Tyrosine-specific Phosphorylation Research Articles

The domain architecture of PtkA, the first tyrosine kinase from Mycobacterium tuberculosis, differs from the conventional kinase architecture

The discovery that MptpA (low-molecular-weight protein tyrosine phosphatase A) from Mycobacterium tuberculosis (Mtb) has an essential role for Mtb virulence has motivated research of tyrosine-specific phosphorylation in Mtb and other pathogenic bacteria. The phosphatase activity of MptpA is regulated via phosphorylation on Tyr128 and Tyr129 Thus far, only a single tyrosine-specific kinase, protein-tyrosine kinase A (PtkA), encoded by the Rv2232 gene has been identified within the Mtb genome. MptpA undergoes phosphorylation by PtkA. PtkA is an atypical bacterial tyrosine kinase, as its sequence differs from the sequence consensus within this family. The lack of structural information on PtkA hampers the detailed characterization of the MptpA-PtkA interaction. Here, using NMR spectroscopy, we provide a detailed structural characterization of the PtkA architecture and describe its intra- and intermolecular interactions with MptpA. We found that PtkA's domain architecture differs from the conventional kinase architecture and is composed of two domains, the N-terminal highly flexible intrinsically disordered domain (IDDPtkA) and the C-terminal rigid kinase core domain (KCDPtkA). The interaction between the two domains, together with the structural model of the complex proposed in this study, reveal that the IDDPtkA is unstructured and highly dynamic, allowing for a "fly-casting-like" mechanism of transient interactions with the rigid KCDPtkA This interaction modulates the accessibility of the KCDPtkA active site. In general, the structural and functional knowledge of PtkA gained in this study is crucial for understanding the MptpA-PtkA interactions, the catalytic mechanism, and the role of the kinase-phosphatase regulatory system in Mtb virulence.

Novel method demonstrates differential ligand activation and phosphatase-mediated deactivation of insulin receptor tyrosine-specific phosphorylation

Insulin receptor signaling is a complex cascade leading to a multitude of intracellular functional responses. Three natural ligands, insulin, IGF1 and IGF2, are each capable of binding with different affinities to the insulin receptor, and result in variable biological responses. However, it is likely these affinity differences alone cannot completely explain the myriad of diverse cellular outcomes. Ligand binding initiates activation of a signaling cascade resulting in phosphorylation of the IR itself and other intracellular proteins. The direct catalytic activity along with the temporally coordinated assembly of signaling proteins is critical for insulin receptor signaling. We hypothesized that determining differential phosphorylation among individual tyrosine sites activated by ligand binding or dephosphorylation by phosphatases could provide valuable insight into insulin receptor signaling. Here, we present a sensitive, novel immunoassay adapted from Meso Scale Discovery technology to quantitatively measure changes in site-specific phosphorylation levels on endogenous insulin receptors from HuH7 cells. We identified insulin receptor phosphorylation patterns generated upon differential ligand activation and phosphatase-mediated deactivation. The data demonstrate that insulin, IGF1 and IGF2 elicit different insulin receptor phosphorylation kinetics and potencies that translate to downstream signaling. Furthermore, we show that insulin receptor deactivation, regulated by tyrosine phosphatases, occurs distinctively across specific tyrosine residues. In summary, we present a novel, quantitative and high-throughput assay that has uncovered differential ligand activation and site-specific deactivation of the insulin receptor. These results may help elucidate some of the insulin signaling mechanisms, discriminate ligand activity and contribute to a better understanding of insulin receptor signaling. We propose this methodology as a powerful approach to characterize agonists and antagonists of the insulin receptor and can be adapted to serve as a platform to evaluate ligands of alternate receptor systems.

