PTK Inhibitors Research Articles

Characterization of Na+-Coupled Glutamate/Aspartate Transport by a Rat Brain Astrocyte Line Expressing GLAST and EAAC1

D-aspartate (D-Asp) uptake by suspensions of cerebral rat brain astrocytes (RBA) maintained in long-term culture was studied as a means of characterizing function and regulation of Glutamate/Aspartate (Glu/Asp) transporter isoforms in the cells. A-asp influx is Na+-dependent with Km = 5 microm and Vmax = 0.7 nmoles x min(-1) x mg protein-1. Influx is sigmoidal as f[Na+] with Na+Km approximately 12 microm and Hill coefficient of 1.9. The cells establish steady-state D-Asp gradients >3,000-fold. Phorbol ester (PMA) enhances uptake, and gradients near 6,000-fold are achieved due to a 2-fold increase in Vmax, with no change in Km. At initial [D-Asp] = 10 microm, RBA take up more than 90% of total D-Asp, and extracellular levels are reduced to levels below 1 microm. Ionophores that dissipate the Delta(mu)Na+ inhibit gradient formation. Genistein (GEN, 100 microm), a PTK inhibitor, causes a 40% decrease in d-Asp. Inactive analogs of PMA (4alpha-PMA) and GEN (daidzein) have no detectable effect, although the stimulatory PMA response still occurs when GEN is present. Further specificity of action is indicated by the fact that PMA has no effect on Na+-coupled ALA uptake, but GEN is stimulatory. d-Asp uptake is strongly inhibited by serine-O-sulfate (S-O-S), threohydroxy-aspartate (THA), L-Asp, and L-Glu, but not by D-Glu, kainic acid (KA), or dihydrokainate (DHK), an inhibition pattern characteristic of GLAST and EAAC1 transporter isoforms. mRNA for both isoforms was detected by RT-PCR, and Western blotting with appropriate antibodies shows that both proteins are expressed in these cells.

Binding of Amadori glucose-modified albumin by the monocytic cell line MonoMac 6 activates protein kinase C epsilon protein tyrosine kinases and the transcription factors AP-1 and NF-kappaB.

An affinity purification procedure is employed for the isolation of FL-specific binding proteins from MM6 cell membranes using magnetobeads coated with glycated polylysine and elution with FL and glycated 6-aminocaproic acid. Two main binding proteins were identified as membrane-bound nucleolin and cellular myosin heavy chain, which are glycosylated. This study shows that in these cells binding of short-term glycated albumin leads to activation of PKC, especially its isoform epsilon and this is linked to translocation of AP-1 and NF-kappaB into the nucleus. Consequently, an increased formation of IL-1ss mRNA is observed. The PKC inhibitor GO6976 prevents all these effects. Glycated albumin also stimulates activation of PTK. The PTK inhibitor genistein prevents activation of AP-1 indicating that PTK is also involved in this process, whereas NF-kappaB translocation is only dependent on PKC activation.

Chronic myeloid lukemia cells: Tyrosine kinase inhibition using ag 957 and arachidonic acid inducing apoptosis

