Detectable Tyrosine Phosphorylation Research Articles

Thrombopoietin Induces Tyrosine Phosphorylation and Activation of the Janus Kinase, JAK2

Thrombopoietin (TPO) is a recently characterized growth and differentiation factor for megakaryocytes and platelets that exerts its effects via the receptor, c-MpI. This receptor is a member of the hematopoietin receptor superfamily and is essential for megakaryocyte maturation; however, the molecular mechanisms of TPO and c-MpI action have not been elucidated. Recently, the Janus kinases have emerged as important elements in signaling via this family of receptors. In this report, we show that, in the M07e megakaryocytic cell line, which expresses c-MpI and proliferates in response to TPO, TPO induces phosphorylation of a number of substrates between 80 and 140 kD. Specifically, we show that stimulation with TPO induces the rapid tyrosine phosphorylation of a 130-kD protein that we identify as the Janus kinase, JAK2. However, no detectable tyrosine phosphorylation of JAK1, JAK3, or TYK2 was observed. TPO also induced activation of JAK2 phosphotransferase activity in vitro. Taken together, these data indicate that JAK2 likely plays a key role in TPO-mediated signal transduction.

Detection of Phosphorylation by Immunological Techniques

Phosphorylation of unlabeled proteins can be detected using immunological or enzymatic techniques. Anti-phosphotyrosine antibodies are used with immunoblots to detect tyrosine phosphorylation. This unit presents a protocol employing anti-phosphotyrosine antibodies with detection by either (125)I-labeled protein A or enhanced chemiluminescence (ECL).

Gp120 ligation of CD4 induces p56lck activation and TCR desensitization independent of TCR tyrosine phosphorylation.

HIV gp120 binding to CD4 suppresses TCR function. The molecular mechanism of this anergizing effect is incompletely understood. Studies reported here reveal that CD4 ligation initiates p56lck activation and renders human peripheral T cells and HPB-ALL cells hyporesponsive to Ag receptor stimulation, as indicated by the failure of TCR binding ligands to induce either protein tyrosine phosphorylation or elevation of intracellular-free calcium concentration. To approach the possibility that p56lck-mediated tyrosine phosphorylation of specific sites within TCR zeta-chain might be involved in the gp120-induced TCR signaling defect, we tested the kinase's ability to phosphorylate various zeta peptides. Kinetics analyses indicate that peptides derived from the in vitro autophosphorylation site of p56lck Y394 and two sites within zeta, Y84, and Y152, are equally effective substrates for p56lck, whereas p56fyn prefers a substrate peptide derived from a different site within zeta, Y142. Although these data are consistent with the possibility that gp120-mediated signal disruption of TCR could be due to p56lck phosphorylation of Y84 and Y152 residues within zeta, further experiments revealed that gp120 does not induce detectable zeta tyrosine phosphorylation under conditions in which it disrupts TCR signaling. These data indicate that gp120 can induce uncoupling of TCR from the earliest events in signal transduction and that this effect can be mediated by a mechanism other than tyrosine phosphorylation of TCR zeta-chain.

Treatment of IM-9 cells with human growth hormone (GH) promotes rapid disulfide linkage of the GH receptor.