Abstract B253: The IGF1R inhibitor NVP-AEW541 disrupts a pro-survival and pro-angiogenic IGF-Stat3-HIF1 pathway in human glioblastoma cells

Abstract Glioblastomas (GBMs) typically develop hypoxic areas within the tumor mass, which for some cells results in necrosis, but for others leads to pleiotropic adaptive changes promoting tumor progression through activation of survival pathways, disruption of apoptosis, increase in invasive potential and neovascularization. The transcription factor HIF1 (hypoxia-inducible factor 1) is known to play a major role in directing this adaptive program, following stabilization of its inducible -subunit under hypoxic conditions; however, HIF1 activity can also be enhanced by oxygen-independent mechanisms upon activation of growth factor receptors and their downstream signal transduction pathways. The present study investigates a) the possible presence of a regulatory loop linking IGF1R and HIF1 in human glioblastoma cells; b) the role played by the signal transducer and activator of transcription 3 (Stat3) in this pathway; and c) the ability of the IGF1R inhibitor NVP-AEW541 (Novartis Pharma, Basel, CH) to disrupt this pathway. We show that activation of IGF1R by its ligand IGF1 causes Stat3 activation, increased levels of HIF1α and increased release of VEGF, the product of one of the major target genes for HIF1 transcriptional activity. Treatment with NVP-AEW541 reverses all these changes, and so does transfection of the cells with a dominant negative form of Stat3. Interestingly, we also observe that IGF-independent activation of HIF1, obtained in the presence of the hypoxia mimic CoCl2, results in IGF1R activation, thereby indicating that a two-way relationship exists between growth factor receptor activation and increase in HIF1 transcriptional activity; NVP-AEW541 treatment significantly reduces both HIF1α protein levels and IGF1R tyrosine-specific phosphorylation in CoCl2-treated cells. Because cells surviving under hypoxic condition often become resistant to radiotherapy and chemotherapy, modulation of the IGF1R-HIF1 pathway could be a viable approach to increase the effectiveness of conventional cytotoxic agents; as GBM typically presents hypoxic areas, this strategy might prove particularly valuable in improving the poor prognosis of patients affected by this tumor type. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B253.

A site-specific, multiplexed kinase activity assay using stable-isotope dilution and high-resolution mass spectrometry

Most kinases are capable of recognizing and phosphorylating peptides containing short, linear sequence motifs. To measure the activation state of many kinases from the same cell lysate, we created a multiplexed, mass-spectrometry-based in vitro kinase assay. Ninety chemically synthesized peptides derived from well-characterized peptide substrates and in vivo phosphorylation sites with either known or previously unidentified upstream kinases were reacted individually in a plate format with crude cell lysates and ATP. Phosphorylation rates were directly measured based on the addition of 90 same-sequence, site-specific phosphopeptides enriched in stable isotopes to act as ideal quantitative internal standards for analysis by liquid chromatography coupled to tandem mass spectrometry. This approach concurrently measured up to 90 site-specific peptide phosphorylation rates, reporting a diagnostic fingerprint for activated kinase pathways. We applied this unique kinome-activity profiling strategy in a variety of cellular settings, including mitogen stimulation, cell cycle, pharmacological inhibition of pathways, and to a panel of breast cancer cell lines. Finally, we identified the source of activity for a peptide (derived from a PI3K regulatory subunit) from our library. This peptide substrate demonstrated mitotic and tyrosine-specific phosphorylation, which was confirmed to be a novel Src family kinase site in vivo.

Pyridopyrimidine derivatives as inhibitors of cyclic nucleotide synthesis: Application for treatment of diarrhea

Acute secretory diarrhea induced by infection with enterotoxigenic strains of Escherichia coli involves binding of stable toxin (STa) to its receptor on the intestinal brush border, guanylyl cyclase type C (GC-C). Intracellular cGMP is elevated, inducing increase in chloride efflux and subsequent accumulation of fluid in the intestinal lumen. We have screened a library of compounds and identified a pyridopyrimidine derivatives {5-(3-bromophenyl)-1,3-dimethyl-5,11-dihydro-1H-indeno[2',1':5,6]pyrido[2,3-d]pyrimidine-2,4,6-trione; BPIPP} as an inhibitor of GC-C that can suppress STa-stimulated cGMP accumulation by decreasing GC-C activation in intact T84 human colorectal carcinoma cells. BPIPP inhibited stimulation of guanylyl cyclases, including types A and B and soluble isoform in various cells. BPIPP suppressed stimulation of adenylyl cyclase and significantly decreased the activities of adenylyl cyclase toxin of Bordetella pertussis and edema toxin of Bacillus anthracis. The effects of BPIPP on cyclic nucleotide synthesis were observed only in intact cells. The mechanism of BPIPP-dependent inhibition appears to be complex and indirect, possibly associated with phospholipase C and tyrosine-specific phosphorylation. BPIPP inhibited chloride-ion transport stimulated by activation of guanylyl or adenylyl cyclases and suppressed STa-induced fluid accumulation in an in vivo rabbit intestinal loop model. Thus, BPIPP may be a promising lead compound for treatment of diarrhea and other diseases.