Chronic myelogenous leukemia (CML) is characterized by Philadelphia chromosome that fuses genetic sequences of the BCR gene on chromosome 22. AG 957, a member of the tyrphostin compound produces a selective inhibition of P 210 BCR/ABL tyrosine phosphorylation. A group of ten patients with CML in chronic phase were treated after CD34 separation with 1 to 100 umol/L of AG 957. All patients were 100% Ph1 positive. Eight patients showed a b3a2 BCR/ABL junction and two cases showed a b2a2 junction when analyzed by RT-PCR. The effect of AG 957 on CML and normal CD34 cells in culture assay (CFU/MIX, BFU-E and CFU-GM) is clearly demonstrated. The inhibition on CML untreated and AG957 treated colonies was from 2%–7% for CFU-MIX, 38%–80% for BFU-E and 31%–18–% for CFU-GM. In the group of normal samples the inhibition ranged from 4%–8% for CFU-MIX, 24%–96% for BFU-E and 4%–64% for CFU-GM. The inhibitory effect of AG 957 on CML progenitors is statistically significant at the dose of 1 umol/L for CFU-MIX and BFU-E, 5 umol/L for CFU-GM. For colonies obtained from normal progenitors the inhibiting dose of AG 957 was more consistent: 5 umol/L for CFU-MIX and BFU-E, 10 umol/L for CFU-GM. These data demonstrated the possibility to select non clonal CML progenitors after incubation with the PTK inhibitor AG 957. This selection of “normal” progenitors from CML marrow could be used for autograft in patients without suitable allogeneic bone marrow donors. The research is in progression to explore the potential therapeutic effect of AG 957 in combination with other agents (IFN - ARA-C) and/or inductors of apoptosis as arachidonic acid in cell culture, to briefly translate the “in vitro” experimental approach to clinical therapeutic application.

Inhibition of the Establishment of Zygotic Polarity by Protein Tyrosine Kinase Inhibitors Leads to an Alteration of Embryo Pattern in Fucus

Fucoid algae, including the genus Fucus and Pelvetia, are recognized as model systems to study early embryogenesis in plants. In particular the zygotes of these fucoid algae are highly suitable experimental systems for investigating the establishment of polarity and its requirement for later embryogenesis. However, the transduction pathways involved in the initiation of polarization are still poorly understood, and the link between the early polarization processes and embryo long-term patterning has never been experimentally demonstrated. We, therefore, have investigated the putative role of protein phosphorylation in the regulation of early embryogenesis, using a combined pharmacological and biochemical approach. Among the various protein kinase inhibitors tested, a subset of well-known PTK inhibitors, including genistein, prevented germination but had no effect on growth of germinated zygotes and embryos. Inhibition of germination appeared to be a direct consequence of prevention of polarization since genistein and other PTK inhibitors specifically inhibited axis formation in a light-independent manner. Genistein inhibited cellular events associated with polarization such as polarized secretion of cell wall sulfated compounds. Anchorage of F-actin at the rhizoid pole was also inhibited and F-actin redistributed in response to a new light vector. Zygotes inhibited in the polarization process over the period of axis formation recovered from the treatment and displayed differentiated cellular structures after a few days. However, they exhibited a deeply disorganized pattern, suggesting that the early polarization process is essential for normal patterning of the embryo. Western blot analysis of protein phosphorylation showed that the patterns of protein phosphorylation changed during development and were disturbed by treatments with genistein. This drug also inhibited in vitro autophosphorylation. The nature of the genistein-sensitive kinases required for polarization and long-term patterning is discussed in light of these data.

Involvement of Ras and MAP kinase (ERK-1) in cisplatin-induced activation of murine bone marrow-derived macrophages.

Cisplatin (cis-diamminedichloroplatinum II), a potent antitumor compound, stimulates immune responses by activating monocytes/macrophages and other cells of the immune system. However, the mechanism by which cisplatin activates these cells is poorly characterised. Our earlier findings indicate that cisplatin treatment stimulates rapid tyrosine phosphorylation in a number of cellular proteins in murine macrophages. This initial tyrosine phosphorylation is an important regulatory mechanism and is followed by activation of several other proteins. In the present study, we report the involvement of other key molecules and the role of tyrosine phosphorylation in their activation in the signaling cascade of cisplatin. We observed the involvement of Ras (a low molecular weight GTP-binding protein) and ERK-1 (a MAP kinase) in this signaling cascade. Cisplatin treatment results in an increase in the expression of both Ras and ERK-1 in a dose-dependent manner, which was dependent upon tyrosine phosphorylation. Genistein a PTK inhibitor inhibited the cisplatin induced expression of Ras and ERK-1. These findings indicate that Ras and ERK-1 are important signaling molecules involved in the tumoricidal activation of macrophages with cisplatin and is dependent on initial tyrosine phosphorylation.