Human GH (hGH) is believed to elicit its signal by promoting dimerization of the hGH receptor (hGHR). In this study, we examined a covalent linkage of receptors induced by hGH treatment of IM-9 cells. hGH induced a time- and concentration-dependent appearance of a disulfide-linked species of 215-230 kilodaltons, designated p215-230, that at 37 C was long-lived (> 1 h). p215-230 was confirmed to contain the hGHR (115-140 kilodaltons) as at least one of its constituents by two-dimensional diagonal sodium dodecyl sulfate-polyacrylamide gel electrophoresis. hGH induction of p215-230 required intact cells and was inhibitable by pretreatment of cells with N-ethylmaleimide (NEM), a sulfhydryl-reactive alkylating agent. NEM pretreatment did not, however, prevent hGH-dependent formation of a nondisulfide-linked p215-230 form, which was detected in NEM-pretreated hGH-stimulated cells by chemical cross-linking of detergent cell extracts. The disulfide-linked form of the hGHR accounted for a substantial fraction of the receptors that became tyrosine phosphorylated early into hGH treatment. However, formation of the disulfide-linked hGHR was not blocked by attenuation of tyrosine kinase activation, in that pretreatment of cells with staurosporine (1.25 microM) prevented detectable hGH-induced tyrosine phosphorylation without preventing the appearance of p215-230. These findings indicate that hGH induces its receptor to form a noncovalently associated complex, which then undergoes a rapid transition to a disulfide-linked form. These processes may have relevance to hGH signaling and/or hGHR trafficking.

Direct stimulation of Vav guanine nucleotide exchange activity for Ras by phorbol esters and diglycerides.

We recently identified Vav as a Ras-activating guanine nucleotide exchange factor (GEF) stimulated by a T-cell antigen receptor-coupled protein tyrosine kinase (PTK). Here, we describe a novel, protein kinase-independent alternative pathway of Vav activation. Phorbol ester, 1,2-diacylglycerol, or ceramide treatment of intact T cells, Vav immunoprecipitates, or partially purified Vav generated by in vitro translation or COS-1 cell transfection stimulated the Ras exchange activity of Vav in the absence of detectable tyrosine phosphorylation. GEF activity of gel-purified Vav was similarly stimulated by phorbol myristate acetate (PMA). Stimulation was resistant to PTK and protein kinase C inhibitors but was blocked by calphostin, a PMA and diacylglycerol antagonist. In vitro-translated Vav lacking its cysteine-rich domain, or mutated at a single cysteine residue within this domain (C528A), was not stimulated by PMA but was fully activated by p56lck. This correlated with increased binding of radiolabeled phorbol ester to COS-1 cells expressing wild-type, but not C528A-mutated, Vav. Thus, Vav itself is a PMA-binding and -activated Ras GEF. Recombinant interleukin-1 alpha stimulated Vav via this pathway, suggesting that diglyceride-mediated Vav activation may couple PTK-independent receptors which stimulate production of lipid second messengers to Ras in hematopoietic cells.

Direct stimulation of Vav guanine nucleotide exchange activity for Ras by phorbol esters and diglycerides.

We recently identified Vav as a Ras-activating guanine nucleotide exchange factor (GEF) stimulated by a T-cell antigen receptor-coupled protein tyrosine kinase (PTK). Here, we describe a novel, protein kinase-independent alternative pathway of Vav activation. Phorbol ester, 1,2-diacylglycerol, or ceramide treatment of intact T cells, Vav immunoprecipitates, or partially purified Vav generated by in vitro translation or COS-1 cell transfection stimulated the Ras exchange activity of Vav in the absence of detectable tyrosine phosphorylation. GEF activity of gel-purified Vav was similarly stimulated by phorbol myristate acetate (PMA). Stimulation was resistant to PTK and protein kinase C inhibitors but was blocked by calphostin, a PMA and diacylglycerol antagonist. In vitro-translated Vav lacking its cysteine-rich domain, or mutated at a single cysteine residue within this domain (C528A), was not stimulated by PMA but was fully activated by p56lck. This correlated with increased binding of radiolabeled phorbol ester to COS-1 cells expressing wild-type, but not C528A-mutated, Vav. Thus, Vav itself is a PMA-binding and -activated Ras GEF. Recombinant interleukin-1 alpha stimulated Vav via this pathway, suggesting that diglyceride-mediated Vav activation may couple PTK-independent receptors which stimulate production of lipid second messengers to Ras in hematopoietic cells.

Tyrosine kinase JAK1 is associated with the granulocyte-colony-stimulating factor receptor and both become tyrosine-phosphorylated after receptor activation.

