Levels Of Tyrosine Kinase Activity Research Articles

Mechanistic Insights into Regulation of JAK2 Tyrosine Kinase.

JAK2 is a member of the Janus kinase (JAKs) family of non-receptor protein tyrosine kinases, which includes JAK1–3 and TYK2. JAKs serve as the cytoplasmic signaling components of cytokine receptors and are activated through cytokine-mediated trans-phosphorylation, which leads to receptor phosphorylation and recruitment and phosphorylation of signal transducer and activator of transcription (STAT) proteins. JAKs are unique among tyrosine kinases in that they possess a pseudokinase domain, which is just upstream of the C-terminal tyrosine kinase domain. A wealth of biochemical and clinical data have established that the pseudokinase domain of JAKs is crucial for maintaining a low basal (absence of cytokine) level of tyrosine kinase activity. In particular, gain-of-function mutations in the JAK genes, most frequently, V617F in the pseudokinase domain of JAK2, have been mapped in patients with blood disorders, including myeloproliferative neoplasms and leukemias. Recent structural and biochemical studies have begun to decipher the molecular mechanisms that maintain the basal, low-activity state of JAKs and that, via mutation, lead to constitutive activity and disease. This review will examine these mechanisms and describe how this knowledge could potentially inform drug development efforts aimed at obtaining a mutant (V617F)-selective inhibitor of JAK2.

Cytokine-dependent activation of JAK-STAT pathway in Saccharomyces cerevisiae.

Protein phosphorylation is an important post-translational modification for intracellular signaling molecules, mostly found in serine and threonine residues. Tyrosine phosphorylations are very few events (less than 0.1% to phosphorylated serine/threonine residues), but capable of governing cell fate decisions involved in proliferation, differentiation, apoptosis, and oncogenic transformation. Hence, it is important for drug discovery and system biology to measure the intracellular level of phosphotyrosine. Although mammalian cells have been conventionally utilized for this purpose, accurate determination of phosphotyrosine level often suffers from high background due to the unexpected crosstalk among endogenous signaling molecules. This situation led us firstly to establish the ligand-induced activation of homomeric receptor tyrosine kinase (i.e., epidermal growth factor receptor) in Saccharomyces cerevisiae, a lower eukaryote possessing organelles similar to higher eukaryote but not showing substantial level of tyrosine kinase activity. In this study, we expressed heteromeric receptor tyrosine kinase (i.e., a complex of interleukin-5 receptor (IL5R) α chain, common β chain, and JAK2 tyrosine kinase) in yeast. When coexpressed with a cell wall-anchored form of IL5, the yeast exerted the autophosphorylation of JAK2, followed by the phosphorylation of transcription factor STAT5a and subsequent nuclear accumulation of phosphorylated STAT5a. Taken together, yeast could be an ideal host for sensitive detection of phosphotyrosine generated by a wide variety of tyrosine kinases. Biotechnol. Bioeng. 2016;113: 1796-1804. © 2016 Wiley Periodicals, Inc.

Inflammation-induced shift in spinal GABAAsignaling is associated with a tyrosine kinase-dependent increase in GABAAcurrent density in nociceptive afferents

To account for benzodiazepine-induced spinal analgesia observed in association with an inflammation-induced shift in the influence of the GABA(A) receptor antagonist gabazine on nociceptive threshold, the present study was designed to determine whether persistent inflammation is associated with the upregulation of high-affinity GABA(A) receptors in primary afferents. The cell bodies of afferents innervating the glabrous skin of the rat hind paw were retrogradely labeled, acutely dissociated, and studied before and after the induction of persistent inflammation. A time-dependent increase in GABA(A) current density was observed that was more than twofold by 72 h after the initiation of inflammation. This increase in current density included both high- and low-affinity currents and was restricted to neurons in which GABA increased intracellular Ca(2+). No increases in GABA(A) receptor subunit mRNA or protein were detected in whole ganglia. In contrast, the increased current density was completely reversed by 20-min preincubation with the tyrosine kinase inhibitor genistein and partially reversed with the Src kinase inhibitor PP2. Genistein reversal was partially blocked by the dynamin inhibitor peptide P4. Changes in nociceptive threshold following spinal administration of genistein and muscimol to inflamed rats indicated that the pronociceptive actions of muscimol observed in the presence of inflammation were reversed by genistein. These results suggest that persistent changes in relative levels of tyrosine kinase activity following inflammation provide not only a sensitive way to dynamically regulate spinal nociceptive signaling but a viable target for the development of novel therapeutic interventions for the treatment of inflammatory pain.