Differential tyrosine phosphorylation/activation of oncogenic proline-directed protein kinase F(A)/GSK-3alpha in well and poorly differentiated human prostate carcinoma cells.

Computer analysis of protein phosphorylation site sequences revealed that transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of the proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of proline-directed protein kinase F(A)/glycogen synthase kinase-3alpha (PDPK F(A)/GSK-3alpha) has been optimized to demonstrate significantly increased (p < 0.01) activity in poorly differentiated human prostate carcinoma PC-3 cells (55.5+/-3.8 units/mg) when compared to well-differentiated LNCaP cells (28.1+/-2.3 units/mg). Immunoblotting analysis revealed that increased activity of this PDPK in PC-3 cells is due not to overexpression of the protein, but to enhanced tyrosine phosphorylation of the kinase. When treated with genistein (a protein tyrosine kinase PTK inhibitor), the enhanced tyrosine phosphorylation/activation of the kinase in PC-3 cells can be blocked. Conversely, when treated with vanadate (a protein tyrosine phosphatase PTP inhibitor), the phosphotyrosine content of PDPK F(A)/GSK-3alpha in LNCaP cells can be promoted to the level of PC-3 cells. In sharp contrast, the PTK inhibitor has little effect on the tyrosine phosphorylation level of the kinase in LNCaP cells, whereas the PTP inhibitor has little effect on the tyrosine phosphorylation level of the kinase in PC-3 cells. Taken together, the results provide initial evidence that the tyrosine phosphorylation/activation levels of this oncogenic PDPK can be differentially regulated in well- and poorly differentiated prostate carcinoma cells.

CD45 and Src-Related Protein Tyrosine Kinases Regulate the T Cell Response to Phorbol Esters

Protein kinase C (PKC)-dependent activation of the Ras signal transduction cascade is essential for induction of the IL-2 promoter during stimulation of T lymphocytes via the T cell receptor (TCR). In this study, the effects of PKC-activating phorbol myristate acetate (PMA) on Ras-dependent activation of transcription from the ets/AP-1 Ras-responsive promoter element were examined in human T cells. Pretreatment of Jurkat cells with the Src-family PTK inhibitor herbimycin A resulted in a 50% inhibition of transactivation of the reporter following incubation with PMA. Evidence was also obtained to suggest the participation of the leukocyte-specific protein tyrosine phosphatase CD45, a regulator of Src-like PTKs, in the PMA-induced activation of the Ras/Raf pathway. First, PMA-induced transactivation of ets/AP-1 is diminished 75% in CD45-negative variants, compared with CD45-positive cells. Second, engagement of CD45 by monoclonal antibodies suppresses the PMA response from the reporter construct. Taken together, these data suggest that Src-related proteins mediate PKC-dependent activation of the Ras/Raf pathway and implicate CD45 in the TCR-independent activation of T lymphocytes induced by agents such as PMA.

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.

Apoptosis induced by HIV-gp120 in a Th1 clone involves the generation of reactive oxygen intermediates downstream CD95 triggering

HIV-gp120 sensitizes Th1 clones from seronegative donors to apoptosis, which occurs through two distinct events: expression of CD95L followed by its interaction with CD95 to trigger cell death. gp120-apoptosis of the Th1 clone 103 was inhibited by Cyclosporin A, the PTK inhibitors Genistein and PNU152518, as well as the anti-oxidants Ascorbic Acid and Glutathione. Cyclosporin A interfered with CD95L expression, Ascorbic Acid and Glutathione inhibited cell death triggered by CD95/CD95L interaction; Genistein and PNU152518 acted on both steps. The occurrence of oxidative stress during CD95-dependent apoptosis was supported by the direct evidence of ROI production.