The differentiation/retrodifferentiation program of human U937 leukemia cells is accompanied by changes of VCP/p97

BackgroundRetrodifferentiation and regained proliferative capacity of growth-arrested human leukemic cells after monocyte-like differentiation requires proteolytic activities together with distinct regulatory factors. The AAA ATPase valosin-containing protein (VCP/p97) contributes to protein degradation and cell cycle regulation, respectively, and it was of interest to study a possible role of VCP/p97 during this myelomonocytic differentiation and retrodifferentiation.ResultsSeparation of autonomously proliferating human U937 myeloid leukemia cells by centrifugal elutriation demonstrated unaltered VCP/p97 expression levels throughout distinct phases of the cell cycle. However, phorbol ester-induced G0/G1 cell cycle arrest in differentiating human U937 leukemia cells was associated with a significantly increased protein and mRNA amount of this AAA ATPase. These elevated VCP/p97 levels progressively decreased again when growth-arrested U937 cells entered a retrodifferentiation program and returned to the tumorigenic phenotype. Whereas VCP/p97 was observed predominantly in the cytosol of U937 tumor and retrodifferentiated cells, a significant nuclear accumulation appeared during differentiation and G0/G1 growth arrest. Analysis of subcellular compartments by immunoprecipitations and 2D Western blots substantiated these findings and revealed furthermore a tyrosine-specific phosphorylation of VCP/p97 in the cytosolic but not in the nuclear fractions. These altered tyrosine phosphorylation levels, according to distinct subcellular distributions, indicated a possible functional involvement of VCP/p97 in the leukemic differentiation process. Indeed, a down-modulation of VCP/p97 protein by siRNA revealed a reduced expression of differentiation-associated genes in subsequent DNA microarray analysis. Moreover, DNA-binding and proliferation-associated genes, which are down-regulated during differentiation of the leukemic cells, demonstrated elevated levels in the VCP/p97 siRNA transfectants.ConclusionThe findings demonstrated that monocytic differentiation and G0/G1 growth arrest in human U937 leukemia cells was accompanied by an increase in VCP/p97 expression and a distinct subcellular distribution to be reverted during retrodifferentiation. Together with a down-modulation of VCP/p97 by siRNA, these results suggested an association of this AAA ATPase in the differentiation/retrodifferentiation program.

Survival Signaling in Type II Pneumocytes Activated by Surfactant Protein-A

Surfactant-associated protein-A (SP-A) is a component of pulmonary surfactant that acts as a cytokine through interaction with a cell-surface receptor (SPAR) on lung epithelial cells. SP-A regulates important physiological processes including surfactant secretion, gene expression, and protection against apoptosis. Tyrosine kinase and PI3K inhibitors block effects of SP-A, suggesting that SPAR may be a receptor tyrosine kinase and activate the PI3K–PKB/Akt pathway. Here we report that SP-A treatment leads to rapid tyrosine-specific phosphorylation of several important proteins in lung epithelial cells including insulin receptor substrate-1 (IRS-1), an upstream activator of PI3K. Analysis of anti-apoptotic signaling species downstream of IRS-1 showed activation of PKB/Akt but not of MAPK. Phosphorylation of IκB was minimally affected by SP-A as was NFκB gel shift activity. However, FKHR was rapidly phosphorylated in response to SP-A and its DNA-binding activity was significantly reduced. Since FKHR is pro-apoptotic, this may play an important role in signaling the anti-apoptotic effects of SP-A. Therefore, we have characterized survival-enhancing signaling activated by SP-A leading from SPAR through IRS-1, PI3K, PKB/Akt, and FKHR. The activity of this pathway may explain, in part, the resilience of type II cells to lung injury and their survival to repopulate alveolar epithelium after peripheral lung damage.