The RhoGEF domain of p210 Bcr-Abl activates RhoA and is required for transformation

The BCR-ABL oncogene encodes an in-frame fusion protein containing N-terminal sequences derived from Bcr and C-terminal sequences derived from Abl. Bcr contains a centrally located Rho-specific guanine nucleotide exchange factor (RhoGEF) domain that is retained within p210 Bcr-Abl. Although this domain is subject to autoinhibition in the context of Bcr, here we show that it is constitutively activated in p210 Bcr-Abl. p210 Bcr-Abl can stimulate RhoA activation independently of its tyrosine kinase activity, and mutations within the RhoGEF domain that are predicted to eliminate RhoGEF activity inhibit RhoA activation. The RhoGEF mutant of p210 Bcr-Abl does not affect the tyrosine kinase activity of the molecule, nor the ability of p210 Bcr-Abl to interact with XPB through the RhoGEF domain. Despite retaining normal levels of tyrosine kinase activity, the RhoGEF mutant of p210 Bcr-Abl is impaired in transforming activity as measured by anchorage-independent growth. However, the mutant is still able to confer the phenotype of growth factor independence in myeloid cells, suggesting that some, but not all parameters of p210 Bcr-Abl transformation, are dependent upon a catalytically active RhoGEF domain. Collectively, these observations identify a gain-of-function activity attributable to the RhoGEF domain of p210 Bcr-Abl that is required to support the transformed phenotype.

Parallel Analysis of v-Src Mutant Protein Function Using Reverse Transfection Cell Arrays

The conversion of the genomic information produced by the recent sequencing projects into a comprehensive understanding of the human proteome has yet to occur. A new technology that represents a potential bridge between genomics and proteomics is reverse transfection. Reverse transfection cell microarrays are produced by overlaying cDNA arrays with mammalian cells, generating localized clusters of transfected cells with each cluster overexpressing a unique protein. This miniaturized cell-based microarray format affords parallel functional analysis of thousands of cDNA constructs in a high throughput format. In this report we document the development of a co-transfection methodology for reverse transfection applications. The demonstrated high co-transfection efficiency with a "marker" plasmid encoding for GFP enables the identification of transfected cells and eliminates the need for epitope-tagged constructs in cell-based high throughput screening applications using reverse transfection. This co-transfection method was used to study in parallel the structure/function of multiple versions of the v-Src protein using automated fluorescence microscopy. The wild-type v-Src protein and four mutants having insertions or deletions in the SH2 or SH3 domains displayed high levels of tyrosine kinase activity in HEK293T cells. Three other mutated v-Src proteins, including a kinase-dead version, were shown to be defective for tyrosine kinase activity. This reverse co-transfection approach is applicable for high throughput screening of both cDNA libraries and positional scanning recombinant protein libraries.

