Major Phosphotyrosine-containing Protein Research Articles

Genomic organization, transcription start sites, and chromosomal location of the Drosophila cortactin gene.

An actin filament binding protein cortactin was initially identified as a major phosphotyrosine-containing protein in v-Src-transformed chicken embryo fibroblast cells. The mouse, human, and Drosophila homologs were independently identified as a signaling molecule involved in a mitogenic response, as a product of a putative oncogene EMS1, and as a molecule interacting with a scaffolding protein ZO-1, respectively. In this report, we describe the cloning of the Drosophila cortactin gene, which consists of four exons and three introns, covering 3 kilobases in length. All exon-intron junctions are well matched with the GT/AG consensus sequence. S1 nuclease mapping revealed one major and several minor transcription start sites. The cytological location of the Drosophila cortactin gene is between chromosome segments 93B3 and 93B7.

Requirement for focal adhesion kinase in tumor cell adhesion.

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase and a major phosphotyrosine-containing protein. FAK is found in cell-matrix attachment sites (focal adhesions), and is activated on integrin-ligand binding and by other signaling pathways. Several roles have been proposed for FAK; here we report a novel function. We observed abundant FAK protein in all human melanoma cell lines tested except COLO839, a line that grows predominantly in suspension and was derived from peripheral blood. Five adherent lines, isolated from solid metastases in the same patient as COLO839, did express FAK. We derived four adherent sublines from COLO839. These did express FAK, even when plated on bacteriological plastic, to which they did not adhere. Thus, substrate attachment was not required for FAK expression. Three of the adherent sublines were then grown in the presence of antisense oligonucleotides to the initial FAK coding sequence. All showed substantially reduced FAK expression and, interestingly, the cells largely detached from the substrate while continuing to grow. Similar results were obtained with an independent melanoma line, DX3. Thus, FAK expression appears to be required by melanoma cells for substrate adhesion.

Characterisation of alpha-dystrobrevin in muscle.

Dystrophin-related and associated proteins are important for the formation and maintenance of the mammalian neuromuscular junction. Initial studies in the electric organ of Torpedo californica showed that the dystrophin-related protein dystrobrevin (87K) co-purifies with the acetylcholine receptors and other postsynaptic proteins. Dystrobrevin is also a major phosphotyrosine-containing protein in the postsynaptic membrane. Since inhibitors of tyrosine protein phosphorylation block acetylcholine receptor clustering in cultured muscle cells, we examined the role of alpha-dystrobrevin during synapse formation and in response to agrin. Using specific antibodies, we show that C2 myoblasts and early myotubes only produce alpha-dystrobrevin-1, the mammalian orthologue of Torpedo dystrobrevin, whereas mature skeletal muscle expresses three distinct alpha-dystrobrevin isoforms. In myotubes, alpha-dystrobrevin-1 is found on the cell surface and also in acetylcholine receptor-rich domains. Following agrin stimulation, alpha-dystrobrevin-1 becomes re-localised beneath the cell surface into macroclusters that contain acetylcholine receptors and another dystrophin-related protein, utrophin. This redistribution is not associated with tyrosine phosphorylation of alpha-dystrobrevin-1 by agrin. Furthermore, we show that alpha-dystrobrevin-1 is associated with both utrophin in C2 cells and dystrophin in mature skeletal muscle. Thus alpha-dystrobrevin-1 is a component of two protein complexes in muscle, one with utrophin at the neuromuscular junction and the other with dystrophin at the sarcolemma. These results indicate that alpha-dystrobrevin-1 is not involved in the phosphorylation-dependent, early stages of receptor clustering, but rather in the stabilisation and maturation of clusters, possibly via an interaction with utrophin.

Hormonal regulation of focal adhesions in bovine adrenocortical cells: induction of paxillin dephosphorylation by adrenocorticotropic hormone.

