region and the linker for activation of T cells adapter protein. J. Immunol. 172(9): 5379-87. 15100278

Ito, T., et al. (2003). Interaction of SAP-1, a transmembrane-type protein-tyrosine phosphatase, with the tyrosine kinase Lck. Roles in regulation of T cell function. J. Biol. Chem. 278(37): 34854-63. 12837766

Sakaguchi, N., et al. (2003). Altered thymic T-cell selection due to a mutation of the ZAP-70 gene causes autoimmune arthritis in mice. Nature 426(6965): 454-60. 14647385

Soede, R. D., Wijnands, Y. M., van Kouteren-Cobzaru, I. and Roos, E. (1998). ZAP-70 tyrosine kinase is required for LFA-1-dependent T cell migration. J. Cell Biol. 142: 1371-1379. 9732296

Suga, H., Koyanagi, M., Hoshiyama, D., Ono, K., Iwabe, N., Kuma, K. and Miyata, T. (1999). Extensive gene duplication in the early evolution of animals before the parazoan-eumetazoan split demonstrated by G proteins and protein tyrosine kinases from sponge and hydra. J. Mol. Evol. 48: 646-653. 10229568

Taniguchi, T., Kobayashi, T., Kondo, J., Takahashi, K., Nakamura, H., Suzuki, J., Nagai, K., Yamada, T., Nakamura, S. and Yamamura, H. (1991). Molecular cloning of a porcine gene syk that encodes a 72-kDa protein-tyrosine kinase showing high susceptibility to proteolysis. J. Biol. Chem. 266: 15790-15796. 1874735

Tran, D. H. and Berg, C. A. (2003). bullwinkle and shark regulate dorsal-appendage morphogenesis in Drosophila oogenesis. Development 130: 6273-6282. 14602681

Woodside, D. G., et al. (2001). Activation of Syk protein tyrosine kinase through interaction with integrin beta cytoplasmic domains. Curr. Biol. 11(22): 1799-804. 11719224

Woodside, D. G., Obergfell, A., Talapatra, A., Calderwood, D. A., Shattil, S. J. and Ginsberg, M. H. (2002). The N-terminal SH2 domains of Syk and ZAP-70 mediate phosphotyrosine-independent binding to integrin beta cytoplasmic domains. J. Biol. Chem. 277(42): 39401-8. 12171941

Yang, Y., Villain, P., Mustelin, T. and Couture, C. (2003). Critical role of Ser-520 phosphorylation for membrane recruitment and activation of the ZAP-70 tyrosine kinase in T cells. Mol. Cell. Biol. 23(21): 7667-77. 14560012

Zeitlinger, J. and D. Bohmann. (1999). Thorax closure in Drosophila: Involvement of Fos and the JNK pathway. Development 126: 3947-3956. 10433922


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date revised: 12 January 2018

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