Granulocyte-colony-stimulating factor (G-CSF) stimulates the proliferation and differentiation of cells of the neutrophil lineage by interaction with a specific receptor. Early signal transduction events following G-CSF receptor activation were studied. We detected tyrosine phosphorylation of both the G-CSF receptor and the protein tyrosine kinase JAK1 following G-CSF binding to the human G-CSF receptor. In vitro, the kinase activity of JAK1 was increased by G-CSF stimulation. Coimmunoprecipitation of JAK1 with the G-CSF receptor suggested a physical association which existed prior to G-CSF stimulation.

Cultured hippocampal neurons show responses to BDNF, NT-3, and NT-4, but not NGF

To investigate the possibility of neurotrophins acting directly on hippocampal neurons, we first examined expression of the trk receptors in sections of adult rat brain and in cultures of embryonic rat hippocampus, and then investigated general and specific responses of cultured hippocampal neurons to each of the neurotrophins. In situ hybridization studies indicated high levels of expression of trkB and trkC but not trkA in pyramidal cells, dentate granule neurons, and scattered interneurons. Cultures of embryonic day 18 (E18) hippocampal neurons were also found by Northern analysis to express trkB and trkC but not trkA, indicating potential responsiveness to brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4, but not NGF. Phosphorylation experiments indeed showed that BDNF, NT-3, and NT-4 produced rapid tyrosine phosphorylation of Trk proteins, as detected by immunoprecipitation using a pan-Trk-specific antibody, whereas NGF produced no detectable tyrosine phosphorylation in hippocampal cultures. Similarly, all of the neurotrophins, except NGF, induced expression of c-fos mRNA and c-fos protein in these cultures. c-Fos protein induction was detectable in approximately 40-50% of the cells. While we observed no major effect of any of the neurotrophins upon the survival of E18 hippocampal neurons, BDNF, NT-3, and NT-4, but not NGF, produced marked increases in the number of neurons expressing detectable levels of either calbindin or AChE. NT-3 produced the greatest increase in the number of calbindin-positive neurons, whereas BDNF and NT-4 produced the greater increase in the number of AChE-positive neurons. Our results suggest that several of the neurotrophins have important effects in the differentiation and maintenance of function of subpopulations of hippocampal neurons.

Specific activity of phosphatidylinositol 3-kinase is increased by insulin stimulation.

We investigated whether phosphatidylinositol 3-kinase (PI3K) is phosphorylated and whether its specific activity is increased by insulin stimulation in vivo using Fao cells and antibodies raised against the 85 kDa subunit of PI3K, insulin-receptor substrate-1 (IRS-1), and phosphotyrosine (pTyr). PI3K activity was detected in the immunoprecipitate produced with anti-PI3K at a basal state. The activity was increased 2-3-fold by insulin stimulation, although the protein concentration of kinase in the anti-PI3K immunoprecipitates was the same before and after insulin stimulation. Both anti-pTyr and anti-IRS-1 antibodies immunoprecipitated the kinase activity only after insulin stimulation. After the first immunoprecipitation with anti-pTyr, the supernatant was immunoprecipitated once more with anti-PI3K. PI3K activity in the second immunoprecipitate revealed little difference between the basal and insulin-stimulated states, suggesting that most of the insulin-activated portion of PI3K was precipitated by anti-pTyr. Both IRS-1 and the insulin-receptor beta-subunit (95 kDa) were phosphorylated on tyrosine residues by insulin stimulation and immunoprecipitated with anti-pTyr. However, phosphorylation of neither subunit of PI3K (85 kDa or 110 kDa) was detectable in the immunoprecipitate produced with anti-pTyr. The 185 kDa pTyr-containing protein was immunoprecipitated with anti-PI3K after insulin stimulation, although there was little phosphorylation of the 85 kDa protein. pTyr in the 110 kDa protein immunoprecipitated with anti-PI3K was below detectable levels. These results indicate that the specific activity of PI3K is increased by insulin stimulation without detectable tyrosine phosphorylation of PI3K itself in Fao cells. The majority of the insulin-activated portion of PI3K is associated with pTyr-containing proteins including IRS-1, which suggests that this is important for activation of PI3K by insulin.

