Computational and experimental analysis reveals a novel Src family kinase in the C. elegans genome

MOTIVATION: The complete genomes of a number of organisms have already been sequenced. However, the vast majority of annotated genes are derived by gene prediction methods. It is important to not only validate the predicted coding regions but also to identify genes that may have been missed by these programs. METHODS: We searched the entire C.elegans genomic sequence database maintained by the Sanger Center using human c-Src sequence in a TBLASN search. We have confirmed one of the predicted regions by isolation of a cDNA and carried out a phylogenetic analysis of Src kinase family members in the worm, fly and several vertebrate species. RESULTS: Our analysis identified a novel tyrosine kinase in the C.elegans genome that contains functional features typical of the Src family kinases that we have designated as Src-1. The open reading frame contains a conserved N-terminal myristoylation site and a tyrosine residue within the C-terminus that is crucial for regulating the activity of Src kinases. Our phylogenetic analysis of Src family members from C. elegans, Drosophila and other higher organisms revealed a relationship among Src kinases from C. elegans and Drosophila.     

The non-receptor tyrosine kinase Syk is a target of Cbl-mediated ubiquitylation upon B-cell receptor stimulation.

The negative regulator Cbl functions as a ubiquitin ligase towards activated receptor tyrosine kinases and facilitates their transport to lysosomes. Whether Cbl ubiquitin ligase activity mediates its negative regulatory effects on cytoplasmic tyrosine kinases of the Syk/ZAP-70 family has not been addressed, nor is it known whether these kinases are regulated via ubiquitylation during lymphocyte B-cell receptor engagement. Here we show that B-cell receptor stimulation in Ramos cells induces the ubiquitylation of Syk tyrosine kinase which is inhibited by a dominant-negative mutant of Cbl. Intact tyrosine kinase-binding and RING finger domains of Cbl were found to be essential for Syk ubiquitylation in 293T cells and for in vitro Syk ubiquitylation. These same domains were also essential for Cbl-mediated negative regulation of Syk as measured using an NFAT-luciferase reporter in a lymphoid cell. Association with Cbl did not alter the kinase activity of Syk. Altogether, our results support an essential role for Cbl ubiquitin ligase activity in the negative regulation of Syk, and establish that ubiquitylation provides a mechanism of Cbl-mediated negative regulation of cytoplasmic targets.     

Drosophila Dok is required for embryonic dorsal closure

Embryonic dorsal closure (DC) in Drosophila is a series of morphogenetic movements involving the bilateral dorsal movement of the epidermis (cell stretching) and dorsal suturing of the leading edge (LE) cells to enclose the viscera. The Syk family tyrosine kinase Shark plays a crucial role in this Jun amino-terminal kinase (JNK)-dependent process, where it acts upstream of JNK in LE cells. Using a yeast two-hybrid screen, the unique Drosophila homolog of the downstream of kinase (Dok) family, Ddok, was identified by its ability to bind Shark SH2 domains in a tyrosine phosphorylation-dependent fashion. In cultured S2 embryonic cells, Ddok tyrosine phosphorylation is Src dependent; Shark associates with Ddok and Ddok localizes at the cell cortex, together with a portion of the Shark protein. The embryonic expression pattern of Ddok resembles the expression pattern of Shark. Ddok loss-of-function mutant (Ddok(PG155)) germ-line clones possess DC defects, including the loss of JNK-dependent expression of dpp mRNA in LE cells, and decreased epidermal F-actin staining and LE actin cable formation. Epistatic analysis indicates that Ddok functions upstream of shark to activate JNK signaling during DC. Consistent with these observations, Ddok mutant embryos exhibit decreased levels of tyrosine phosphorylated Shark at the cell periphery of LE and epidermal cells. As there are six mammalian Dok family members that exhibit some functional redundancy, analysis of the regulation of DC by Ddok is expected to provide novel insights into the function of the Dok adapter proteins.     

The Caenorhabditis elegans homologue of thioredoxin reductase contains a selenocysteine insertion sequence (SECIS) element that differs from mammalian SECIS elements but directs selenocysteine incorporation.

