Total Tyrosine Kinase Activity Research Articles

Hypoglycemic activity of Phaseolus vulgaris (L.) aqueous extract in type 1 diabetic rats

Abstract The aim of the present study was to evaluate the hypoglycemic activity of the aqueous extract from the fruit walls of Phaseolus vulgaris pods and to examine the potential mechanism underlying the improvement of the glycemic level. In the course of the study, diabetes mellitus was induced in rats with a single intraperitoneal injection of streptozotocin (45 mg·kg−1 b.w.). Diabetic and control rats were then orally administered with a single-dose or repeated-dose (28 day) of P. vulgaris extract (200 mg·kg−1). Results show that the extract was found to possess significant hypoglycemic activity, and the study of glucose utilization by isolated rat hemidiaphragm suggests that the aqueous extract may enhance the peripheral utilization of glucose. The subsequent experiments have revealed that the P. vulgaris extract could increase glucose transporter 4 (GLUT-4) content in skeletal muscle cells of control and diabetic rats. Our data also indicate that the P. vulgaris extract did not affect the content of the insulin receptor, but significantly reduced the total tyrosine kinase activity in skeletal muscle cells of both experimental groups of rats. The present results clearly indicated that P. vulgaris extract may be beneficial for reducing hyperglycemia through its potency in regulation of glucose utilization via GLUT-4, but the current mechanism remains to be unidentified.

Modulation of protein tyrosine phosphorylation in gastric mucosa during re-epithelization processes

To investigate the role of protein tyrosine phosphorylation in gastric wound formation and repair following ulceration. Gastric lesions were induced in rats using restraint cold stress. To investigate the effect of oxidative and nitrosative cell stress on tyrosine phosphorylation during wound repair, total activity of protein tyrosine kinase (PTK), protein tyrosine phosphatase (PTP), antioxidant enzymes, nitric oxide synthase (NOS), 2',5'-oligoadenylate synthetase, hydroxyl radical and zinc levels were assayed in parallel. Ulcer provocation induced an immediate decrease in tyrosine kinase (40% in plasma membranes and 56% in cytosol, P < 0.05) and phosphatase activity (threefold in plasma membranes and 3.3-fold in cytosol), followed by 2.3-2.4-fold decrease (P < 0.05) in protein phosphotyrosine content in the gastric mucosa. Ulceration induced no immediate change in superoxide dismutase (SOD) activity, 30% increase (P < 0.05) in catalase activity, 2.3-fold inhibition (P < 0.05) of glutathione peroxidase, 3.3-fold increase (P < 0.05) in hydroxyl radical content, and 2.3-fold decrease (P < 0.05) in zinc level in gastric mucosa. NOS activity was three times higher in gastric mucosa cells after cold stress. Following ulceration, PTK activity increased in plasma membranes and reached a maximum on day 4 after stress (twofold increase, P < 0.05), but remained inhibited (1.6-3-fold decrease on days 3, 4 and 5, P < 0.05) in the cytosol. Tyrosine phosphatases remained inhibited both in membranes and cytosol (1.5-2.4-fold, P < 0.05). NOS activity remained increased on days 1, 2 and 3 (3.8-, 2.6-, 2.2-fold, respectively, P < 0.05). Activity of SOD increased 1.6 times (P < 0.05) days 4 and 5 after stress. Catalase activity normalized after day 2. Glutathione peroxidase activity and zinc level decreased (3.3- and 2-fold, respectively, P < 0.05) on the last day. Activity of 2',5'-oligoadenylate synthethase increased 2.8-fold (P < 0.05) at the beginning, and 1.6-2.3-fold (P < 0.05) during ulcer recuperation, and normalized on day 5, consistent with slowing of inflammation processes. These studies show diverse changes in total tyrosine kinase activity in gastric mucosa during the recovery process. Oxidative and nitrosative stress during lesion formation might lead to the observed reduction in tyrosine phosphorylation during ulceration.