A study of bovine adrenocortical cell shape on adrenocorticotropic hormone (ACTH) challenge showed that the cells round up and develop arborized processes. This effect was found to be (1) specific for ACTH because angiotensin II and basic fibroblast growth factor have no effect; (2) mediated by a cAMP-dependent pathway because forskolin reproduces the effect of the hormone; (3) inhibited by sodium orthovanadate, a phosphotyrosine phosphatase inhibitor, but unchanged by okadaic acid, a serine/threonine phosphatase inhibitor; and (4) correlated with a complete loss of focal adhesions. Biochemical studies of the focal-adhesion-associated proteins showed that pp125fak, vinculin (110 kDa) and paxillin (70 kDa) were detected in the Triton X-100-insoluble fraction from adrenocortical cells. During cell adhesion on fibronectin as substratum, two major phosphotyrosine-containing proteins of molecular masses 125 and 68 kDa were immunodetected in the same fraction. A dramatic decrease in the extent of tyrosine phosphorylation of these proteins was observed within 60 min after treatment with ACTH. No change in pp125fak tyrosine phosphorylation nor in Src activity was detected. In contrast, paxillin was found to be tyrosine-dephosphorylated in a time-dependent manner in ACTH-treated cells. Sodium orthovanadate completely prevented the effect of ACTH. These observations suggest a possible role for phosphotyrosine phosphatases in hormone-dependent cellular regulatory processes.

Regulation of Protein-Tyrosine Phosphorylation and Human Sperm Capacitation by Reactive Oxygen Derivatives

Spermatozoa undergoing capacitation, a necessary prerequisite event to successful fertilization that can be induced in vitro by reactive oxygen species (ROS), generate superoxide anion (O2−). Because, in neutrophils, the generation of O2− is associated with tyrosine phosphorylation of several proteins, the aim of the present study was to investigate the association between protein-tyrosine phosphorylation and ROS-induced human sperm capacitation. Human spermatozoa express two major phosphotyrosine-containing proteins of 105 and 81 kDa, the phosphotyrosine content of which is increased when spermatozoa are incubated under capacitating conditions. Superoxide dismutase and catalase abolish both sperm capacitation and tyrosine phosphorylation of p105 and p81, suggesting the involvement of O2− and hydrogen peroxide in these two processes. Inhibitors of NADPH oxidase, the enzyme responsible for the neutrophil's respiratory burst, decrease both p105 and p81 tyrosine phosphorylation and sperm capacitation while hydrogen peroxide stimulates these two processes. Tyrosine phosphorylation of p105 and p81 occurs through a herbimycin A-sensitive tyrosine kinase, and sperm incubation with phosphotyrosine-protein phosphatase inhibitors results in an increase in phosphotyrosine content of these two proteins. Indirect immunocytochemical studies reveal phosphotyrosine-containing proteins mostly in the principal piece of the flagellum, in agreement with the localization of p105 and p81 in the human sperm fibrous sheath. Although tyrosine phosphorylation of p105 and p81 and sperm capacitation are related in a time-dependent fashion, some discrepancies are observed in the regulation of these two processes according to the redox status of the spermatozoa.Copyright © 1997 Elsevier Science Inc.

Cyclic adenosine 3',5'monophosphate-dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility.

The involvement of cAMP in the process of sperm capacitation has been the subject of several studies. In addition, the importance of protein-tyrosine phosphorylation in this process has been investigated, although only a few studies have been reported in the human. Since agents regulating the intracellular concentrations of cAMP affect sperm capacitation rates, the role of cAMP on the expression of phosphotyrosine-containing proteins was investigated during human sperm capacitation. Fetal cord serum ultrafiltrate, a known capacitation inducer in human spermatozoa, caused an increase in the phosphotyrosine content of 105- and 81-kDa proteins (p105 and p81), the two major phosphotyrosine-containing proteins of human spermatozoa. Similar effects were observed when spermatozoa were incubated with phosphodiesterase inhibitors or cell-permeant cAMP analogs, suggesting that cAMP is involved in these two processes. Forskolin, an adenylyl cyclase activator, also caused an increase in both sperm capacitation rates and tyrosine phosphorylation of p105 and p81, while 12-O-tetradecanoyl phorbol 13-acetate stimulated both capacitation and tyrosine phosphorylation of p105 and p81 only when spermatozoa were incubated in the presence of bicarbonate, in agreement with its reported effects on cAMP production and hamster sperm capacitation. The inhibition of these phenomena by cAMP-dependent protein kinase inhibitors, and the stimulation by protein phosphatase inhibitors, suggest that Ser/Thr protein phosphorylation plays an important role in the regulation of both sperm capacitation and protein-tyrosine phosphorylation pathways. However, observations that both calyculin A and okadaic acid stimulated sperm capacitation, whereas only calyculin A increased p105 and p81 phosphotyrosine content and sperm velocity, suggest that protein phosphatase PP1 is involved in the two latter phenomena while PP2A mediates sperm capacitation. These results suggest that divergent pathways might regulate tyrosine phosphorylation of p105 and p81 and sperm capacitation after cAMP-dependent phosphorylation of an intermediate protein.