Thioredoxin reductases (TRR) serve critical roles in maintaining cellular redox states. Two isoforms of TRR have been identified in mammals: both contain a penultimate selenocysteine residue that is essential for catalytic activity. A search of the genome of the invertebrate, Caenorhabditis elegans, reveals a gene highly homologous to mammalian TRR, with a TGA selenocysteine codon at the corresponding position. A selenocysteyl-tRNA was identified in this organism several years ago, but no selenoproteins have been identified experimentally. Herein we report the first identification of a C. elegans selenoprotein. By (75)Se labeling of C. elegans, one major band was identified, which migrated with the predicted mobility of the C. elegans TRR homologue. Western analysis with an antibody against human TRR provides strong evidence for identification of the C. elegans selenoprotein as a member of the TRR family. The 3'-untranslated region of this gene contains a selenocysteine insertion sequence (SECIS) element that deviates at one position from the previously invariant consensus "AUGA." Nonetheless, this element functions to direct selenocysteine incorporation in mammalian cells, suggesting conservation of the factors recognizing SECIS elements from worm to man.     

The Src,Syk, and Tec family kinases: Distinct types of molecular switches

The Src, Syk, and Tec family kinases are three of the most well characterized tyrosine kinase families found in the human genome. Members of these kinase families function downstream of antigen and F_c receptors in hematopoietic cells and transduce signals leading to calcium mobilization, altered gene expression, cytokine production, and cell proliferation. Over the last several years, structural and biochemical studies have begun to uncover the molecular mechanisms regulating activation of these kinases. It appears that each kinase family functions as a distinct type of molecular switch. This review discusses the activation of the Src, Syk, and Tec kinases from the perspective of structure, phosphorylation, allosteric regulation, and kinetics. The multiple factors that regulate the Src, Syk, and Tec families illustrate the important role played by each of these kinases in immune cell signaling.     

Nonreceptor Protein-Tyrosine Kinases in Neutrophil Activation

Nonreceptor protein-tyrosine kinases are involved in the regulation of almost all neutrophil responses such as adhesion, chemotaxis, priming, oxidative burst, and degranulation. Here, we show that phagocytosis is also regulated by protein-tyrosine kinase activity. Using various protein-tyrosine kinase inhibitors, we further demonstrate that opsonized zymosan-induced degranulation of specific and azurophil granules is regulated by protein-tyrosine kinase activity, whereas phorbol ester-induced degranulation is not. Several of the nonreceptor protein-tyrosine kinases involving in neutrophil signal transduction are known, including Fgr, Hck, Lyn, Yes, and Syk. Among these, Hck and Fgr are localized on the azurophil and specific granules, suggesting the involvement of these two protein-tyrosine kinases in the regulation of degranulation. In this report, we characterize some of the molecular properties of Hck and Fgr. We discuss the methods generally used for the measurement of protein-tyrosine kinase activities in neutrophils highlighting precautions against proteolysis. In addition, we show that in subcellular fractions of retinoic acid-differentiated neutrophil-like NB4 cells, the 59- and 61-kDa forms of Hck are attached to the membranes of their respective compartments by different mechanisms. Finally, we discuss the functional roles of protein-tyrosine kinases in the regulation of neutrophil activation and speculate on the importance of their subcellular localization.     

Molecular features underlying the sequential phosphorylation of HS1 protein and its association with c-Fgr protein-tyrosine kinase

The hematopoietic lineage cell-specific protein HS1 was shown to undergo a process of sequential phosphorylation both in vitro and in vivo, which is synergistically mediated by Syk and Src family protein-tyrosine kinases and essential for B cell antigen receptor-mediated apoptosis. We have now identified tyrosine 222 as the HS1 residue phosphorylated by the Src family protein kinases c-Fgr and Lyn, and we show that a truncated form of HS1 (HS1-208-401) lacking the N-terminal putative DNA binding region and the C-terminal Src homology 3 (SH3) domain is still able to undergo all the steps of sequential phosphorylation as efficiently as full-length HS1. We also show that a stable association of phospho-HS1 with c-Fgr through its SH2 domain requires previous autophosphorylation of the kinase and is prevented by subsequent phosphorylation of Tyr-222. Kinetic studies with HS1 and its truncated forms previously phosphorylated by Syk and with a peptide substrate reproducing the sequence around tyrosine 222 support the view that efficient phosphorylation of HS1 by Src family protein kinases entirely relies on TyrP-SH2 domain interaction with negligible, if any, contribution of local specificity determinants. Our data indicate that the proline-rich region of HS1 bordered by tyrosyl residues affected by Syk and Src family kinases represents a functional domain designed to undergo a process of sequential phosphorylation.     