Role of GTP‐binding proteins in reversing the antiproliferative effects of tocotrienols in preneoplastic mammary epithelial cells

Tocotrienols are a subclass of vitamin E compounds that display potent anticancer activity. Determining the anticancer mechanism of action of tocotrienols will provide essential information necessary for understanding the potential health benefits of these compounds in reducing the risk of breast cancer in women. Epidermal growth factor (EGF) is a potent mitogen for normal and neoplastic mammary epithelial cells. Initial events in EGF-receptor (EGF-R) mitogenic-signalling are G-protein activation, stimulation of adenylyl cyclase and cyclic AMP (cAMP) production. Studies were conducted to determine if the antiproliferative effects of tocotrienols are associated with reduced EGF-induced G-protein and cAMP-dependent mitogenic signalling. Preneoplastic CL-S1 mouse mammary epithelial cells were grown in culture and maintained on serum-free media containing 0-25 micro mol/L tocotrienol-rich fraction of palm oil and/or different doses of pharmacological agents that alter intracellular cAMP levels. Tocotrienol-induced effects on EGF-receptor levels of tyrosine kinase activity, as well as EGF-dependent mitogen-activated pathway kinase (MAPK) and Akt activation, were determined by western blot analysis. Results demonstrate that the antiproliferative effects of tocotrienols in preneoplastic mammary epithelial cells do not reflect a reduction in EGF-receptor mitogenic responsiveness, but rather, result from an inhibition in early post-receptor events involved in cAMP production upstream from EGF-dependent MAPK and phosphoinositide 3-kinase/Akt mitogenic signalling. In summary, these data further characterise the mechanism of action of tocotrienols in suppressing preneoplastic mammary epithelial cell proliferation, and advance the current understanding of the potential health benefits of these compounds in reducing the risk of breast cancer in women.

The level of tyrosine kinase activity regulates the expression of p21/WAF1 in cancer cells.

It is well documented that epidermal growth factor (EGF) inhibits proliferation of A431 and MDA-468 cells via activation of p21/WAF1. In the present study, we have shown that treatment of MDA-468 and A431 cells that express high levels of EGFR with 100 ng/ml of EGF leads to 14.9-fold increase in epidermal growth factor receptor (EGFR) autophosphorylation and high levels of p21/WAF1-induction (6. 7-fold), down regulation of cdk2 activity and growth arrest. When MDA-468 or A431 cells were simultaneously treated with 100 ng/ml EGF and RG13022, a relatively specific tyrosine kinase inhibitor, there was a significant reduction in p21/WAF1 levels. In contrast, when MDA-468, A432 cells that are treated with low levels of EGF (10 ng/ml) or other cells which express low to moderate levels of EGFRs such as MCF-7, MCF-10A, MDA-231 and SKBR-3 breast cells were exposed to 100 ng/ml of EGF there was a 3.8-fold increase in EGFR autophosphorylation, leading to 1.6-fold induction of p21/WAF1 and increased cell proliferation. These results suggest that the level of EGFR tyrosine kinase activity may regulate p21/WAF1 induction in cancer cells.

IL-3 induces apoptosis in a ras-transformed myeloid cell line.

Growth factors promote cell survival and proliferation. Homeostasis is maintained by programmed cell death which occurs when the growth stimulus is withdrawn, in response to negative growth regulators such as interferons, TNF-alpha and CD95 ligand, or following differentiation. Although acutely-transforming oncogenes often overcome the need for growth factors, growth regulatory cytokines can influence proliferative responses of transformed cells. In this study we investigated the effects of IL-3 on the proliferative responses of parental bone marrow-derived 32D cells and cells transformed with ras and abl oncogenes. We show that treatment of ras-transformed 32D cells with IL-3 reduced proliferative responses and decreased colony-forming ability. These effects were exacerbated in the absence of serum and associated with inhibition of tyrosine kinase activity, down-regulation of RAS and MYC expression, and induction of apoptosis as indicated by DNA fragmentation. In contrast, treatment of parental 32D cells with IL-3, which is obligatory for cell survival and proliferation, increased tyrosine kinase activity, upregulated MYC and RAS expression and maintained DNA integrity. With abl-transformed cells, proliferation and colony-forming ability were also inhibited by IL-3. Tyrosine kinase activity and MYC expression were reduced, but early apoptosis was not evident. Calcium uptake however, was stimulated by IL-3 in both parental and oncogene-transformed cells. These results suggest that threshold levels of tyrosine kinase activity are necessary for cell survival and proliferation and that with ras-transformed cells, IL-3 treatment may result in this threshold being breached. We conclude that in some situations, growth-promoting cytokines can inhibit proliferation of transformed cells and induce cell death by apoptosis.