Cellular Activation Induced by BCG Is a PTK-Dependent Event

Mycobacterial antigens including BCG stimulate human peripheral blood mononuclear cells resulting in cellular proliferation and the release of inflammatory cytokines such as TNF-α. However, the signal transduction mechanisms responsible for the BCG-induced cell activation are not completely understood. In this study, we investigated the role of PTK as a signal transduction pathway in BCG-induced cell activation, with the use of two PTK inhibitors (genistein and tyrphostin). Our results indicated that genistein significantly inhibited BCG-induced cell growth determined by thymidine uptake in a dose-dependent manner. BCG-induced TNF-α secretion was completely suppressed by genistein in a dose-dependent manner, producing 92% inhibition at a concentration of 50 μM.In addition, strong inhibition (81%) of BCG-induced TNF-α secretion was observed with tyrphostin (30 μM), another specific protein tyrosine kinase with a different mechanism of action. These inhibitory effects were not attributed to an alteration in cell viability as judged by trypan blue staining, and were not due to LPS contamination. On the other hand, monoclonal antibodies directed against HLA-DR and DQ inhibited the BCG-induced secretion of TNF-α. Taken together, these findings suggest that PTK may play an essential role in BCG-induced cellular activation.

NOREPINEPHRINE (NE) SUPPRESSES AND MAP KINASE KINASE (MEK1) INHIBITOR ENHANCES PROTEIN TYROSINE KINASE (PTK) ACTIVITY IN NEWBORN LAMB MIDDLE CEREBRAL ARTERIES (MCA). † 355

Protein Tyrosine Kinase plays a pivotal role in intracellular signal transduction pathways leading to complex expressions of cellular responses. NE is an important neurotransmitter engaging in varied arterial vascular pathophysiology. However, its post-receptor signalling pathway(s) in cerebral circulation is poorly understood. We have preliminarily studied regulatory aspects of NE on PTK of isolated MCA preparations of newborn lambs. PTK activities [PTK] were measured on soluble fraction [sPTK] and particulate fraction [pPTK] (100,000xgx1h) using a synthetic peptide (MW = 1,520) substrate RR-SRC with a known amino acid sequence (RRLIQDAEYAARG) surrounding the phosphorylation site in pp60src(Krebs, E.G., 1985). NE challenge(5μM) suppressed impressively both [sPTK](86%) and [pPTK](75%). In NE naive preparations, the enzyme activity was preferentially localized in [sPTK](≈ 160%/[pPTK]). Genistein(PTK inhibitor, 20 μM) stimulated [pPTK] over 40%. Interestingly, PD098059 (MEK1 inhibitor, 10 μM) robustly heightened[pPTK](80%). Taken together, it is concluded that NE exerted a suppressive effect on [PTK] and MEK1 inhibitor strengthened [pPTK]. Hence, it is proposed that interplay between PTK and MEK1 is implicated in NE-associated modification of intracellular machinery and that the link of the two signal transducing enzyme systems may present a characteristic biochemical basis for altered cerebral circulation and related clinical manifestations. (Inst Korean-American Studies;American Heart Association national council #95009270; Allegheny-Singer Res Inst #960181SCHC)

CONTRACTILE RESPONSE OF NEWBORN LAMB PULMONARY ARTERY (PA) TO SEROTONIN AND HISTAMINE IS ASSOCIATED WITH PROTEIN TYROSINE KINASE- AND NITRIC OXIDE-DEPENDENT MECHANISMS † 2062