Oridonin induces human epidermoid carcinoma A431 cell apoptosis through tyrosine kinase and mitochondrial pathway

Oridonin, a diterpenoid isolated from the plant Rabdosia rubescens, induces human epidermoid carcinoma A431 cell death through apoptosis and tyrosine kinase pathway. To examine the pathway of oridonin-induced A431 cell death, morphologic observation, lactate dehydrogenase activity-based assay, DNA agarose gel electrophoresis and Western blot analysis were carried out. When A431 cells, which overexpress epidermal growth factor receptor (EGFR), were treated with oridonin, caspase-3 was activated followed by the degradation of caspase-3 substrates, inhibitor of caspase-activated DNase (ICAD) and poly(ADP-ribose) polymerase (PARP) in a time-dependent manner. Oridonin promoted the release of cytochrome c and the down-regulation of mitochondrial transmembrane potential (ΔΨm). Oridonin up-regulated the expression ratio of mitochondrial proteins, Bax/Bcl-2. In addition, the total tyrosine kinase activity of A431 cellular proteins and the expression of EGFR were markedly reduced after oridonin treatment. Taken together, oridonin induced apoptosis in A431 cells via mitochondrial pathway, activation of caspase-3 and inhibition of tyrosine kinase activities.

Oridonin-Induced A431 Cell Apoptosis Partially Through Blockage of the Ras/Raf/ERK Signal Pathway

We have reported that oridonin, a diterpenoid isolated from the plant Rabdosia rubescens, had apoptosis-inducing activities in many cell lines (e.g., human melanoma A375-S2, human cervical cancer HeLa, human breast adenocarcinoma MCF-7, and murine fibrosarcoma L929). In this study, we further investigated signaling events involved in oridonin-induced apoptosis in human epidermoid carcinoma A431 cells. It was found that the total tyrosine kinase activity was inhibited and the protein expressions of epidermal growth factor receptor (EGFR) and phosphorylated EGFR were decreased in oridonin-induced A431 cell apoptosis. Expression of EGFR downstream effector proteins, Grb2, Ras, Raf-1, and extracellular signal-regulated kinase (ERK), was also downregulated by oridonin. Moreover, the oridonin-induced apoptosis was augmented by the Ras inhibitor manumycin A, Raf-1 inhibitor GW5074, or ERK inhibitor PD98059, suggesting that inactivation of Ras, Raf, or ERK participates in oridonin-induced apoptosis. Taken together, oridonin-induced apoptosis in A431 cells might be through blocking EGFR and its downstream Ras/Raf/ERK signal pathway.

EFFECT OF TYROSINE KINASE INHIBITORS ON TYROSINE PHOSPHORYLATION AND MOTILITY PARAMETERS IN HUMAN SPERM

Tyrosine phosphorylation has recently been associated with capacitation and suggested as a regulator of sperm movement, especially characterizing hyperactivation. The objective of this study was to verify if tyrosine phosphorylation of human sperm proteins was essentially required for the maintenance of motility as well as the development of hyperactivation. Washed sperm were incubated for 6 h in Ham's F10 + 0.35% HSA at 37°C in 5% CO 2, with and without the tyrosine kinase inhibitors genistein, tyrphostin, erbstatin, or herbimycin A and the wide-spectrum kinase inhibitor staurosporin. The concentrations of the inhibitors used in the experiments did not induce sperm toxicity, as measured by membrane integrity and mitochondrial function assays. Samples incubated without the inhibitors (control), increased their tyrosine kinase activity (ELISA), the number and intensity of tyrosine-phosphorylated (PY) protein bands (Western blot), the incidence of PY-immunoreactive sperm (immunofluorescence), and some of the sperm motion characteristics (CASA), such as velocity (VEL), amplitude of lateral head displacement (ALH), and hyperactivation. Among the selective protein tyrosine kinase inhibitors, genistein was the most active and consistent, inhibiting sperm tyrosine kinase activity, PY proteins, incidence of PY sperm, and sperm motility and motion parameters, such as VEL, ALH, and hyperactivation. The rest of the kinase inhibitors decreased motion characteristics to a varied extent and had different effects on phosphorylation parameters. In general, they decreased PY phosphorylation of 2 proteins (83 and 54 kDa) present in whole sperm extracts, and two sets of proteins of low (39-49 kDa) and medium (55-87 kDa) molecular weight present in the Triton X-100-solubilized sperm protein fraction. Thisinhibition was evident regardless of the total tyrosine kinase activity of the samples or the incidence of PY-immunoreactive sperm. The described findings further support the association between motility and protein tyrosine phosphorylation in human sperm and point to certain proteins as the main linkers.

