Involvement of protein tyrosine phosphorylation and reduction of cellular sulfhydryl groups in cell death induced by 1' -acetoxychavicol acetate in Ehrlich ascites tumor cells

Elucidation of the mechanisms underlying potential anticancer drugs continues and unraveling these mechanisms would not only provide a conceptual framework for drug design but also promote use of natural products for chemotherapy. To further evaluate the efficacy of the anticancer activity of 1'-acetoxychavicol acetate (ACA), this study investigates the underlying mechanisms by which ACA induces death of Ehrlich ascites tumor cells. ACA treatment induced loss of cell viability, and Western blotting analysis revealed that the compound stimulated tyrosine phosphorylation of several proteins with 27 and 70 kDa proteins being regulated in both dose- and time-dependent manner prior to loss of viability. Protein tyrosine kinase inhibitor herbimycin A moderately protected cells from ACA-induced toxicity. In addition, cellular glutathione and protein sulfydryl groups were also significantly reduced both dose- and time-dependently during evidence of cell death. Replenishing thiol levels by antioxidant, N-acetylcysteine (NAC), an excellent supplier of glutathione and precursor of glutathione, substantially recovered the viability loss, but the recovery being time-dependent, as late addition of NAC (at least 30 min after ACA addition to cultures) was, however, ineffective. Addition of NAC to ACA treated cultures also abolished tyrosine phosphorylation of the 27 kDa protein. These results, at least partly, identify cellular sulfhydryl groups and protein tyrosine phosphorylation as targets of ACA cytotoxicity in tumor cells.     

In vitro substrate specificity of protein tyrosine kinases

Synthetic peptides such as P60^stc autophosphorylation site peptides and angiotensin are indiscriminately phosphorylated by protein tyrosine kinases. The observation has led to the general belief that protein tyrosine kinases are highly promiscuous, displaying littlein vitro site specificity. In recent years, evidence has been accumulating to indicate that such a belief requires close examination. Synthetic peptides showing high substrate activity for specific groups of protein tyrosine kinases have been obtained. Systematic modification of certain substrate peptides suggests that kinase substrate determinants reside with specific amino acid residues proximal to the target tyrosine. A number of protein kinases have been shown to be regulated by tyrosine phosphorylation at specific sites by highly specific protein tyrosine kinases. These and other selected biochemical studies that contribute to the evolving view ofin vitro substrate specificity of protein tyrosine kinases are reviewed.     

Regulation of protein kinases activity by quercetin in Ehrlich ascites tumor cells

Cyclic AMP-independent protein kinase activities from Ehrlich ascites tumor cells, partially purified by DEAE-cellulose and phosphocellulose chromatography were inhibited by quercetin. The cyclic AMP in the tumor ascites cells and the cyclic AMP-dependent protein kinase activity from this tumor and from bovine and mouse tissues were unaffected by this drug. Since we reported that quercetin elevates cyclic AMP level in Ehrlich ascites tumor cells, this bioflavonoid may have a dual effect on the protein kinae activities in these cells, thus, increasing the cyclic AMP-dependent and decreasing the cyclic AMP-independent protein kinase activities.     

Pamlin-induced tyrosine phosphorylation of SUp62 protein in primary mesenchyme cells during early embryogenesis in the sea urchin, Hemicentrotus pulcherrimus

Ingression of primary mesenchyme cells (PMC) is associated with the encounter of basal lamina including pamlin. It was found that sea urchin embryos have a protein that binds antihuman focal adhesion kinase (FAK) antibodies, yet it has a 62 kDa homo-dimeric structure. Thus, this protein was distinctive from known FAK, and was named SUp62. In mesenchyme blastulae, one of the subunits increased its apparent molecular mass slightly but distinctively, then restored the original molecular mass in early gastrulae. This temporal and stage-specific shifting of the molecular mass was associated with the occurrence of tyrosine phosphorylation of a subunit that did not increase the apparent molecular mass. Herbimycin A induced the hyperphosphorylation of tyrosine residues of SUp62, and inhibited the occurrence of molecular mass shifting. Immunohistochemistry showed a strong positive signal of SUp62 and phosphotyrosine in PMC. Herbimycin A also severely but reversibly inhibited PMC dissociation, migration and gastrulation. Tyrosine phosphorylation of SUp62 was induced when PMC were incubated with pamlin in vitro, and it was initiated within 10 min after onset of the incubation. It reached its peak in 1 h, and declined gradually in the next 1 h, indicating that pamlin-induced tyrosine phosphorylation of SUp62 occurs closely associated with acquiring PMC migration activity.     