Antiserum raised against a synthetic phosphotyrosine-containing peptide selectively recognizes p185neu/erbB-2 and the epidermal growth factor receptor.

Rabbits were immunized with a synthetic phosphopeptide corresponding to a major autophosphorylation site of p185neu/erbB2 to determine the feasibility of producing tyrosine-phosphopeptide-specific antibodies. A series of adsorption and affinity chromatography steps were used to select antibodies with the desired reactivity. Immunoblot experiments showed that the resulting serum is highly specific for tyrosine-phosphorylated forms of p185 and the related epidermal growth factor receptor. The serum recognized these two receptors selectively when compared to five other receptor tyrosine kinases and several phosphorylated substrates. The serum is compatible with tissue-based assays since it detected tyrosine phosphorylation of the epidermal growth factor receptor in immunofluorescence experiments on permeabilized cells. The generality of the procedures used means that similar anti-tyrosine phosphopeptide sera can be produced that recognize other tyrosine kinases and substrates. Such sera will have numerous applications in research and clinical settings.

Role of protein kinase activation in the induction of B cell adhesion by MHC class II ligands.

Engagement of MHC class II (Ia) molecules on B cells induces tyrosine phosphorylation, phosphoinositide turnover, elevation of intracellular calcium concentrations, and a rise in cAMP levels. However, a role for these biochemical signals in mediating functional responses induced by Ia ligands remains largely undefined. In this study, we utilized the induction of B cell adhesion by Ia ligands to demonstrate a role for signals transduced via Ia molecules in the generation of a functional response. Ia ligands that induced B cell aggregation induced tyrosine phosphorylation, whereas Ia ligands that did not induce B cell aggregation failed to induce any detectable tyrosine phosphorylation. Ia-induced B cell aggregation and tyrosine phosphorylation were inhibited by genistein and by herbimycin A, inhibitors of tyrosine kinases (PTK). Sphingosine and calphostin C, inhibitors of protein kinase C (PKC), also inhibited Ia-induced adhesion whereas HA1004, an inhibitor of cyclic nucleotide-dependent kinases, did not. Ia ligands induced both LFA-1-dependent and LFA-1-independent B cell adhesion. These two pathways of cell adhesion differed in their requirement for activation signals. PKC activation was sufficient for LFA-1-dependent adhesion, whereas LFA-1-independent adhesion required independent phosphorylation events mediated by PKC and by PTK. These results provide functional relevance for biochemical signals transduced via Ia molecules by demonstrating that Ia-induced B cell adhesion is mediated by the activation of PKC and by one or more PTK.

Tyrosine phosphorylation of phospholipase C-gamma 2 upon cross-linking of membrane Ig on murine B lymphocytes.