Effect of tyrosine kinase inhibitors on tyrosine phosphorylation and motility parameters in human sperm.

Tyrosine phosphorylation has recently been associated with capacitation and suggested as a regulator of sperm movement, especially characterizing hyperactivation. The objective of this study was to verify if tyrosine phosphorylation of human sperm proteins was essentially required for the maintenance of motility as well as the development of hyperactivation. Washed sperm were incubated for 6 h in Ham's F10 + 0.35% HSA at 37 degrees C in 5% CO(2), with and without the tyrosine kinase inhibitors genistein, tyrphostin, erbstatin, or herbimycin A and the wide-spectrum kinase inhibitor staurosporin. The concentrations of the inhibitors used in the experiments did not induce sperm toxicity, as measured by membrane integrity and mitochondrial function assays. Samples incubated without the inhibitors (control), increased their tyrosine kinase activity (ELISA), the number and intensity of tyrosine-phosphorylated (PY) protein bands (Western blot), the incidence of PY-immunoreactive sperm (immunofluorescence), and some of the sperm motion characteristics (CASA), such as velocity (VEL), amplitude of lateral head displacement (ALH), and hyperactivation. Among the selective protein tyrosine kinase inhibitors, genistein was the most active and consistent, inhibiting sperm tyrosine kinase activity, PY proteins, incidence of PY sperm, and sperm motility and motion parameters, such as VEL, ALH, and hyperactivation. The rest of the kinase inhibitors decreased motion characteristics to a varied extent and had different effects on phosphorylation parameters. In general, they decreased PY phosphorylation of 2 proteins (83 and 54 kDa) present in whole sperm extracts, and two sets of proteins of low (39-49 kDa) and medium (55-87 kDa) molecular weight present in the Triton X-100-solubilized sperm protein fraction. This inhibition was evident regardless of the total tyrosine kinase activity of the samples or the incidence of PY-immunoreactive sperm. The described findings further support the association between motility and protein tyrosine phosphorylation in human sperm and point to certain proteins as the main linkers.

Identification of a tyrosine kinase-phosphorylated protein in arachidonic acid- and Prostaglandin A2-treated cells in vitro

The effects of 20 μg/ml exogenous arachidonic acid (AA) and prostaglandin A2 (PGA2) were evaluated on total tyrosine kinase (TK) activity and tyrosine phosphorylation status in HeLa and MCF-7 cells. AA and PGA2 increased TK activity in both HeLa and MCF-7 cells. Western blotting employing an anti-phosphotyrosine antibody showed only one protein of approximately 55 kDa (∼55 kDa) to be phosphorylated in the MCF-7 cells, while a variety of proteins were phosphorylated in the HeLa cells, including the ∼55 kDa protein. Amino acid analyses as well as Matrix Assisted Laser Desorption Ionization were conducted on this protein from different cell lines and it was shown to be similar. Comparison to p53 did not show similarities. The identity of this protein needs to be further characterized to help elucidate the signal transduction pathways of AA and PGA2.

Central role of leukemia-derived factors in the development of leukemia-associated immune dysfunction.