Activating mutations in the extracellular domain of the fibroblast growth factor receptor 2 function by disruption of the disulfide bond in the third immunoglobulin-like domain

Multiple human skeletal and craniosynostosis disorders, including Crouzon, Pfeiffer, Jackson-Weiss, and Apert syndromes, result from numerous point mutations in the extracellular region of fibroblast growth factor receptor 2 (FGFR2). Many of these mutations create a free cysteine residue that potentially leads to abnormal disulfide bond formation and receptor activation; however, for noncysteine mutations, the mechanism of receptor activation remains unclear. We examined the effect of two of these mutations, W290G and T341P, on receptor dimerization and activation. These mutations resulted in cellular transformation when expressed as FGFR2/Neu chimeric receptors. Additionally, in full-length FGFR2, the mutations induced receptor dimerization and elevated levels of tyrosine kinase activity. Interestingly, transformation by the chimeric receptors, dimerization, and enhanced kinase activity were all abolished if either the W290G or the T341P mutation was expressed in conjunction with mutations that eliminate the disulfide bond in the third immunoglobulin-like domain (Ig-3). These results demonstrate a requirement for the Ig-3 cysteine residues in the activation of FGFR2 by noncysteine mutations. Molecular modeling also reveals that noncysteine mutations may activate FGFR2 by altering the conformation of the Ig-3 domain near the disulfide bond, preventing the formation of an intramolecular bond. This allows the unbonded cysteine residues to participate in intermolecular disulfide bonding, resulting in constitutive activation of the receptor.

Protein tyrosine phosphorylation: Implications for synaptic function

The phosphorylation of proteins on tyrosine residues, initially believed to be primarily involved in cell growth and differentiation, is now recognized as having a critical role in regulating the function of mature cells. The brain exhibits one of the highest levels of tyrosine kinase activity in the adult animal and the synaptic region is particularly rich in tyrosine kinases and phosphorylated proteins. Recent studies have described the effects of tyrosine phosphorylation on the activities of a number of proteins which are potentially involved in the regulation of synaptic function. Furthermore, it is becoming apparent that tyrosine phosphorylation is involved in the modification of synaptic activity, such as occurs during depolarization, the induction of long-term potentiation or long-term depression, and ischemia. Changes in the activities of tyrosine kinases and/or protein tyrosine phosphatases which are associated with synaptic structures may result in altered tyrosine phosphorylation of proteins located at the synapse leading to both short-term and long-lasting changes in synaptic and neuronal function.

Characterization of the kinase activity essential for tyrosine phosphorylation of p130Cas in fibroblasts.

The cellular transformation by v-Src or v-Crk induces tyrosine phosphorylation of a common substrate molecule, p130Cas (Cas), which tightly binds these oncoproteins in vivo. From its structure, Cas is deduced to be an ideal substrate for Src family kinases and Abl kinase. The tyrosine kinase activity associated with Cas was analysed using mouse variant fibroblasts lacking at least one of tyrosine kinases. In normal fibroblasts, Cas is associated with a significant level of tyrosine kinase activity which efficiently phosphorylates Cas in vitro. The Cas-associated tyrosine kinase activity was remarkably elevated in Csk-/- cells, which resulted in hyperphosphorylation of cellular Cas. The associated kinase activity was slightly increased in Src-/- cells whereas not significantly changed in Abl-/- nor Fak-/- cells. On the contrary, the Cas-associated kinase activity was remarkably decreased in Fyn-/- cells. At the same time, association of Cas with Fyn kinase in vitro was most obviously detected in normal fibroblasts as well as Csk-/- cells. Transient expression of v-Crk induced elevation of the Cas-associated kinase activity in all of these cell lines except the primary culture of Fyn-/- fibroblasts. These results indicate that Fyn kinase plays an essential role in v-Crk-mediated phosphorylation of Cas.