Role of Tyrosine Phosphorylation in Ligand-independent Sequestration of CXCR4 in Human Primary Monocytes-Macrophages

The chemokine stromal cell-derived factor (SDF)-1 and its receptor, CXCR4, play important roles in human immunodeficiency virus type 1 (HIV-1) pathophysiology, leukocyte trafficking, inflammation, hematopoiesis, embryogenesis, angiogenesis, and cancer metastasis. The effects of cytokines on the regulation of CXCR4 function were investigated in human primary monocytes-macrophages. The expression of functional CXCR4 on the cell surface was demonstrated by the detection of ligand-induced Ca(2+) mobilization, chemotaxis, and ligand-induced receptor endocytosis. Surface CXCR4 expression was down-regulated by cytokines interleukin-4 (IL-4), IL-13, and granulocyte-macrophage colony-stimulating factor (GM-CSF) and up-regulated by IL-10 and transforming growth factor-beta 1. Down-regulation was mediated post-translationally, in the absence of protein degradation, through an endocytotic mechanism. In contrast to SDF-1 alpha-induced CXCR4 endocytosis, cytokine-induced endocytosis of this receptor was independent of actin filament polymerization. GM-CSF increased the expression of G protein-coupled receptor kinase 3 (GRK3), beta-arrestin-1, Pyk2, and focal adhesion kinase (FAK). Cytokine treatment also increased the total and tyrosine-specific phosphorylation of CXCR4 as well as the phosphorylation of FAK on tyrosine 397. It also induced the formation of GRK3.CXCR4 or FAK.CXCR4 complexes. Infection of macrophages by primary R5X4 and X4 isolates of HIV-1 was inhibited by IL-4, IL-13, and GM-CSF, an effect that was associated with down-regulation of surface CXCR4 expression. These data indicate that ligand-dependent and ligand-independent endocytoses of CXCR4 are mediated by different mechanisms. Cytokine-induced endocytosis of chemokine receptors may be of therapeutic value in HIV-1 infection, inflammation, tumor metastasis, and defective hematopoiesis.

Interleukin 6 and Haemostasis

Interleukin 6 and Haemostasis

Tyrosine kinase expression in the inner ear of mice

Tyrosine-specific phosphorylation events serve to modulate cell to cell interactions during the normal growth and development of many vertebrate tissues. Numerous studies have been performed on the involvement of growth factor receptor tyrosine kinases (RTKs) in inner ear function. Many RTKs, however, have only been recently identified, and their expression patterns have not yet been systematically examined in all tissues. In this study, we surveyed mouse inner ear transcript sequences for the presence of RTKs using a degenerate-PCR based methodology. Isolates encoding RTKs that had and had not been previously identified in the inner ear were obtained. Our data reveals the presence of numerous RTKs in the inner ear that have not yet been studied in the context of inner ear and demonstrates the usefulness of RT-PCR analysis in identifying genes involved in inner ear function.

SV40-immortalized human keratinocytes as an in vitro model system for studying environmental carcinogens

We have extensively characterized the properties of an in vitro model system of human epidermal keratinocytes immortalized by the oncogenic virus, SV40. Here we show how the unique features of this system can be employed as a tool for studying the effects of both mutagenic and nongenotoxic carcinogens on human epithelial cells. 1. SV40-infected human keratinocyte lines at low serial passage have been used as an in vitro system to study the effects of arsenic. Arsenic as both arsenate and arsenite was found to stimulate cell growth over test periods of 5 and 7 days at submicromolar concentrations. Tyrosine specific phosphorylation was examined in immunoprecipitates from cells exposed to arsenic at growth stimulatory concentrations. A number of phosphorylated species which were found to be present in both arsenite and arsenate treated cells were not observed in the untreated cells. Concomitantly, analyses of cell cycle kinetics by fluorescence activated cell sorting (FACS) revealed that the stimulation of cell growth might be accounted for by a relative shortening of the G 1 phase of the cell cycle. 2. The mutagenic effects of the methylating carcinogen, MNNG have been studied using the integrated viral origin of replication in a line of SV40-immortalized human keratinocytes as a target. Sequence data from this system revealed an unusual spectrum of base alterations as compared with data obtained with bacteria and other mammalian cell types including single nucleotide insertions and deletions and base-pair inversions.