Nerve Growth Factor Stimulates the Tyrosine Phosphorylation of Endogenous Crk-II and Augments Its Association with p130Cas in PC-12 Cells

The cellular homologs of the v-Crk oncogene product consist primarily of Src homology region 2 (SH2) and 3 (SH3) domains. v-Crk overexpression causes cell transformation and elevation of tyrosine phosphorylation in fibroblasts and accelerates differentiation of PC-12 cells in response to nerve growth factor (NGF). To further explore the role of Crk in NGF-induced PC-12 cell differentiation, we found that both NGF and epidermal growth factor stimulate the tyrosine phosphorylation of endogenous Crk II. Moreover, hormone stimulation enhanced the specific association of Crk proteins with the tyrosine-phosphorylated p130Cas, the major phosphotyrosine-containing protein in cells transformed with v-Crk. This interaction is mediated by the SH2 domain of Crk and can be inhibited with a phosphopeptide containing the Crk-SH2 binding motif. Furthermore, the Crk-SH2 domain binds tyrosine-phosphorylated paxillin, a cytoskeletal protein, following treatment of PC-12 cells with NGF or epidermal growth factor. These data suggest that Crk functions in a number of signaling processes in PC-12 cells.

Tyrosine protein phosphorylation in plasma membranes of rat kidney cortex.

The endogenous tyrosine protein kinase activity (TPKA) associated with brush-border (BBM) and basolateral (BLM) membranes of rat kidney cortex was studied with an anti-phosphotyrosine monoclonal antibody (PY20). Distinct major phosphotyrosine-containing proteins were associated with BBM (50, 54, and 120 kDa) and BLM (37, 90, 130, and 170 kDa). For both plasma membranes, tyrosine phosphorylation leveled off after 10 min of incubation. Endogenous phosphotyrosine-specific protein phosphatases (PT-Pases) were active in both membranes, since the presence of sodium vanadate or ammonium molybdate, which are inhibitors of PTPases, was essential to detect endogenous phosphorylation. Substrates and/or tyrosine protein kinases (TPKs) seem to be differently distributed in these plasma membranes, since phosphorylation of endogenous substrates in BLM and BBM was differently sensitive to competitive inhibitors of TPKs. Moreover, insulin- and insulin-like growth factor I-stimulated tyrosine phosphorylation of a 90-kDa substrate was only observed in solubilized BLM proteins. However, similar p60v-src-related TPKs appear to be present in the BBM and BLM, since an antibody raised against p60v-src recognized proteins of 52, 58, and 75 kDa by immunoblotting and could immunoprecipitate the TPKs associated with both plasma membranes. These data provide evidence that the endogenous tyrosine protein phosphorylation observed in the BLM is catalyzed by nonreceptor TPKs as well as receptor TPKs, whereas that observed in the BBM is exclusively due to nonreceptor TPKs.

Tyrosine and serine phosphorylation of dystrophin and the 58-kDa protein in the postsynaptic membrane of Torpedo electric organ.

Dystrophin associates with a 58-kDa and an 87-kDa protein in the postsynaptic membrane of the Torpedo electric organ. We have previously shown that the 87-kDa protein is a major phosphotyrosine-containing protein in these membranes. Immunoprecipitation of the 87-kDa protein from phosphorylated postsynaptic membranes results in coimmunoprecipitation of additional phosphorproteins. These phosphorproteins are identified as dystrophin and the 58-kDa protein. Monoclonal antibodies to dystrophin and the 58-kDa protein immunoprecipitate phosphorylated forms of these proteins from postsynaptic membranes phosphorylated in vitro. Phosphoamino acid analysis reveals that dystrophin and the 58-kDa protein are phosphorylated on serine and tyrosine residues. In addition, both dystrophin and the 58-kDa protein are shown to be phosphorylated on tyrosine residues in vivo. These results suggest that the synaptic function of dystrophin and its associated proteins, the 58-kDa and 87-kDa proteins, may be modulated by tyrosine and serine protein phosphorylation.

P95, the major phosphotyrosine-containing protein in mouse spermatozoa, is a hexokinase with unique properties.