Ornithine Decarboxylase- and ras-Induced Cell Transformations: Reversal by Protein Tyrosine Kinase Inhibitors and Role of pp130CAS

We have found that overexpression of human ornithine decarboxylase (ODC) induces cell transformation in NIH 3T3 and Rat-1 cells (M. Auvinen, A. Paasinen, L. C. Andersson, and E. Hölttä, Nature (London) 360:355-358, 1992). The ODC-transformed cells display increased levels of tyrosine phosphorylation, in particular of a cluster of 130-kDa proteins. Here we show that one of the proteins with enhanced levels of tyrosine phosphorylation in ODC-overexpressing cells is the previously described p130 substrate of pp60v-src, known to associate also with v-Crk and designated p130CAS. We also studied the role of protein tyrosine phosphorylation in the ODC-induced cell transformation by exposing the cells to herbimycin A, a potent inhibitor of Src-family kinases, and to other inhibitors of protein tyrosine kinases. Treatment with the inhibitors reversed the phenotype of ODC-transformed cells to normal, with an organized, filamentous actin cytoskeleton. Coincidentally, the tyrosine hyperphosphorylation of p130 was markedly reduced, while the level of activity of ODC remained highly elevated. A similar reduction in pp130 phosphorylation and reversion of morphology by herbimycin A were observed in v-src- and c-Ha-ras-transformed cells. In addition, we show that expression of antisense mRNA for p130CAS resulted in reversion of the transformed phenotype of all these cell lines. An increased level of tyrosine kinase activity, not caused by c-Src or c-Abl, was further detected in the cytoplasmic fraction of ODC-transformed cells. Preliminary characteristics of this kinase are shown. These data indicate that p130CAS is involved in cell transformation by ODC, c-ras, and v-src oncogenes, raise the intriguing possibility that p130CAS may be generally required for transformation, and imply that there is at least one protein tyrosine kinase downstream of ODC that is instrumental for cell transformation.

Mechanism of insulin receptor kinase inhibition in non-insulin-dependent diabetes mellitus patients. Phosphorylation of serine 1327 or threonine 1348 is unaltered.

The tyrosine kinase activity of insulin receptor isolated from the skeletal muscle of NIDDM patients has previously been found to be decreased compared with the activity of receptor from nondiabetic subjects but the mechanism underlying this defect is unknown. Phosphorylation of receptor serine/threonine residues has been proposed to exert an inhibitory influence on receptor tyrosine kinase activity and Ser 1327 and Thr 1348 have been identified as specific sites of phosphorylation in the insulin receptor COOH terminal domain. To address the potential negative regulatory role of phosphorylation of these residues in vivo, we assessed the extent of phosphorylation of each site in insulin receptor isolated from the skeletal muscle of 12 NIDDM patients and 13 nondiabetic, control subjects. Phosphorylation of Ser 1327 and Thr 1348 was determined using antibodies that specifically recognize insulin receptor phosphorylated at these sites. In addition, a phosphotyrosine-specific antibody was used to monitor receptor tyrosine phosphorylation. The extent of insulin-induced tyrosine autophosphorylation was decreased in receptor isolated from diabetic versus nondiabetic muscle, thus confirming earlier reports. In contrast, there was no significant difference in the extent of phosphorylation of either Ser 1327 or Thr 1348 in receptor isolated from diabetic or nondiabetic muscle as assessed by immunoprecipitation (Ser 1327: 5.6 +/- 1.6% diabetics vs. 4.7 +/- 2.0% control; Thr 1348: 3.8 +/- 1.0% diabetics vs. 3.2 +/- 1.2% control). Moreover, within each group there was no correlation between the level of tyrosine kinase activity and the extent of serine/threonine phosphorylation. It is concluded that the stoichiometry of serine/threonine phosphorylation of insulin receptor in vivo is low, and that increased phosphorylation of Ser 1327 or Thr 1348 is not responsible for the decreased insulin receptor tyrosine kinase activity observed in the skeletal muscle of NIDDM patients.