When membrane Ig (mIg) on the surface of B lymphocytes is cross-linked using anti-Ig antibodies, the enzyme phospholipase C (PLC) is activated to cleave inositol phospholipids. Tyrosine kinase inhibitors have been reported to inhibit this event. Therefore, we investigated the effect of cross-linking of mIg on the state of tyrosine phosphorylation of PLC activity in two murine B cell lines and in normal resting mouse B cells. Proteins from lysates of stimulated or unstimulated cells were immunoprecipitated with an antiphosphotyrosine antibody and subsequently assayed for PLC activity. Treatment of the B cell line WEHI-231 with anti-IgM led within 15 to 30 s to a 10- to 20-fold increase in tyrosine-phosphorylated PLC activity. Inositol trisphosphate generation by WEHI-231 cells stimulated under the same conditions demonstrated similar kinetics. Normal resting B cells treated with anti-IgM or anti-IgD demonstrated 2.5- and 4-fold increases, respectively, of tyrosine-phosphorylated PLC activity. To identify the isozyme of PLC that was phosphorylated, we immunoprecipitated PLC-gamma 1 or PLC-gamma 2 with specific antibodies and assessed the amount of tyrosine phosphorylation of these proteins by antiphosphotyrosine immunoblotting. Treatment of WEHI-231 or Bal17 cells with anti-IgM induced an increase in PLC-gamma 2 tyrosine phosphorylation over background levels. There was no detectable tyrosine phosphorylation of PLC-gamma 1 in treated or untreated WEHI-231 cells, whereas anti-IgM-treated Bal17 cells did exhibit low but detectable levels of tyrosine phosphorylation of PLC-gamma 1. In normal resting mouse B cells, there was no detectable PLC-gamma 1, but PLC-gamma 2 was abundant. These observations suggest that PLC-gamma 2 is a significant substrate for the mIg-activated protein tyrosine kinase and may be responsible for mediating mIg stimulation of inositol phospholipid hydrolysis in murine B cells.

Monoclonal antibodies directed to different epitopes in the CD3-TCR complex induce different states of competence in resting human T cells.

After the initial stages of activation, T cells are not able to proliferate on their own but become competent to proliferate in response to exogenously added lymphokines. In the present study we compared the capacity of mAb directed to CD3 (OKT3, Leu4, UCHT1) or to common epitopes on the alpha/beta T-cell receptor (BMA 031, BMA 032) to induce competence in purified resting T cells. Stimulation with either soluble anti-CD3 or anti-alpha/beta TCR mAb rendered cells competent to progress to DNA synthesis in response to exogenous IL-2. In contrast, only soluble BMA 031 and BMA 032 were able to induce responsiveness to IL-4; anti-CD3 mAb had either to be immobilized or used in combination with anti-CD28 mAb to induce responsiveness to IL-4. Further, BMA 031-induced IL-4 responsiveness was selectively found in the CD45RA+ T cell subset. Analysis of early activation events revealed that the capacity of soluble BMA 031 and BMA 032 to induce responsiveness to IL-4 did not correlate with the ability of these mAb to increase the level of cytosolic Ca2+ or to induce detectable tyrosine phosphorylation. On the other hand, soluble Leu4 (anti-CD3) triggered an increase in both intracellular Ca2+ and tyrosine phosphorylation but was unable to induce IL-4 responsiveness. These data indicate that the induction of IL-2 and IL-4 responsiveness requires different sets of activation signals which can be induced by stimulating different epitopes in the CD3-TCR complex. This supports the concept that distinct activation pathways are coupled to the CD3-TCR complex.

Activation of tyrosine phosphorylation in human T cells via the CD2 pathway. Regulation by the CD45 tyrosine phosphatase.

In this study we compare the effect of CD3 and CD2 ligation on tyrosine kinase activation in human peripheral blood T cells. Using antiphosphotyrosine antibody to detect tyrosine phosphorylation of cellular substrates, we demonstrate that mAb stimulation of either CD3 or CD2 results in tyrosine phosphorylation of the TCR-zeta chain and 135- and 100-kDa proteins. However, differences are observed between CD3 and CD2 ligation; only the former results in rapid tyrosine phosphorylation of 72-, 65-, and 40-kDa substrates. Co-aggregation of CD2 and CD45, a tyrosine phosphatase, results in inhibition of intracellular calcium elevation and T cell proliferation. We demonstrate in this study that this manipulation also inhibits polyphosphoinositide hydrolysis and tyrosine phosphorylation of the 100-kDa substrate. The failure of tyrosine phosphorylation of the 100-kDa substrate is specific in that phosphorylation of the 135-kDa protein is not inhibited. Similar results are observed when CD2 and CD45 are independently cross-linked rather than co-aggregated. The observation that CD45 cross-linking alters tyrosine phosphorylation of T cell substrates and effects polyphosphoinositide hydrolysis is further evidence that tyrosine phosphorylation regulates early events in T cell activation including, perhaps, phospholipase C activity.