The underlying mechanism of tumor-associated T-cell dysfunction has been suggested to be due to an acquired abnormality in the T-cell receptor signaling complex, however, tumor-derived inhibitory factors acting on immune effectors could also explain the observed immunosuppression. Here, in a murine acute leukemia model, responses (proliferation and IL-2 secretion) to mitogens were suppressed in the early stages of leukemic progression due to a leukemia-derived soluble factor(s). Correlations between the development of immunosuppression and changes in expression of TCR signaling proteins (CD3-zeta, p56(lck), p59(fyn)), calcium mobilization, and total tyrosine kinase activity following TCR stimulation were examined. In contrast to reduced responses to mitogens seen as early as 1 week following injection of leukemic cells, signaling abnormalities were only seen in advanced disease (ie four weeks). The only significant alteration in signaling protein expression was loss of CD3-zeta for four weeks following the initiation of leukemia. Importantly, a direct role of a leukemia-derived soluble factor(s) in the loss of CD3-zeta could be shown in vitro by co-culturing splenocytes with leukemic cells separated by a transwell for seven to 10 days. Further studies demonstrated that the leukemia-derived factor(s) stimulated splenic macrophages to secrete a second soluble factor(s) that caused the loss of CD3-zeta. These data indicate that soluble leukemia-derived factor(s) have both immediate immunosuppressive actions and a later effect acting indirectly to induce intrinsic defects in T-cell signaling pathways via accessory cells. In some tumors, there is evidence that the soluble factor is Fas-ligand, which induces apoptosis in tumor-infiltrating T cells and the action of activated caspases cleave CD3-zeta. In this model, the soluble factor prevents apoptosis indicating a second mechanism is responsible for the loss of T-cell signaling proteins.

Tri-iodothyronine induces proliferation in cultured bovine thyroid cells: evidence for the involvement of epidermal growth factor-associated tyrosine kinase activity.

The effects of the tri-iodothyronine (T(3)) secreted by thyroid cells on the growth of the thyrocyte are poorly known. In this study we analyzed the effects of T(3) on the proliferation of bovine thyroid follicles in primary culture previously depleted of endogenous T(3). Cellular deoxiribonucleic acid (DNA) synthesis, determined by [(3)H]thymidine incorporation, was stimulated by T(3) (0.1-5.0 nM) for 24 h in a concentration-dependent fashion with a maximal effect at 1.0 nM T(3) (P<0.01). This T(3) action was time-dependent when assayed from 12 to 72 h. The induction of mitogenic activity was corroborated by the increase in proliferating cell nuclear antigen (PCNA) measured by Western blot analysis. PCNA increased after treatment with T(3) (0.1-5.0 nM) in a concentration-dependent manner. Since T(3) modifies the activity of growth factors whose actions are mainly mediated by tyrosine kinase (TK) activation in diverse cellular types, we assayed the effects of genistein, a general TK inhibitor, and tyrphostin A25, a specific epidermal growth factor (EGF)-receptor (EGFR)-dependent TK activity inhibitor, on the proliferative effects of T(3). The T(3)-induced [(3)H]thymidine incorporation was inhibited by both agents in a concentration-dependent manner. A significant increase in the total TK activity measured in cellular protein extracts was induced by 0.5 and 1.0 nM T(3) (P<0.001). Tyrosine phosphorylation of the EGFR was also stimulated by T(3) (P<0.001) with no change in the EGFR expression as determined by Western blot analysis. Both, the T(3)-stimulated [(3)H]thymidine incorporation and the TK activity were inhibited by a anti-mouse EGF antibody. These results lead us to propose that T(3) could operate as a proliferative agent in bovine thyroid cells through a mechanism involving an autocrine/paracrine EGF/EGFR-dependent regulation.