Mouse sperm contain a major phosphotyrosine-containing protein of M(r) 95,000 (nonreducing conditions) which has been implicated as a sperm membrane receptor for the egg zona pellucida glycoprotein, ZP3 (Leyton, L., and Saling, P. (1989) Cell 57, 1123-1130; Leyton, L., LeGuen, P., Bunch, D., and Saling, P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 11692-11695). This protein was purified and subjected to limited tryptic digestion and subsequent amino acid analysis. Three sequenced peptides revealed 100% amino acid identity to a mouse hepatoma hexokinase (Arora, K. K., Fanciulli, M., and Pederson, P. L. (1990) J. Biol. Chem. 265, 6481-6488). The purified protein, which migrated at M(r) 116,000 under reducing conditions (p95/116), reacted with an antiserum to the purified rat brain hexokinase, type 1, and comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the purified rat brain enzyme under both nonreducing and reducing conditions. Unlike p95/116, the rat brain enzyme was not a phosphotyrosine-containing protein. The p95/116 protein could be immunoprecipitated with the hexokinase antiserum or an O-phosphotyrosine antibody. Limited tryptic digestion of the purified p95/116 and the rat brain enzyme generated subsets of identical peptides which reacted with the hexokinase antiserum. However, p95/116 also contained phosphotyrosine-containing peptides that were not present in the rat brain hexokinase. When different mouse tissues were probed with the hexokinase antiserum all tissues, with the exception of liver, contained immunoreactive protein. In contrast, only sperm and testis possessed a phosphotyrosine-containing form of hexokinase. These data suggest that the germ cell component of the testis possesses a unique tyrosine-phosphorylated form of hexokinase.

Association of neuronal pp60c-src with growth cone glycoproteins of rat brain.

Tyrosine phosphorylation and protein tyrosine kinase (PTK) activity in the growth cone membrane-associated glycoprotein (GCGP) fraction of 1-day-old rat brain were examined. Using immunoblotting and immunoprecipitation techniques, pp60c-src was identified as one of the major PTKs associated with GCGPs. Furthermore, only GCGP-associated src that was also tyrosine phosphorylated was active. Immunoprecipitation experiments using various src antibodies revealed that pp60c-src contributed partially to the PTK activity detected in GCGPs, and that it is associated with several proteins of Mr 140 K, 120 K, 85 K and 50 K. This association of src protein with GCGPs was specific, and another src family member p59fyn, which is also abundant in the brain, did not exhibit such an association. In addition to pp60c-src, the GCGP fraction contained several major phosphotyrosine-containing proteins of Mr 140 K, and a 97/90 K doublet that corresponded to the beta subunits of IGF-I/insulin receptors. These studies show that pp60c-src associated with GCGPs is an active PTK that could be involved in neuronal growth and development, transmembrane signalling, and in recognition and/or adhesive events.

Tyrosine-phosphorylated epidermal growth factor receptor and cellular p130 provide high affinity binding substrates to analyze Crk-phosphotyrosine-dependent interactions in vitro.

The genome of CT10 avian sarcoma virus encodes a 47-kDa fusion protein that consists of viral gag sequences fused to a cell-derived sequence containing SH2 and SH3 domains (v-crk). Genetic and biochemical evidence suggests that v-Crk can induce transformation of chicken embryo fibroblasts by influencing the activity of cellular proteins involved in growth regulation. In this report, we have developed an in vitro microtiter assay to study the binding of bacterially expressed glutathione S-transferase-fusion proteins of v-Crk and its cellular homolog, c-Crk, to the phosphorylated epidermal growth factor receptor (EGFR). Competitive binding data are presented that compare the abilities of heterologous glutathione S-transferase-fusion proteins containing GAPSH2[N], AblSH2, SrcSH2, and PLC-gamma SH2[N] sequences to inhibit Crk binding. Results indicate that both full-length Crk and GAPSH2[N] bind the phosphorylated EGFR with high affinity and can quantitatively compete the binding of each other by competitive enzyme-linked immunosorbent assay. Binding of full-length Crk or the isolated SH2 domains of GAP or Abl resulted in a significant protection of phosphorylated EGFR against dephosphorylation by cellular phosphatase activity, but did not appear to stimulate the intrinsic tyrosine kinase activity of the EGFR. To extend these findings to p130, the major phosphotyrosine-containing protein in CT10-transformed cells, we utilized a nitrocellulose filter binding assay. Results demonstrate high affinity binding of Crk toward denatured p130 and, as is the case for phosphorylated EGFR, Crk binding can partially protect p130 from phosphatase activity. However, no apparent competition of Crk binding was noted with heterologous SH2-containing proteins including GAPSH2[N], suggesting a possible specificity of Crk-p130 binding. These data are consistent with a direct role of SH2 in the modulation of cellular phosphotyrosine status in vivo.

Paxillin is a major phosphotyrosine-containing protein during embryonic development.