Increased Zap-70 Association with CD3ζ in CD4 T Cells from Old Mice

Aging diminishes the amount of phosphotyrosine in the CD3ζ chains of resting and activated mouse CD4 T cells by about threefold and might therefore be expected to a corresponding decline in Zap-70 association with CD3ζ and in Zap-70 kinase function in CD3ζ complexes. We show here that aging leads, unexpectedly, to an approximately twofold increase in the amount of Zap-70 associated with CD3ζ in resting CD4 T cells. There is, however, no effect of age on total intracellular Zap-70 content. Cross-linking CD3 to CD4 leads to an increase of only 50% in the functional activity of Zap-70 in CD3ζ complexes from freshly isolated CD4 T cells of young donors. Compared to Jurkat and HT-2 cells, fresh T cells show both higher baseline levels and lower induced levels of Zap-70 function in CD3ζ complexes. CD4 T cells from old mice have baseline levels of Zap-70 activity similar to those seen in activated T cells from young mice, and these levels do not increase after CD3/CD4 cross-linking. Tyrosine-specific phosphorylation of Zap-70 is also higher at rest in old T cells than in young T cells and inducible only in cells from young donors. These data suggest that age-related defects in T cell activation are not likely to be attributable simply to a decline in Zap-70 association with CD3ζ or to diminished Zap-70 phosphorylation. The increase with age in CD3ζ-Zap association, despite the loss with age in CD3ζ tyrosine phosphorylation, suggests that the pattern of tyrosine phosphate groups among CD3ζ ITAM groups may be different in T cells from young and old donors or that access to ITAM regions within CD3ζ may be blocked by inter- or intramolecular steric hindrance in young CD4 T cells.

Effects of the protein tyrosine kinase inhibitor, herbimycin A, on prolactin gene expression in GH 3 and 235-1 pituitary tumor cells

The high basal level of prolactin (PRL) gene expression in rat pituitary GH 3 cells is maintained through the spontaneous activity of voltage-sensitive calcium channels (VSCCs). This can be observed experimentally by addition of 0.5 mM CaCl 2 to GH 3 cells cultured in a low calcium, serum-free medium. CaCl 2 specifically induces PRL gene expression and this induction is inhibited by VSCC blockers. PRL gene expression is also stimulated by several hormones and growth factors. In the present study, we examined the effects of tyrosine kinase inhibitors on the ability of CaCl 2, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and thryrotropin-releasing hormone (TRH) to increase PRL mRNA levels. Of several PTK inhibitors used, one PTK inhibitor, herbimycin A, specifically inhibited the CaCl 2-induced increase in cytoplasmic and nuclear prolactin (PRL) mRNA without affecting cell viability, cell-cell and cell-matrix adhesion, or the expression of several other genes. The effects of herbimycin A were reversible. In cells pretreated with herbimycin A, PRL mRNA levels were reduced by 69±12% ( P<0.001; n=4).Western blot analysis using anti-phosphotyrosine antibody revealed a decrease of 91±1% ( P<0.001; n=4) in the phosphotyrosine content of proteins in the molecular weight range of 130–160 kDa. After changing the medium back to SFM plus 0.5 mM CaCl 2, levels of PRL mRNA increased over a period of several hours, and this increase was accompanied by the tyrosine phosphorylation of two or more proteins in the approximate size range of 130–160 kDa. Herbimycin A also inhibited PRL gene expression in the independently-derived 235-1 lactotrope cell line and lowered the tyrosine specific phosphorylation of protein(s) in a similar size range. Herbimycin A inhibited the ability of bFGF, EGF and TRH to stimulate PRL gene expression in GH 3 cells. Again, in cells pretreated with herbimycin A, bFGF induced a reappearance of tyrosine-specific phosphorylation, followed by a reappearance of PRL mRNA. These findings provide evidence for a role for at least one PTK which is necessary for basal and stimulated PRL gene expression.

Tyrosine kinase signaling pathways control the expression of retinoic acid receptor-alpha in SK-BR-3 breast cancer cells.