The purpose of the present study is to determine if contractile property of PA is regulated by protein tyrosine kinase [PTK])- and nitric oxide[NO]-dependent signaling systems. We preliminarily evaluated, using tissue chamber technique, effects of genistein[G], a PTK inhibitor, and SNAP[(+/-)-S-Nitroso-N-acetylpenicillamine], an NO donor, in histamine[H]](100 μM)- and serotonin [S](1 μM, 10 μM)-challenged PA preparations (wet weight ≈0.75mg, initial load 1g) as a model. [G](20μM, 80 μM) dose-dependently relaxed [S](10 μM)-induced maximum contraction (0.73±0.03 g/mg tissue) to (0.57±0.01, 0.01±0.01 g/mg, respectively; equal to 22%, 98% relaxation) and[H]-induced maximum contraction (0.16±0.09 g/mg) to (-0.05±0.04,-0.09±0.07 g/mg, respectively; equal to 160%, 286% relaxation). SNAP(1μM, 10 μM) relaxed [S](10 μM)-induced maximum contraction(0.92±0.03 g/mg) to (0.38±0.14, 0.17±0.02 g/mg, respectively; equal to 58%, 78% relaxation) in dose-dependent manner. Interestingly, [G](20 μM)-induced relaxation of both [S]- and [H]-induced contraction had a slow onset but was steady and sustaining while SNAP caused rapid relaxation of both [S]- and [H]-induced contraction. SNAP-induced relaxation of [S]-contraction was partially and dose-dependently recovered. LY-83583, an inhibitor of NO-induced activation of soluble guanylate cyclase, reversed SNAP-induced relaxation of both [S]- and [H]-contraction. In contrast, SOV reversed [G]-induced relaxation of both [S]- and[H]-contraction. L-NAME (100 μM), NO synthase (NOS) inhibitor, did not effect SNAP- or Zaprinast [Z](c-GMP phosphodiesterase inhibitor)-induced relaxation. [Z](10 μM) further enhanced SNAP-induced relaxation of[S](1μM)-contraction. Diphenyleneiodonium chloride (DPI) (5 μM), another NOS inhibitor, showed a similar effect of [Z]. Based on these data, we conclude that contractile property of isolated newborn lamb PA is regulated by PTK mediated signal transduction pathway(s) and that [S]- and [H]-induced contraction in this paradigm is associated with PTK-dependent mechanisms and linked to NO-responsive element(s). {supported in part by Institute for Korean-American Studies; American Heart Association, national council#95009270}

PLD activation in Chinese hamster ovary (CHO) cells transfected with PGF2α receptor cDNA

Pertussis toxin-insensitive GTP-binding protein was observed to be involved in prostaglandin F2 α(PGF2 α)-induced phosphoinositide metabolism in Chinese hamster ovary (CHO) cells transfected with PGF2 α receptor cDNA (CHO-PGF2 α·R cells) (Ito, S. et al. Biochem. Biophys. Res. Commun. 200: 756, 1994). In the present study, we investigated PGF2 α-induced PLD activation in CHO-PGF2 α·R cells. PLD activation was examined by measuring the production of [ 3H]phosphatidylbutanol ([ 3H]PBut), a specific product of the PLD-catalyzed transphosphatidylation reaction. PGF2 α-induced [ 3H]PBut formation was concentration-dependent with the maximal level obtained at 1 μM PGF2 α. The maximal [ 3H]PBut formation was observed at 2 min after addition of PGF2 α. Depletion of extracellular Ca 2+ with EGTA suppressed PGF2 α-induced PLD activation by 50%. PKC inhibitors Ro31–8425 and calphostin C inhibited PGF2 α-induced [ 3H]PBut formation by 50%. PTK inhibitors genistein and herbimycin A failed to inhibit PGF2 α-induced PLD activation. A combination of maximal effective concentrations of PGF2 α (1 μM) and PMA (100 nM) enhanced PLD activation in an additive manner. Pretreatment of the cells with PMA for 2 h down-regulated PKC α and decreased PGF2 α-induced PLD activation. These results suggest that PLD activation by PGF2 α is mediated by both PKC-dependent and -independent pathways and that PKC α is involved in the former pathway.