Phosphotyrosine-containing proteins were immunoprecipitated from embryonic chicken tissue extracts using anti-phosphotyrosine antibody coupled to agarose beads. Major phosphotyrosine-containing proteins of 110, 70, and 50 kD were observed following blotting with anti-phosphotyrosine antibody. The 70-kD band was selectively removed from the samples by precipitation with antibodies to the focal adhesion protein paxillin, therefore identifying paxillin as one of the major tyrosine kinase substrates during chick embryonic organogenesis. The tyrosine phosphorylation of paxillin is regulated developmentally: during embryogenesis, a marked decrease in its phosphotyrosine content was observed, although the total level of paxillin remained essentially constant. Approximately 20% of the paxillin was phosphorylated on tyrosine in the early embryo. In contrast, tyrosine phosphorylation of paxillin was undetectable in the adult. A similar profile of phosphotyrosine-containing proteins was identified in rat embryos. Paxillin was also found to be a major phosphotyrosine-containing protein in the rat embryo. These data suggest that the regulated phosphorylation of tyrosine residues on paxillin may perform a critical role in controlling cell and tissue cytoarchitecture rearrangement during vertebrate development.

Postnatal Age and Protein Tyrosine Phosphorylation at Synapses in the Developing Rat Brain

The relationship between postnatal age and protein tyrosine kinase activity in synaptosomes prepared from the rat forebrain was studied. Synaptosomal particulate and soluble fractions, as well as total homogenates, the cell soluble fraction, and P3, were prepared from rats ranging in postnatal age from 5 to 60 days and analyzed for (a) tyrosine kinase activity using polyglutamyltyrosine (4:1) as the substrate, (b) the presence of endogenous substrates for tyrosine phosphorylation using polyclonal antibodies specific for phosphotyrosine, and (c) levels of pp60src. Enzyme activity, expressed per milligram of protein, in the total homogenate, P3, and both the cell and synaptosomal soluble fractions was highest in the brains of young animals (postnatal days 5-10) and decreased thereafter to adult levels. In contrast, tyrosine kinase activity in the synaptosomal particulate fraction exhibited a unique biphasic developmental profile, increasing to maxima at postnatal days 10 and 20 before decreasing to adult values. Endogenous substrates for tyrosine phosphorylation were identified by incubating subcellular fractions with 2 mM ATP in the presence of sodium orthovanadate and probing nitrocellulose blots of proteins separated by gel electrophoresis with antiphosphotyrosine antibodies. Several phosphotyrosine-containing proteins were detected in the synaptosomal particulate and P3 fractions, including proteins of Mr 180K, 145K, 120K, 100K, 77K, 68K, 62K, 54K, 52K, and 42K. In the cell soluble fraction a protein doublet of Mr 54/52K and a 120K protein were the major phosphotyrosine-containing proteins. The 54/52K doublet was the major protein tyrosine kinase substrate in the synaptosomal soluble fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphotyrosine-containing proteins in bovine chromaffin cells: effects of insulin-like growth factor I (IGF-I).

1. Antiphosphotyrosine antibodies were used to detect phosphotyrosine-containing proteins in immunoblots of bovine chromaffin cell proteins. 2. Unstimulated cells exhibited two major phosphotyrosine-containing proteins, which had Mr's of 121,000 and 70,000. Insulin-like growth factor I (IGF-I) had little effect on the phosphotyrosine content of these two proteins but greatly increased the phosphotyrosine content of three other proteins of Mr 185,000, 170,000, and 96,000. These proteins were found predominantly in the particulate fraction of cell homogenates. 3. The effects of the IGF-I were time and concentration dependent, with maximal increases in phosphorylation occurring after 1 min of treatment with 10 nM IGF-I. Na3VO4, an inhibitor of phosphotyrosine phosphatases, potentiated the effects of IGF-I. 4. Thus, the IGF-I receptor appears to function as an IGF-I-activated protein tyrosine kinase in chromaffin cells. The tyrosine kinase activity of the IGF-I receptor presumably mediates the effects of IGF-I on chromaffin cell function.