Breast carcinomas are frequently characterized by hyperactivated c-erbB receptor tyrosine kinase signaling. Combination of anti-proliferative retinoids with growth-inhibitory c-erbB-specific agents might induce therapeutic benefit. We demonstrate close interactions between the c-erbB and the retinoic acid receptor system in SK-BR-3 breast cancer cells. Epidermal growth factor and heregulin-beta1 activate c-erbB receptors and dose- and time-dependently up-regulate retinoic acid receptor-alpha (RAR-alpha) mRNA. Similar effects have been found for the growth-inhibitory c-erbB-2 receptor tyrosine kinase-activating antibody 4D5 and the tyrosine phosphatase inhibitor orthovanadate. In contrast, the tyrosine kinase-inhibitor herbimycin A reduces tyrosine-specific protein phosphorylation and down-regulates RAR-alpha. Our data demonstrate that the expression of RAR-alpha, which represents a key mediator of the anti-proliferative effects of retinoids in breast cancer cells, is regulated by modulators of tyrosine kinase signaling. The levels of RAR-beta and -gamma mRNAs, however, are not affected by such agents.

Role of tyrosine specific phosphorylation of cellular proteins, especially EGF receptor and p125FAK in human lung cancer cells

To clarify the role of tyrosine phosphorylation of cellular proteins in human lung cancer cells, phosphotyrosine (PTYR)-containing proteins in lung cancer cell lines and in paired tissues resected from cancerous and normal lungs were studied by immunoblotting with an anti-PTYR antibody. We found that the profiles of protein phosphorylation were very similar among those cell lines which had different histological features, The major PTYR-containing proteins (180–190 KDa, 120–130 KD, and 95–100 KDa) were detected in lung cancer cell lines. The expression of EGF receptor (EGF-r) (p185) and c-erb B2 protein, and tyrosine phosphorylation of p125FAK were examined in cancerous lung tissues and normal lung tissues. In surgical specimens, approximately half of the samples of lung cancer tissues showed clear elevation of tyrosine phosphorylation. In these cancerous tissues, no clear amplification of EGF-r and c-erb B2 protein expression was observed. However, elevation of tyrosine phosphorylation of p125FAK was observed in cancerous lung tissues but not in normal lung tissues, and its phosphorylation was closely correlated with the nodal involvement of cancer and disease-free survival time. These results suggested that the intracellular signaling pathway via tyrosine phosphorylation plays a role in the generation and immortalization of lung cancer, and assessment of tyrosine phosphorylation of cellular proteins, especially p125FAK, may be available clinically as a prognostic factor.

Acidification and recovery results in nuclear accumulation of supravital dyes during interphase.

Recent studies using real time imaging demonstrated relative nuclear insulation for ion-size particles. We show here that acidification and recovery converted the insulated interphase nuclei of KB carcinoma and nontumorigenic Chang cells into intense nuclear accumulating states marked by sequestration of the exogenous supravital dyes neutral red, methylene blue, and brilliant cresyl blue. The phenomenon was not affected by Na(+)-free and HCO3(-)-free conditions nor by the presence of cationic and anionic antiport regulators of cytosolic pH. Cytological, microspectrophotometric, and flow cytometric evaluation of whole cell populations showed that the nuclear influx was abolished by omitting the pH recovery response, and by modulating the recovery response. The abolition of nuclear influx in the presence of the P-ATPase and Fzero-ATPase inhibitors, vanadyl(IV) ions and oligomycin, respectively, suggest that H(+)-translocating ATPase pumps are involved in regulating cytosolic acidification in Na(+)-free and HCO3-conditions vanadyl(IV) inhibited nuclear uptake of supravital dyes in a dose dependent manner. Nuclear uptake of dyes, however, was not affected by up to 1 mM of genistein even though tyrosine-specific phosphorylation and DNA synthesis were abolished. Upgradient nuclear influx involving proton pump is novel. KB cancer cells and nontumorigenic Chang cells had differential dye accumulations induced by acidification and recovery.