Exposure of B-lineage Lymphoid Cells to Low Energy Electromagnetic Fields Stimulates Lyn Kinase

Here, we present evidence that exposure of B-lineage lymphoid cells to low energy electromagnetic fields (EMF) stimulates the protein tyrosine kinases Lyn and Syk, results in tyrosine phosphorylation of multiple electrophoretically distinct substrates, and leads to downstream activation of protein kinase C (PKC). EMF exposure enhances protein tyrosine phosphorylation in Syk deficient but not in Lyn-deficient B-lineage lymphoid cells and stimulates Lyn kinase activity in wild-type as well as Syk-deficient B-lineage lymphoid cells. These results indicate that activation of Lyn kinase is sufficient and mandatory for EMF-induced tyrosine phosphorylation in B-lineage lymphoid cells. The PKC activity increases later than the Lyn activity and pretreatment with the PTK inhibitors genistein or herbimycin A abrogates the EMF-induced PKC signal. Thus, stimulation of Lyn is a proximal and mandatory step in EMF-induced activation of PKC in B-lineage lymphoid cells. Our observations prompt the hypothesis that a delicate growth regulatory balance might be altered in B-lineage lymphoid cells by EMF-induced activation of Lyn.

Analysis of Phosphorylhydroxyamino Acids Present in Hydrolyzed Cell Extracts Using Dabsyl Derivatization

We have developed a new method for the quantification of phosphoserine, phosphothreonine, and phosphotyrosine as dabsyl derivatives in acid-hydrolyzed extracts of 32P-labeled A431 cells. In the first step the phosphoamino acids are concentrated using a disposable anion-exchange column. Subsequently, the phosphoamino acid fraction is treated with dabsyl reagent (28.8 mM) for 10 min at 70°C. After cleanup with a second anion-exchange column followed by separation on a disposable C18 column, the covalently modified phosphoamino acids are separated on silica TLC sheets using a one-dimensional solvent system. The major advantages of this method are the complete separation of dabsylated P-Ser, P-Thr, and P-Tyr on silica aluminum sheets in a very reproducible way without the interference of 32P contaminants originating from hydrolyzed cell extracts. Very clean chromatograms are obtained, enabling the fast and unambiguous quantification of the phosphoamino acids by simply cutting out the relevant spots from the aluminum sheets. A high sensitivity is achieved by the removal of the amino acids before derivatization of the sample. This allows the use of relatively low amounts of [32P]orthophosphate to load up the cells. Most important, the method allows the simultaneous analysis of dozens of samples within 1 day, making it a very convenient technique for routine analysis of the phosphorylation state of cultured cells. Consequently the method is well suited to implementation in large screenings for inhibitors of protein kinases, e.g., PTK inhibitors, in whole-cell-studies.

Modulation of potassium channels by protein tyrosine kinase inhibitors.

Exposure of non-excitatory cells to the tyrosine kinase (PTK) inhibitors, genistein, herbimycin A, and tyrphostin, induced at least two families of K+ currents. The first, a TEA-insensitive slow-inactivating K+ current, is induced within 3 min following treatment with 140 mM genistein or 100 nM herbimycin A. The second current, a TEA-sensitive delayed rectifier, is induced within 30 min following treatment with 50 mM genistein or 10 nM herbimycin A. Currents with similar biophysical and pharmacological characteristics are induced in these cells following exposure to ionizing radiation. The radiation-induced currents are inhibited by pretreatment with the free radical scavenger, N-Acetyl L-Cysteine, or by pretreatment with the protein kinase C inhibitor, staurosporine; those induced by PTK inhibitors are not. The latter, therefore, do not appear to be mediated through free radicals or require serine/threonine phosphorylation for activation. Once the channels are activated by the PTK inhibitors, phosphorylation of the channel at serine/threonine residues results in slower inactivation of the induced current. We propose that protein tyrosine phosphorylation of the K+ channel protein itself or of a factor that interacts with it maintains the K+ channels of non-excitatory cells in a closed state. Following exposure to ionizing radiation, free radical-induced activation of serine/threonine kinase(s) results in phosphorylation of the channel and/or inactivation of a tyrosine kinase that in turn leads to activation of the K+ channels.