Paxillin is a major phosphotyrosine-containing protein during embryonic development

Paxillin is a major phosphotyrosine-containing protein during embryonic development

Insulin-Like Growth Factor-Mediated Phosphorylation and Protooncogene Induction in Madin-Darby Canine Kidney Cells

We have characterized the role of tyrosine phosphorylation in protooncogene induction mediated by insulin-like growth factors I and II (IGF-I and IGF-II) in the Madin-Darby canine kidney (MDCK) cell line. These cells possess few, if any, insulin receptors, thus allowing determination of the effects of these growth factors in the absence of any secondary signal mediated through the insulin receptor. We found that IGF-I produced a specific stimulation of tyrosine kinase activity of the 97-kDa beta-subunit of the IGF-I receptor, resulting in autophosphorylation of the receptor and an increase in kinase activity toward a synthetic peptide substrate. This was associated with a gradual decrease in the level of phosphorylation of pp120, the major constitutive phosphotyrosine-containing protein of MDCK cells, and an increase in the ratio of serine to tyrosine phosphorylation. This was followed by a rapid, but transient, induction of c-fos gene expression, with no change in the levels of c-myc mRNA. Cycloheximide treatment resulted in a superinduction of both c-fos and c-myc and prevented any further stimulation by IGF-I. IGF-II did not stimulate tyrosine phosphorylation of its own receptor, but was 25% as active as IGF-I in stimulating phosphorylation of the IGF-I receptor. Despite this, IGF-II did not significantly enhance the expression of either nuclear protooncogene. Insulin also produced a delayed stimulation of IGF-I receptor phosphorylation, but was unable to stimulate biological effects in these cells. Under these conditions neither of the IGFs nor insulin produced any significant stimulation of thymidine incorporation into DNA. These data indicate that the IGF-I receptor can be activated upon binding of IGF-I, and to a lesser extent IGF-II, in intact cells to mediate cellular events. The nature of the signal generated by the IGF-I receptor appears to vary depending on the ligand that occupies it.

Association of the v-crk oncogene product with phosphotyrosine-containing proteins and protein kinase activity.

An antiserum specific for P47gag-crk, the oncogene product of avian sarcoma virus CT10, was used to investigate possible crk-binding proteins. In in vitro kinase assays, four proteins were phosphorylated in anti-crk immunoprecipitates. Tyrosine, serine, and threonine residues were phosphorylated. A number of tyrosine-phosphorylated proteins were identified in anti-crk immunoprecipitates of 32P-labeled cells, including the three major phosphotyrosine-containing proteins of CT10-infected cells. These three proteins also bound to bacterially synthesized crk protein. These results suggest that the crk transforming protein can stably associate with both endogenous kinases and cellular kinase substrates.

Structure and Expression of STK, a src-Related Gene in the Simple Metazoan Hydra attenuata

Both cDNA clones and a genomic DNA clone encoding a 509-amino-acid protein that is 64% similar to chicken pp60c-src were isolated from the simple metazoan Hydra attenuata. We have designated this gene STK, for src-type kinase. Features of the amino acid sequence of the protein encoded by the STK gene suggest that it is likely to be myristoylated and regulated by phosphorylation in a manner similar to that found for pp60c-src. The genomic sequence encoding the protein was found to be interrupted by at least two introns, one of which was located in a position identical to that of one of the introns in the chicken src gene. The STK gene was expressed during early development of H. attenuata and at high levels in the epithelial cells of adult polyps. Probing of Hydra proteins with an antibody to phosphotyrosine indicated that the major phosphotyrosine-containing protein in H. attenuata may be the STK protein itself. H. attenuata is the simplest organism from which a protein-tyrosine kinase gene has been isolated. The presence of such a gene in the evolutionarily ancient phylum Cnidaria suggests that protein-tyrosine kinase genes arose concomitantly with or shortly after the appearance of multicellular organisms.

Structure and expression of STK, a src-related gene in the simple metazoan Hydra attenuata.

Both cDNA clones and a genomic DNA clone encoding a 509-amino-acid protein that is 64% similar to chicken pp60c-src were isolated from the simple metazoan Hydra attenuata. We have designated this gene STK, for src-type kinase. Features of the amino acid sequence of the protein encoded by the STK gene suggest that it is likely to be myristoylated and regulated by phosphorylation in a manner similar to that found for pp60c-src. The genomic sequence encoding the protein was found to be interrupted by at least two introns, one of which was located in a position identical to that of one of the introns in the chicken src gene. The STK gene was expressed during early development of H. attenuata and at high levels in the epithelial cells of adult polyps. Probing of Hydra proteins with an antibody to phosphotyrosine indicated that the major phosphotyrosine-containing protein in H. attenuata may be the STK protein itself. H. attenuata is the simplest organism from which a protein-tyrosine kinase gene has been isolated. The presence of such a gene in the evolutionarily ancient phylum Cnidaria suggests that protein-tyrosine kinase genes arose concomitantly with or shortly after the appearance of multicellular organisms.