Point Mutations in the Conserved Box 1 Region Inactivate the Human Granulocyte Colony-Stimulating Factor Receptor for Growth Signal Transduction and Tyrosine Phosphorylation of p75c-rel

The human granulocyte colony-stimulating factor receptor (hG-CSFR) belongs to the cytokine receptor superfamily. As with other members of this family, the cytoplasmic domain of hG-CSFR lacks intrinsic tyrosine kinase activity. To identify critical regions mediating growth signal transduction by hG-CSFR, deletions or site-directed amino acid substitutions were introduced into the cytoplasmic domain of hG-CSFR, and the mutant cDNAs were transfected into the murine interleukin-3 (IL-3)-dependent Ba/F3 and FDCP cell lines. Truncation of the carboxy-terminal end of the receptor to the membrane-proximal 53 amino acids of the cytoplasmic domain, which retained the conserved Box 1 and Box 2 sequence motifs, decreased the ability of hG-CSFR to transduce G-CSF-mediated growth signals without an associated loss in receptor binding affinity. Substitution of proline by alanine at amino acid positions 639 and 641 within Box 1 completely abolished the G-CSF-mediated growth signal. Rapid induction of tyrosine phosphorylation of several cellular proteins, including a 75-kD protein (p75) identified as c-rel, was an early event associated with transduction of proliferative signals by hG-CSFR in Ba/F3 transfectants. Mutant receptors containing Pro-to-Ala substitutions that inactivated the receptor for mitogenic activity also inactivated the receptor for tyrosine-specific phosphorylation of p75. These results show that the conserved Box 1 sequence motif (amino acids 634 to 641) is critical for mitogenesis and activation of cellular tyrosine kinases by hG-CSFR.

The tyrosine specific phosphorylation of a 14 KDA protein seems to be associated with growth suppression of the jejunal epithelium in diabetic rats: [formula omitted], J.L. Hoffman, T.C. Stephen and M.K. Younoszai, Depts of Pediatrics Gastroenterology and Biochemistry, University of Louisville, KY

The tyrosine specific phosphorylation of a 14 KDA protein seems to be associated with growth suppression of the jejunal epithelium in diabetic rats: [formula omitted], J.L. Hoffman, T.C. Stephen and M.K. Younoszai, Depts of Pediatrics Gastroenterology and Biochemistry, University of Louisville, KY

Tyrosine phosphorylation of protein kinase C-delta in response to its activation.

Retroviral vectors containing five different protein kinase C (PKC) isoenzymes (alpha, delta, epsilon, eta, zeta) were expressed in 32D hematopoietic cells and NIH-3T3 fibroblasts. In an effort to investigate signaling events regulated by PKC activation, we analyzed whether tyrosine phosphorylation of cellular proteins would occur after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of the various transfectants. While no detectable tyrosine-specific phosphorylation was observed after treatment of the majority of the transfectants, pronounced TPA-dependent tyrosine phosphorylation of an 82-kDa protein was detected in the 32D/PKC-delta and NIH-3T3/PKC-delta lines. Interestingly, the 82-kDa substrate proved to be PKC-delta itself. Tyrosine phosphorylation of purified PKC-delta by src family or receptor tyrosine kinases in vitro enhanced PKC-delta activity, suggesting that tyrosine phosphorylation of PKC-delta may positively affect its function.

Suramin, an Experimental Chemotherapeutic Drug, Irreversibly Blocks T Cell CD45-Protein Tyrosine Phosphatase in Vitro

Suramin has long been used for the treatment of Gambian and Rhodesian trypanosomiasis and oncocerciasis. More recently, the demonstration that suramin inhibits DNA polymerases, reverse transcriptase and the lymphocyte terminal deoxynucleotidyl transferase has led to its clinical trials for the treatment of AIDS and cancer. The precise nature of suramin′s anti-neoplastic action is not clear at this time. Suramin rapidly alters the tyrosine-specific phosphorylation of cellular proteins in many cancer cell lines. Here we demonstrate that suramin strongly inhibits the activity of CD45, the principal tyrosine specific protein phosphatase of T lymphocytes. Suramin-induced inactivation of CD45 is noncompetitive, irreversible and complete within 10 min. The ability of suramin to block CD45 mediated phosphatase function provides both new insight into the mechanism of action of this agent and a useful new probe for studies of T cell activation.