Long-Term Inositol Phosphate Release, but Not Tyrosine Kinase Activity, Correlates with IL-2 Secretion and NF-AT Induction in Anti-CD3-Activated Peripheral Human T Lymphocytes

The cascade of events within the first few minutes of T cell stimulation has been well characterized. Although many second messengers have been shown to be necessary and sufficient for T cell activation in a number of model systems, the rate-limiting step in peripheral T cells has not been demonstrated. To model effective versus ineffective CD3-mediated stimulation in peripheral T cells, we used two anti-CD3 mAbs that differ in their ability to stimulate purified T cells: OKT3, which causes early second messenger generation but is unable to activate T cells without a second signal, and 64.1, which stimulates T cell proliferation on its own. We found that tyrosine kinase activity was similar for both mAbs over a period of hours. However, the inositol phosphate response was stronger for 64.1 than for OKT3. To tie these events to gene activation, we measured NF-κB and NF-AT activity in the nucleus after anti-CD3 stimulation. Both stimuli induced the appearance of the NF-κB components (c-Rel, p65 (RelA), and p50 (NF-κB1)) and NF-κB DNA binding activity in the nucleus. However, only 64.1 induced NF-AT in the nucleus, correlating with its ability to activate T cells. Thus, NF-AT induction and IL-2 secretion were correlated with the levels of inositol phosphate release but not with gross levels of tyrosine kinase activity induced late following the response. On the other hand, NF-κB induction and IL-2 receptor expression occurred even with the smaller second messenger response generated by OKT3.

Substitution of the erbB-2 oncoprotein transmembrane domain activates the insulin receptor and modulates the action of insulin and insulin-receptor substrate 1.

The mechanism through which insulin binding to the extracellular domain of the insulin receptor activates the intrinsic tyrosine kinase in the intracellular domain of the protein is unknown. For the c-neu/erbB-2 (c-erbB-2) protooncogene, a single point mutation within the transmembrane (TM) domain converting Val-664 to Glu (erbB-2V-->E) results in elevated levels of tyrosine kinase activity and cellular transformation. We report the construction of a chimeric insulin receptor in which the TM domain of the receptor has been substituted with that encoded by erbB-2V-->E. When expressed in Chinese hamster ovary cells this chimeric receptor displays maximal levels of autophosphorylation and kinase activity in the absence of insulin. This activity results in an increase in the level of insulin-receptor substrate 1 phosphorylation but a down-regulation in insulin-receptor substrate 1 protein and desensitization to insulin stimulation of glycogen synthesis. By contrast, basal levels of DNA synthesis are elevated to levels approximately 60% of those observed in serum-stimulated cells. Over-expression of chimeric insulin receptors containing the c-erbB-2 TM domain or a single point mutation in the insulin receptor TM domain of Val-938-->Asp, on the other hand, shows none of these alterations. Thus, the TM domain encoded by erbB-2V-->E contains structural features that can confer ligand-independent activation in a heterologous protein. Constitutive activation of the insulin receptor results in a relative increase in basal levels of DNA synthesis, but an apparent resistance to the metabolic effects of insulin.

Tissue-dependent regulation of protein tyrosine kinase activity during embryonic development.