Protein tyrosine kinase activation is required for lipopolysaccharide induction of cytokines in human blood monocytes.

Bacterial LPS induce production of cytokines such as IL-1, IL-6, and TNF in mononuclear phagocytes, and this represents a central component in the pathogenesis of septic shock syndrome. However, the mechanisms by which LPS activates these cells to express cytokines are not completely characterized. The present study addressed the role of different protein kinases in the LPS induction of cytokines. It is shown that LPS induced a 12- to 16-fold increase in IL-1 beta, IL-6, and TNF-alpha mRNA levels, and this was completely or more than 80% blocked by the protein tyrosine kinase specific inhibitors herbimycin A and genistein at the concentrations of 1.7 and 37 microM, respectively. Protein kinase C inhibition by staurosporine reduced LPS induction of TNF-alpha, whereas it had no effects on IL-6 and IL-1 beta. Inhibition of protein kinase A by H89 reduced IL-6 mRNA levels but did not detectably change IL-1 beta or TNF-alpha mRNA levels. In contrast, LPS did not increase leukemia inhibitory factor mRNA, which was constitutively expressed and not significantly reduced by these inhibitors. In addition to cytokine mRNA levels, LPS-induced IL-6 protein synthesis and IL-6 bioactivity were also reduced to baseline levels by the PTK inhibitors herbimycin A and genistein. Both PTK inhibitors also reduced the LPS activation of nuclear factor-kappa B (NF-kappa B), which is a transcription factor involved in the expression of cytokine genes such as IL-6 and TNF-alpha. The activation of NF-kappa B was also reduced by H89, whereas staurosporine had no effect on this response. In summary, these findings suggest that protein kinase C and protein kinase A appear to have selective effects in the LPS induction of cytokines, whereas PTK is required for LPS induction of a broad spectrum of cytokines and NF-kappa B activation in monocytes.

Involvement of protein kinase C and protein tyrosine kinase pathways in tumor necrosis factor-α-induced clustering of ovarian theca-interstitial cells

Tumor necrosis factor-alpha (TNF) induces clustering of theca-interstitial cells (TIC) isolated from immature, hypophysectomized rats, while inhibiting luteinizing hormone (LH)-stimulated androstenedione in vitro. Stimulators of PKC, l-oleoyl-2-acetyl- sn-glycerol (OAG, 50 and 100 μM) and phorbol-12-myristate-13-acetate (PMA, 50 nM), caused TIC clustering by 6 days in vitro. Clustering induced by these compounds resembled that induced by TNF. The protein kinase inhibitor, staurosporine at 1 and 10 nM, impaired TNF-induced TIC clustering for 6 days, as did the protein kinase inhibitor, l-(5-isoquinolinesulfonyl)-2-methylpiperizine dihydrochloride (H-7); conversely, the protein kinase inhibitor, chelerythrine chloride (0.1, 1.0 or 10 μM), did not attenuate TNF-directed clustering. The protein kinase inhibitors did not reverse the suppression of LH-stimulated androstenedione by TNF. Inhibitors of the EGF receptor PTK, A23 (10, 50, or 100 μM) and A46 (0.1, 1.0, 10, or 50 μM), impaired TNF-induced TIC clustering, while TNF suppression of LH-directed androstenedione was unaffected. EGF-induced TIC clustering was also impaired by A46, while A23 was less effective. Both A23 and A46 blocked EGF attenuation of LH-directed androstenedione after 4 days. When challenged with TNF (1 ng/ml) or PMA (50 nM), PKC activity increased in TIC. A23 (50 μM) and A46 (10 μM) each alone blocked the TNF-associated increase in PKC activity; however, PKC activity attributable to PMA was unaffected by A46. Together, these results suggest that TNF-induced TIC clustering involves activation of PTK which directs subsequent increases in PKC activity; however, mechanisms by which TNF inhibits LH-stimulated steroidogenesis remains elusive.