Phosphorylation and recruitment of Syk by immunoreceptor tyrosine-based activation motif-based phosphorylation of tamalin

Tamalin is a scaffold protein that forms a multiple protein assembly including metabotropic glutamate receptors (mGluRs) and several postsynaptic and protein-trafficking scaffold proteins in distinct mode of protein-protein association. In the present investigation, we report that tamalin possesses a typical immunoreceptor tyrosine-based activation motif (ITAM), which enables Syk kinase to be recruited and phosphorylated by the Src family kinases. Coimmunoprecipitation analysis of rat brain membrane fractions showed that tamalin is present in a multimolecular protein assembly comprising not only mGluR1 but also c-Src, Fyn, and a protein phosphatase, SHP-2. The protein association of both tamalin and c-Src, as determined by truncation analysis of mGluR1 in COS-7 cells, occurred at the carboxyl-terminal tail of mGluR1. Mutation analysis of tyrosine with phenylalanine in COS-7 cells revealed that paired tyrosines at the ITAM sequence of tamalin are phosphorylated preferentially by c-Src and Fyn, and this phosphorylation can recruit Syk kinase and enables it to be phosphorylated by the Src family kinases. The phosphorylated tyrosines at the ITAM sequence of tamalin were highly susceptible to dephosphorylation by protein-tyrosine phosphatases in COS-7 cells. Importantly, tamalin was endogenously phosphorylated and associated with Syk in retinoic acid-treated P19 embryonal carcinoma cells that undergo neuron-like differentiation. The present investigation demonstrates that tamalin is a novel signaling molecule that possesses a PDZ domain and a PDZ binding motif and mediates Syk signaling in an ITAM-based fashion.     

Molecular basis for a direct interaction between the Syk protein-tyrosine kinase and phosphoinositide 3-kinase

After engagement of the B cell receptor for antigen, the Syk protein-tyrosine kinase becomes phosphorylated on multiple tyrosines, some of which serve as docking sites for downstream effectors with SH2 or other phosphotyrosine binding domains. The most frequently identified binding partner for catalytically active Syk identified in a yeast two-hybrid screen was the p85 regulatory subunit of phosphoinositide 3-kinase. The C-terminal SH2 domain of p85 was sufficient for mediating an interaction with tyrosine-phosphorylated Syk. Interestingly, this domain interacted with Syk at phosphotyrosine 317, a site phosphorylated in trans by the Src family kinase, Lyn, and identified previously as a binding site for c-Cbl. This site interacted preferentially with the p85 C-terminal SH2 domain compared with the c-Cbl tyrosine kinase binding domain. Molecular modeling studies showed a good fit between the p85 SH2 domain and a peptide containing phosphotyrosine 317. Tyr-317 was found to be essential for Syk to support phagocytosis mediated by FcgammaRIIA receptors expressed in a heterologous system. These studies establish a new type of p85 binding site that can exist on proteins that serve as substrates for Src family kinases and provide a molecular explanation for observations on direct interactions between Syk and phosphoinositide 3-kinase.     

Reconstitution of Btk signaling by the atypical tec family tyrosine kinases Bmx and Txk

Bruton's tyrosine kinase (Btk) is mutated in X-linked agammaglobulinemia patients and plays an essential role in B cell receptor signal transduction. Btk is a member of the Tec family of nonreceptor protein-tyrosine kinases that includes Bmx, Itk, Tec, and Txk. Cell lines deficient for Btk are impaired in phospholipase C-gamma2 (PLCgamma2)-dependent signaling. Itk and Tec have recently been shown to reconstitute PLCgamma2-dependent signaling in Btk-deficient human cells, but it is not known whether the atypical Tec family members, Bmx and Txk, can reconstitute function. Here we reconstitute Btk-deficient DT40 B cells with Bmx and Txk to compare their function with other Tec kinases. We show that in common with Itk and Tec, Bmx reconstituted PLCgamma2-dependent responses including calcium mobilization, extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activation, and apoptosis. Txk also restored PLCgamma2/calcium signaling but, unlike other Tec kinases, functioned in a phosphatidylinositol 3-kinase-independent manner and failed to reconstitute apoptosis. These results are consistent with a common role for Tec kinases as amplifiers of PLCgamma2-dependent signal transduction, but suggest that the pleckstrin homology domain of Tec kinases, absent in Txk, is essential for apoptosis.     

The FER gene is evolutionarily conserved and encodes a widely expressed member of the FPS/FES protein-tyrosine kinase family.