Comparative study of the effects of polyunsaturated fatty acids and their metabolites on cell growth and tyrosine kinase activity in oesophageal carcinoma cells

The effects of exogenous γ-linolenic acid (GLA), arachidonic acid (AA), prostaglandin E2(PGE2) and prostaglandin A2(PGA2) were evaluated on cell growth in two squamous oesophageal carcinoma cell lines, WHCO1 and WHCO3 and normal monkey kidney (NMK) cells. In both cancer cell lines all four compounds inhibited cell growth significantly. Indomethacin (I) alone, or in combination with either GLA or AA, caused marked inhibition of cell growth in WHCO3. Total tyrosine kinase (TK) activity was determined after exposure of all three cell types to the lipid compounds. Negligible differences were observed in TK activity between treated and untreated NMK cells. Small increases were noticed in WHCO1. Marked TK stimulation was observed in WHCO3. Addition of indomethacin to WHCO3 also increased TK activity above control value. Tyrosine phosphorylation status of exposed cells indicated that a band of approximately 55kDa (55kDa) was primarily influenced in both WHCO3 and WHCO1. PGA2caused a decrease in tyrosine phosphorylation of the 55kDa protein in all three cell types. Negligible differences were observed in the tyrosine phosphorylation status of the 55kDa in NMK cells exposed to GLA, AA and PGE2respectively. However, tyrosine phosphorylation of a number of other proteins (21.5-97.4kDa) was observed in NMK cells. Flow cytometry studies showed an increase in S phase and decrease in G1 phase in WHCO3 exposed to PGE2and PGA2. Indomethacin alone, or in combination with GLA and AA, respectively, lead to an increase in G1and a decrease in S phase. Induction of p53 levels was observed in WHCO3 cells exposed to GLA, AA, PGA2, indomethacin and the combination of indomethacin and GLA or AA.

PROTEIN TYROSINE KINASE ACTIVATION BY POLYCYCLIC AROMATIC HYDROCARBONS IN HUMAN HPB-ALL T CELLS

It has been well established that certain polycyclic aromatic hydrocarbons (PAHs), such as 7,12-dimethylbenz\\[a]anthracene (DMBA), 3-methylcholanthrene (3MC), and benzo\\[a]pyrene (BaP), produce immunotoxicity and cancer in rodents and that these effects are also likely seen in humans. Our laboratory has found that polycyclic aromatic hydrocarbons (PAHs) produce an increase in intracellular Ca2+ in lymphocytes that appears to correlate with their immunotoxicity. Specifically, immunotoxic PAHs, such as DMBA and BaP, have been shown to produce a sustained increase in intracellular Ca2+ in lymphocytes, whereas nonimmunosuppressive PAHs, such as benzo\\[e]pyrene (BeP) and anthracene, do not. Our studies previously demonstrated that the rapid increase in intracellular Ca2+ produced by DMBA in HPB-ALL T cells was caused by protein tyrosine kinase (PTK) activation in human T cells, leading to tyrosine phosphorylation of phospholipase C (PLC) and IP-dependent Ca2+ mobilization. However, the specificity of PTK activa1 3 tion by PAHs was not established. In the present studies, we extend our observations of PTK activation by examining a number of PAHs for their effects on total and specific (Fyn and ZAP-70) PTK activity. We show that 10 mu M concentrations of PAHs nonspecifically and rapidly (within 5 min) stimulate PTKs in the HPB-ALL human T cell line. BeP and anthracene were found to be nearly as effective at increasing total tyrosine kinase activity as DMBA, 3MC, and BaP, observed 5 min after exposure. We found that only immunotoxic PAHs activated the Fyn and ZAP-70 PTKs at 10 min, but total PTK activity was still increased by nonimmunotoxic PAHs, BeP, or anthracene after 10 min of exposure. These studies demonstrate that immunotoxic PAHs increase total and specific PTK activity in the human HPB-ALL T cell line. Thus the rapid increase in PTK activity produced by PAHs may not correlate with the immunotoxicity of these agents.