A cyclic adenosine 3',5'-monophosphate-induced tyrosine phosphorylation of Syk protein tyrosine kinase in the flagella of boar spermatozoa

A cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein tyrosine phosphorylation is involved in the expression of fertilizing ability in mammalian spermatozoa. However, there are only limited data concerning the identification of protein tyrosine kinase (PTK) that is activated by the cAMP signaling. In this study, we have shown data supporting that boar sperm flagellum possesses a unique cAMP-protein kinase A (PKA) signaling cascade leading to phosphorylation of Syk PTK at the tyrosine residues of the activation loop. Ejaculated spermatozoa were washed and then incubated in a modified Krebs-Ringer HEPES medium (mKRH) containing polyvinyl alcohol (PVA) plus 0.1 mM cBiMPS (a cell-permeable cAMP analog), 0.25 mM sodium orthovanadate (Na3VO4) (a protein tyrosine phosphatase (PTP) inhibitor) or both at 38.5 degrees C for 180 min. Aliquots of the sperm suspensions were recovered before and after incubation and then used to detect sperm tyrosine-phosphorylated proteins by Western blotting and indirect immunofluorescence. In the Western blotting, the anti-phosphotyrosine monoclonal antibody (4G10) recognized several bands including 72-kDa protein in the protein extracts from spermatozoa that were incubated solely with cBiMPS. The tyrosine phosphorylation in these sperm proteins was dependent on cBiMPS and enhanced by the addition of Na3VO4. The 72-kDa tyrosine-phosphorylated protein was apparently reacted with the anti-phospho-Syk antibody (Tyr525/526). Indirect immunofluorescence revealed that the connecting and principal pieces of spermatozoa incubated with cBiMPS and Na3VO4 were stained with the anti-phospho-Syk antibody. However, the reactivity of the 72-kDa protein with the anti-phospho-Syk antibody was reduced by the addition of H-89 (a PKA inhibitor, 0.01-0.1 mM) to the sperm suspensions but not affected by the pretreatment of spermatozoa with BAPTA-AM (an intracellular Ca2+ chelator, 0.1 mM). Fractionation of phosphorylated proteins from the spermatozoa with a detergent Nonidet P-40 suggested that the 72-kDa tyrosine-phosphorylated protein might be a cytoskeletal component. Based on these findings, we have concluded that the cAMP-PKA signaling is linked to the Ca2+-independent tyrosine phosphorylation of Syk in the connecting and principal pieces of boar spermatozoa.     

Cellular thiols status and cell death in the effect of green tea polyphenols in Ehrlich ascites tumor cells.

Epidemiological studies suggest that the consumption of green tea may help prevent cancers in humans, and also breast and prostate cancers in animal models are reduced by green tea, and several mechanisms have been proposed for these effects. In this study the relationship between cellular sulfhydryl (SH) groups and the cytotoxicity of green tea polyphenols in Ehrlich ascites tumor cells was examined. It was found that in the presence of green tea extract (GTE) (100 microg/ml) and one of its polyphenolic components, epigallocatechin (EGC; 100 microM), both cellular non-protein (GSH) and protein-sulfhydryl (PSH) levels were significantly decreased and this was associated with a decrease in cell viability. Replenishing the thiol levels by using N-acetylcysteine (NAC) caused a recovery in cell viability, but this recovery was dependent on the time of thiol replenishment in the presence of EGC (initial 15 min). These results identify SH groups as a novel target of green tea polyphenols cytotoxicity in tumor cells, and a regulatory role for green tea in terms of reducing sulfhydryls in tumor inhibition.     

Large‐scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis

Protein phosphorylation regulates a wide range of cellular processes. Here, we report the proteome‐wide mapping of in vivo phosphorylation sites in Arabidopsis by using complementary phosphopeptide enrichment techniques coupled with high‐accuracy mass spectrometry. Using unfractionated whole cell lysates of Arabidopsis, we identified 2597 phosphopeptides with 2172 high‐confidence, unique phosphorylation sites from 1346 proteins. The distribution of phosphoserine, phosphothreonine, and phosphotyrosine sites was 85.0, 10.7, and 4.3%. Although typical tyrosine‐specific protein kinases are absent in Arabidopsis, the proportion of phosphotyrosines among the phospho‐residues in Arabidopsis is similar to that in humans, where over 90 tyrosine‐specific protein kinases have been identified. In addition, the tyrosine phosphoproteome shows features distinct from those of the serine and threonine phosphoproteomes. Taken together, we highlight the extent and contribution of tyrosine phosphorylation in plants.     

Binucleate cells in the Ehrlich ascites tumor. Autoradiographic labeling

An autoradiographic study was performed on binucleate and mitotic cells in the Ehrlich ascites tumor (EAT) untreated and after treatment with 5-fluorouracil (FU). The number of binucleate cells was greater in the treated tumor than in the controls. It was also observed that the number of labeled mitoses was greater in the Fu-treated tumor. Autoradiographic labeling showed that the cells that proved to be binucleate had previously passed through S-phase; thus, these cells belonged to the proliferative compartment.     