Protein tyrosine kinase activity was assayed in a variety of chicken tissues during embryonic development and in the adult. In some tissues protein tyrosine kinase activity decreased during embryonic development; however, in other tissues it remained high throughout development, it contrast to the level of protein tyrosine phosphorylation, which decreased during development. The highest levels of tyrosine kinase activity were detected in 17-d embryonic brain although only low levels of protein tyrosine phosphorylation were observed in this tissue. Several alternatives were examined in an effort to determine the mechanism responsible for the low levels of tyrosine phosphorylated proteins in most older embryonic and adult chicken tissues despite the presence of highly active tyrosine kinases. The results show that the regulation of protein tyrosine phosphorylation during embryonic development is complex and varies from tissue to tissue. Furthermore, the results suggest that protein tyrosine phosphatases play an important role in regulating the level of phosphotyrosine in proteins of many older embryonic and adult tissues.

Altered protein tyrosine kinase levels in human colon carcinoma

To further understand the molecular mechanisms and the biological indicators of colonic tumorigenesis, the authors examined tyrosine kinase activity in the cytosol and in the particulate fraction of the homogenates of specimens from 20 human colonic carcinomas and compared them with the adjacent normal mucosal tissues. Total protein tyrosine kinase activity could be precisely detected using miniphosphocellulose column purification and a synthetic peptide, Glu-asparagine (Asp)-alanine (Ala)-Glu-tyrosine (Tyr)-Ala-Ala-arginine (Arg)-Arg-Arg-glycine (Gly) (E11-G1), as an artificial substrate. Tyrosine kinase activity of colonic carcinoma and normal mucosa was reduced in the cytosol fraction whereas activity in the particulate fraction was elevated with respect to protein concentration. The average specific activity ratios were 1.95 +/- 0.27 (normal cytosolic/carcinoma cytosolic) and 0.57 +/- 0.01 (normal particulate/carcinoma particulate) for tyrosine kinase activity. Cellular distribution (% cytosol) of tyrosine kinase activity in normal mucosa and in carcinoma varied from 21.0% to 91.2% and from 7.0% to 61.4%, respectively. In nearly all cases the percentage of cytosolic tyrosine kinase activity in carcinoma tissues was lower than in normal tissues. There was no difference due to histologic type or the presence of adenomatous components. A significant decrease of cytosolic tyrosine kinases was correlated with Dukes' Stage A. With advancing Dukes' stage, the average specific activity ratios (normal cytosol/carcinoma cytosol) were decreased. This study indicates that colonic carcinogenesis might be associated with alterations in cellular levels of tyrosine kinase activity and that the average specific activity ratio (normal cytosol/carcinoma cytosol) had a possible correlation with colonic tumor growth.

Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity.

Extracts of bakers' yeast (Saccharomyces cerevisiae) contain protein-tyrosine kinase activity that can be detected with a synthetic Glu-Tyr copolymer as substrate (G. Schieven, J. Thorner, and G.S. Martin, Science 231:390-393, 1986). By using this assay in conjunction with ion-exchange and affinity chromatography, a soluble tyrosine kinase activity was purified over 8,000-fold from yeast extracts. The purified activity did not utilize typical substrates for mammalian protein-tyrosine kinases (enolase, casein, and histones). The level of tyrosine kinase activity at all steps of each preparation correlated with the content of a 40-kDa protein (p40). Upon incubation of the most highly purified fractions with Mn-ATP or Mg-ATP, p40 was the only protein phosphorylated on tyrosine. Immunoblotting of purified p40 or total yeast extracts with antiphosphotyrosine antibodies and phosphoamino acid analysis of 32P-labeled yeast proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 40-kDa protein is normally phosphorylated at tyrosine in vivo. 32P-labeled p40 immunoprecipitated from extracts of metabolically labeled cells by affinity-purified anti-p40 antibodies contained both phosphoserine and phosphotyrosine. The gene encoding p40 (YPK1) was cloned from a yeast genomic library by using oligonucleotide probes designed on the basis of the sequence of purified peptides. As deduced from the nucleotide sequence of YPK1, p40 is homologous to known protein kinases, with features that resemble known protein-serine kinases more than known protein-tyrosine kinases. Thus, p40 is a protein kinase which is phosphorylated in vivo and in vitro at both tyrosine and serine residues; it may be a novel type of autophosphorylating tyrosine kinase, a bifunctional (serine/tyrosine-specific) protein kinase, or a serine kinase that is a substrate for an associated tyrosine kinase.