Tyrosine phosphorylation in early embryonic growth cones

A large and growing body of evidence suggests that the regulation of tyrosine phosphorylation is important in the induction of axon growth. We have examined the subcellular distribution of enzymes regulating tyrosine phosphorylation in early embryonic brain, employing a preparation of isolated growth cone particles (GCPs). Because of the early developmental age and well-characterized nature of our tissue source, our GCP preparation offers some advantages over those described previously. As was found with other GCPs, our GCPs had relatively high levels of both the growth-associated protein GAP-43 and the intracellular tyrosine kinase pp60c-arc. In addition, we found that both total tyrosine kinase activity and total tyrosine phosphatase activity were concentrated two- to threefold in the GCPs relative to a neuronal membrane fraction. Two other nonreceptor tyrosine kinases, YES and FYN, were concentrated in the GCPs to a similar degree as that seen for SRC. In addition, we examined the developmental expression in brain of the three tyrosine kinases, using both a quantitative ELISA and Western blot analysis. Our results show that FYN, like SRC, reaches a peak of expression early in development, and declines thereafter. In contrast, expression of YES peaks later, and remains high in the adult brain. Immunofluorescence staining suggests that FYN is expressed both by neurons and by glia, and possibly by neuronal precursor cells. Our results implicate multiple tyrosine kinases as well as tyrosine phosphatases in growth cone function. In addition, the concentration of FYN in early embryonic growth cones combined with its early peak of expression suggests an important role for FYN in early neuronal development.

Phosphorylation of proteins of the postsynaptic density: Effect of development on protein tyrosine kinase and phosphorylation of the postsynaptic density glycoprotein, PSD‐GP180

The effect of development on the tyrosine kinase activity of postsynaptic densities (PSDs) has been determined. PSDs were prepared from the forebrains of rats ranging in postnatal age from 13 to 90 days and the phosphorylation of both exogenous and endogenous substrates by tyrosine kinase measured. PSDs exhibited tyrosine kinase activity at all ages examined. Phosphorylation of the exogenous substrates polyglutamyltyrosine (4:1) and [val5] angiotensin II increased twofold between days 17 and 22 and then decreased between days 30 and 90 to levels slightly lower than those present at 13 days. The phosphorylation of endogenous PSD proteins on tyrosine residues, assessed by alkali digestion of polyacrylamide gels of 32P-labelled PSD proteins and by measuring the formation of [32P] phosphotyrosine by PSDs incubated in the presence of [gamma-32P] ATP, closely paralleled the changes in total tyrosine kinase activity. Tyrosine phosphorylation of the PSD-specific glycoprotein, PSD-GP180, also showed a transient increase between days 22 and 30, although its concentration within the PSD continued to increase slowly up to 90 days. The results indicate that the tyrosine kinase activity of PSDs is developmentally regulated and that tyrosine phosphorylation of PSD proteins is limited by enzyme rather than substrate availability.

Regulation of protein phosphotyrosine content by changes in tyrosine kinase and protein phosphotyrosine phosphatase activities during induced granulocytic and monocytic differentiation of HL-60 leukemia cells

About 1.5% of phosphorylated amino acid residues of HL-60 promyelocytic leukemia cells are phosphotyrosine. Induction of granulocytic differentiation by exposure to dimethylsulfoxide decreased tyrosine phosphorylation to 0.2%. A maximum 3-fold increase in tyrosine kinase activity and a 7-fold increase in protein phosphotyrosine phosphatase activity accompanied this change. Monocytic differentiation induced by 12-O-tetradecanoylphorbol-13-acetate, caused a decrease in phosphotyrosine levels to 0.1%; tyrosine kinase activity maximally increased 2-fold, and protein phosphotyrosine phosphatase activity increased 11-fold in these differentiated cells. Thus, although total tyrosine kinase activity markedly increased during differentiation, this was counteracted by an even greater elevation in protein phosphotyrosine phosphatase activity. The findings support the concept that tyrosine phosphorylation is important in the regulation of growth and differentiation of leukemia cells.