We have recently isolated human and rat cDNAs (designated FER and flk, respectively) which encode nonreceptor protein-tyrosine kinases which are very similar to one another and related in sequence and domain structure to the c-fps/fes gene product. We show that FER and flk are human and rat counterparts of an evolutionarily conserved gene, hereafter termed FER regardless of species. The human and rat FER genes encode a widely expressed 94-kilodalton protein-tyrosine kinase which is antigenically related to the fps/fes protein-tyrosine kinase. The structural and antigenic similarities between the FER and fps/fes proteins suggest that they are members of a new family of nonreceptor protein-tyrosine kinases.     

cDNA cloning and characterization of eck, an epithelial cell receptor protein-tyrosine kinase in the eph/elk family of protein kinases.

A human epithelial (HeLa) cDNA library was screened with degenerate oligonucleotides designed to hybridize to highly conserved regions of protein-tyrosine kinases. One cDNA from this screen was shown to contain a putative protein-tyrosine kinase catalytic domain and subsequently used to isolate another cDNA from a human keratinocyte library that encompasses the entire coding region of a 976-amino-acid polypeptide. The predicted protein has an external domain of 534 amino acids with a presumptive N-terminal signal peptide, a transmembrane domain, and a cytoplasmic domain of 418 amino acids that includes a canonical protein-tyrosine kinase catalytic domain. Molecular phylogeny indicates that this protein kinase is closely related to eph and elk and that this receptor family is more closely related to the non-receptor protein-tyrosine kinase families than to other receptor protein-tyrosine kinases. Antibodies raised against a TrpE fusion protein immunoprecipitated a 130-kDa protein that became phosphorylated on tyrosine in immune complex kinase assays, indicating that this protein is a bona fide protein-tyrosine kinase. Analysis of RNA from 13 adult rat organs showed that the eck gene is expressed most highly in tissues that contain a high proportion of epithelial cells, e.g., skin, intestine, lung, and ovary. Several cell lines of epithelial origin were found to express the eck protein kinase at the protein and RNA levels. Immunohistochemical analysis of several rat organs also showed staining in epithelial cells. These observations prompted us to name this protein kinase eck, for epithelial cell kinase.     

Src-dependent Syk activation controls CD69-mediated signaling and function on human NK cells

CD69 C-type lectin receptor represents a functional triggering molecule on activated NK cells, capable of directing their natural killing function. The receptor-proximal signaling pathways activated by CD69 cross-linking and involved in CD69-mediated cytotoxic activity are still poorly understood. Here we show that CD69 engagement leads to the rapid and selective activation of the tyrosine kinase Syk, but not of the closely related member of the same family, ZAP70, in IL-2-activated human NK cells. Our results indicate the requirement for Src family kinases in the CD69-triggered activation of Syk and suggest a role for Lck in this event. We also demonstrate that Syk and Src family tyrosine kinases control the CD69-triggered tyrosine phosphorylation and activation of phospholipase Cgamma2 and the Rho family-specific exchange factor Vav1 and are responsible for CD69-triggered cytotoxicity of activated NK cells. The same CD69-activated signaling pathways are also observed in an RBL transfectant clone, constitutively expressing the receptor. These data demonstrate for the first time that the CD69 receptor functionally couples to the activation of Src family tyrosine kinases, which, by inducing Syk activation, initiate downstream signaling pathways and regulate CD69-triggered functions on human NK cells.     

Neurotrophic activities of trk receptors conserved over 600 million years of evolution

The trk family of receptor tyrosine kinases is crucial for neuronal survival in the vertebrate nervous system, however both C. elegans and Drosophila lack genes encoding trks or their ligands. The only invertebrate representative of this gene family identified to date is Ltrk from the mollusk Lymnaea. Did trophic functions of trk receptors originate early in evolution, or were they an innovation of the vertebrates? Here we show that the Ltrk gene conserves a similar exon/intron order as mammalian trk genes in the region encoding defined extracellular motifs, including one exon encoding a putative variant immunoglobulin-like domain. Chimeric receptors containing the intracellular and transmembrane domains of Ltrk undergo ligand-induced autophosphorylation followed by MAP kinase activation in transfected cells. The chimeras are internalized similarly to TrkA in PC12 cells, and their stimulation leads to differentiation and neurite extension. Knock-down of endogenous Ltrk expression compromises outgrowth and survival of Lymnaea neurons cultured in CNS-conditioned medium. Thus, Ltrk is required for neuronal survival, suggesting that trophic activities of the trk receptor family originated before the divergence of molluscan and vertebrate lineages approximately 600 million years ago.