A pertussis toxin-sensitive G-protein mediates some aspects of insulin action in BC3H-1 murine myocytes.

The involvement of G-proteins in the insulin signal transduction system has been studied in detail using the murine BC3H-1 myocyte system. Pertussis toxin (PT) treatment, previously shown to attenuate some of the metabolic effects of insulin in this cell line (Luttrell, L.M., Hewlett, E.L., Romero, G., and Rogol, A.D. (1988) J. Biol. Chem. 263, 6134-6141), abolished insulin-induced generation of diacylglycerol and inositolglycan mediators with no effects on either the autophosphorylation of the insulin receptor or the phosphorylation of the major endogenous substrates for insulin-stimulated tyrosine kinase activity (pp185 and pp42-45). In vitro ADP-ribosylation and immunoblotting studies suggest that the major PT substrate is a 40-kDa protein of the G alpha family. This protein band did not exhibit detectable tyrosine phosphorylation upon stimulation of either intact cells or cell membranes with insulin. In the presence of low concentrations of GTP, insulin treatment of isolated myocyte plasma membranes resulted in a small (30-40%) but significant stimulation of GTP hydrolysis. This effect was best observed in the presence of small concentrations of sodium dodecyl sulfate. The rate of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding to BC3H-1 membranes was also significantly increased in the presence of insulin. The effects of insulin on GTP hydrolysis and GTP gamma S binding were found to be dependent on the concentration of insulin. These effects were not detected in plasma membranes prepared from PT-pretreated BC3H-1 myocytes. In contrast, pretreatment with the B (inactive) subunit of PT did not alter the response of myocyte membranes to insulin. High affinity binding of [125I]iodoinsulin to myocyte plasma membranes was reduced by 60-70% in the presence of guanine nucleotides. Similar effects on insulin binding were produced by PT pretreatment of the cells. In contrast, adenine nucleotides had no effect on insulin binding. Scatchard analysis of the binding data showed that the observed effects of guanine nucleotides and PT on insulin binding resulted either from a reduction in the number of high affinity insulin binding sites or from a significant reduction of the affinity of insulin for its receptor. Low affinity binding sites did not appear to be affected by either guanine nucleotides nor PT pretreatment. These results provide substantial evidence suggestive of a noncovalent interaction between the insulin receptor and a regulatory G-protein system during the process of insulin signaling.

Analysis of deletions of the carboxyl terminus of the epidermal growth factor receptor reveals self-phosphorylation at tyrosine 992 and enhanced in vivo tyrosine phosphorylation of cell substrates.

The human epidermal growth factor receptor (EGFR) contains a large C' terminus distal to the protein tyrosine kinase domain that is conserved among members of its extended gene family. To investigate the C' terminus, a series of mutant EGFR cDNAs encoding progressive C'-terminal deletions were prepared and expressed in null recipient B82L cells. In vivo self-phosphorylation was retained in receptors truncated to residues 1052 and 1022 which lack the three identified sites of tyrosine self-phosphorylation. Receptors truncated to residue 991 did not undergo in vivo self-phosphorylation. Purified 1022 truncated receptor was self-phosphorylated to the extent of 1 mol of phosphate/mol of receptor protein. The deduced additional site of tyrosine self-phosphorylation at residue 992 was confirmed by tryptic phosphopeptide mapping and protein sequencing. EGFRs deleted to give C'-terminal residues 1052, 1022, 991, and 973 exhibited enhanced EGF-stimulated tyrosine phosphorylation of cell substrates in vivo, whereas deletion at residue 944 abolished all detectable EGF-stimulated protein tyrosine phosphorylation. These results indicate that ligand-induced self-phosphorylation is limited to the C' terminus of the EGFR and suggest that this region of the holoreceptor has an inhibitory function.