Novel Yeast Protein Kinase (YPK1 Gene Product) Is a 40-Kilodalton Phosphotyrosyl Protein Associated with Protein-Tyrosine Kinase Activity

Extracts of bakers' yeast (Saccharomyces cerevisiae) contain protein-tyrosine kinase activity that can be detected with a synthetic Glu-Tyr copolymer as substrate (G. Schieven, J. Thorner, and G.S. Martin, Science 231:390-393, 1986). By using this assay in conjunction with ion-exchange and affinity chromatography, a soluble tyrosine kinase activity was purified over 8,000-fold from yeast extracts. The purified activity did not utilize typical substrates for mammalian protein-tyrosine kinases (enolase, casein, and histones). The level of tyrosine kinase activity at all steps of each preparation correlated with the content of a 40-kDa protein (p40). Upon incubation of the most highly purified fractions with Mn-ATP or Mg-ATP, p40 was the only protein phosphorylated on tyrosine. Immunoblotting of purified p40 or total yeast extracts with antiphosphotyrosine antibodies and phosphoamino acid analysis of 32P-labeled yeast proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 40-kDa protein is normally phosphorylated at tyrosine in vivo. 32P-labeled p40 immunoprecipitated from extracts of metabolically labeled cells by affinity-purified anti-p40 antibodies contained both phosphoserine and phosphotyrosine. The gene encoding p40 (YPK1) was cloned from a yeast genomic library by using oligonucleotide probes designed on the basis of the sequence of purified peptides. As deduced from the nucleotide sequence of YPK1, p40 is homologous to known protein kinases, with features that resemble known protein-serine kinases more than known protein-tyrosine kinases. Thus, p40 is a protein kinase which is phosphorylated in vivo and in vitro at both tyrosine and serine residues; it may be a novel type of autophosphorylating tyrosine kinase, a bifunctional (serine/tyrosine-specific) protein kinase, or a serine kinase that is a substrate for an associated tyrosine kinase.

The Carboxy-Terminal Domains of erbB-2 and Epidermal Growth Factor Receptor Exert Different Regulatory Effects on Intrinsic Receptor Tyrosine Kinase Function and Transforming Activity

The erbB-2 gene product, gp185erbB-2, displays a potent transforming effect when overexpressed in NIH 3T3 cells. In addition, it possesses constitutively high levels of tyrosine kinase activity in the absence of exogenously added ligand. In this study, we demonstrate that its carboxy-terminal domain exerts an enhancing effect on erbB-2 kinase and transforming activities. A premature termination mutant of the erbB-2 protein, lacking the entire carboxy-terminal domain (erbB-2 delta 1050), showed a 40-fold reduction in transforming ability and a lowered in vivo kinase activity for intracellular substrates. When the carboxy-terminal domain of erbB-2 was substituted for its analogous region in the epidermal growth factor receptor (EGFR) (EGFR/erbB-2COOH chimera), it conferred erbB-2-like properties to the EGFR, including transforming ability in the absence of epidermal growth factor, elevated constitutive autokinase activity in vivo and in vitro, and constitutive ability to phosphorylate phospholipase C-gamma. Conversely, a chimeric erbB-2 molecule bearing an EGFR carboxy-terminal domain (erbB-2/EGFRCOOH chimera) showed reduced transforming and kinase activity with respect to the wild-type erbB-2 and was only slightly more efficient than the erbB-2 delta 1050 mutant. Thus, we conclude that the carboxy-terminal domains of erbB-2 and EGFR exert different regulatory effects on receptor kinase function and biological activity. The up regulation of gp185erbB-2 enzymatic activity exerted by its carboxy-terminal domain can explain, at least in part, its constitutive level of kinase activity.