Protein tyrosine phosphorylation is mandatory for CD40‐mediated rescue of germinal center B cells from apoptosis

Spontaneous apoptosis in germinal center (GC) B cells can be arrested either by engaging cell surface immunoglobulin (Ig) with immobilized ligand or, more effectively, by treatment with soluble monoclonal antibody (mAb) directed against CD40. The present study examines the intracellular signal transduction pathways through which rescue from spontaneous apoptosis is engendered in GC B cells following ligation of surface CD40. Cross-linking the surface CD40 of GC B cells with mAb consistently resulted in enhanced tyrosine phosphorylation on a number of distinct substrates: this process could be blocked, in a dose-dependent fashion, by pre-treating GC B cells with the selective protein tyrosine kinase(s) (PTK) inhibitor, herbimycin A. Moreover, the pattern of phosphorylation on tyrosine observed following treatment with anti-CD40 was remarkably similar to that triggered by polyvalent anti-Ig. By contrast, anti-CD40 failed to stimulate the increase in inositol 1,4,5-trisphosphate and cytosolic free calcium observed in both GC B cells and resting B lymphocytes following ligation of surface Ig. The involvement of the signaling pathways generated in the rescue of GC B cells from apoptosis was studied by using selective inhibitors of PTK and of extracellular and intracellular Ca2+. Pre-incubation with the PTK inhibitor herbimycin A (5 microM) abrogated anti-CD40-mediated rescue of GC B cells from apoptosis, while genistein (40 microM) and the tyrphostins AG490 (10 microM) and AG814 (25 microM) significantly inhibited this process. Consistent with these results, herbimycin A (5 microM) abolished the expression of the 26 kDa bcl-2 protooncogene product, which confers resistance to apoptosis, normally observed following culture with anti-CD40. The Ca2+ chelators BAPTA and EGTA did not significantly affect CD40-promoted rescue. Taken together, these results indicate that CD40 of GC B cells is coupled to functional PTK but not to the phosphatidylinositol signaling pathway and that tyrosine phosphorylation is mandatory for CD40-mediated rescue of GC B cells from apoptosis.

Signal transduction through the T cell antigen receptor. Activation of phospholipase C through a G protein-independent coupling mechanism.

The T cell Ag (Ti-CD3) receptor complex has been proposed to regulate phosphoinositide-specific phospholipase C (PLC) through a cholera toxin (CTX)-sensitive guanine nucleotide-binding (G) protein. In this study, we have used CTX and staurosporine as pharmacologic probes to further define the linkage between the Ti-CD3 receptor and PLC activity in the human T cell line, Jurkat. CTX pretreatment inhibited Ti-CD3 receptor-dependent phosphoinositide hydrolysis and, concomitantly, protein tyrosine kinase activation in intact cells. Studies with electrically permeabilized Jurkat cells revealed that guanosine 5'-(3-O-thio) triphosphate stimulated an increase in PLC activity, that unlike the response to Ti-CD3 receptor ligation, was not affected by cellular pretreatment with CTX. In contrast, the phosphotyrosine phosphatase inhibitors, orthovanadate and molybdate anions, stimulated phosphoinositide hydrolysis in permeabilized cells through a CTX-sensitive mechanism of PLC activation. Additional studies with a known PTK inhibitor, staurosporine, supported the results obtained with CTX. Staurosporine pretreatment inhibited the phosphoinositide hydrolysis induced by anti-CD3 antibodies or phosphotyrosine phosphatase inhibitors, but failed to alter the G protein-dependent PLC activation response to guanosine 5'-(3-O-thio) triphosphate. The results of this study indicate that PLC activity(s) in Jurkat cells are regulated by both G protein- and PTK-dependent coupling mechanisms. However, the differential inhibitory effects of CTX and staurosporine on these PLC activation pathways strongly suggest that a protein tyrosine kinase activation event, rather than a G protein, mediates the functional linkage between the Ti-CD3 receptor and PLC activity in Jurkat cells.