Involvement of the N-terminal unique domain of Chk tyrosine kinase in Chk-induced tyrosine phosphorylation in the nucleus

Chk tyrosine kinase phosphorylates Src-family kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. In this study, we explored the role of the N-terminal unique domain of Chk in nuclear localization and Chk-induced tyrosine phosphorylation in the nucleus. In situ binding experiments showed that the N-terminal domain of Chk was associated with the nucleus and the nuclear matrix. The presence of the N-terminal domain of Chk led to a fourfold increase in cell population exhibiting Chk-induced tyrosine phosphorylation in the nucleus. Expression of Chk but not kinase-deficient Chk induced tyrosine phosphorylation of a variety of proteins ranging from 23 kDa to approximately 200 kDa, especially in Triton X-100-insoluble fraction that included chromatin and the nuclear matrix. Intriguingly, in situ subnuclear fractionations revealed that Chk induced tyrosine phosphorylation of proteins that were associated with the nuclear matrix. These results suggest that various unidentified substrates of Chk, besides Src-family kinases, may be present in the nucleus. Thus, our findings indicate that the importance of the N-terminal domain to Chk-induced tyrosine phosphorylation in the nucleus, implicating that these nuclear tyrosine-phosphorylated proteins may contribute to inhibition of cell proliferation.     

Juvenile hormone stimulated tyrosine kinase-mediated protein phosphorylation in the CNS of the silk worm,Bombyx mori

In vitro studies with the larval CNS of the silkworm, Bombyx mori revealed the phosphorylation of a 48-kDa protein, which was not dependent on cyclic nucleotides. Studies also revealed modest phosphorylation of this protein by a calcium-dependent but calmodulin-independent mechanism. However, phosphorylation of this protein was greatly enhanced in the presence of juvenile hormone (JH) I by a calcium-independent mechanism. This stimulatory effect of JH was seen in both homogenates as well as in intact CNS of Bombyx. Immunoblotting studies revealed the cross-reaction of this 48-kDa protein with phosphotyrosine monoclonal antibody and the phosphorylation of this protein was inhibited by genistein. This study suggests that the 48-kDa protein is a substrate for tyrosine kinase. The phosphorylation of this protein was also observed in other larval tissues such as salivary gland, fat body, and epidermis of Bombyx.     

Insulin-stimulated tyrosine phosphorylation of a 43 kDa protein in rat liver membranes

The insulin receptor (IR) tyrosine kinase is essential for the regulation of different cellular functions by insulin. This may occur by a direct phosphorylation of membrane and/or cytoplasmic proteins by the IR tyrosine kinase. Hence it is important to identify putative physiological substrates for the IR tyrosine kinase. In this study we found that the glycoprotein fraction from rat liver membranes contain a 43 kDa protein (pp43) which, like the -subunit of IR, is phosphorylated in an insulin-dependent manner. A 25-fold enhancement of ³²P incorporation into pp43 by insulin was found under optimal conditions. Half-maximal phosphorylation of pp43 and the -subunit of IR were attained at 66 nM and 60 nM insulin, respectively. Mn²⁺ (K_a = 1.0 mM) was much better than Mg²⁺ (K_a = 6.3 mM) in supporting pp43 phosphorylation. Insulin-stimulated phosphorylation of pp43 (t_1/2 = 3.6 min) proceeded at a much slower rate compared to that of the -subunit of IR (t_1/2 = 1.2 min). Phosphoamino acid analysis of pp43 revealed that both tyrosine and serine are phosphorylated in the ratio 4 : 1. Tyrosine, but not serine, phosphorylation was increased 12-fold by insulin. Phosphorylation of pp43 occurred on 4 major tryptic peptides. Comparison to the tryptic phosphopeptides from IR -subunit suggest that pp43 was not derived from IR -subunit by proteolysis. Our results suggest that pp43 may be an endogenous substrate for the IR tyrosine kinase.     

Inhibition of protein kinase C reduces ischemia-induced tyrosine phosphorylation of the N-methyl-d-aspartate receptor

The role of protein kinase C (PKC) in tyrosine phosphorylation of the N‐methyl‐d ‐aspartate receptor (NMDAR) following transient cerebral ischemia was investigated. Transient (15 min) cerebral ischemia was produced in adult rats by four‐vessel occlusion and animals allowed to recover for 15 or 45 min. Following ischemia, tyrosine phosphorylation of NR2A and NR2B and activated Src‐family kinases (SFKs) and Pyk2 were increased in post‐synaptic densities (PSDs). Phosphorylation of NR2B on Y1472 by PSDs isolated from post‐ischemic forebrains was inhibited by the SFK specific inhibitor PP2, and by the PKC inhibitors GF109203X (GF), Gö6976 and calphostin C. Intravenous injection of GF immediately following the ischemic challenge resulted in decreased phosphorylation of NR1 on PKC phosphorylation sites and reduced ischemia‐induced increases in tyrosine phosphorylation of NR2A and NR2B without affecting the increase in total tyrosine phosphorylation of hippocampal proteins. Ischemia‐induced increases in activated Pyk2 and SFKs in PSDs, but not the translocation of PKC, Pyk2 or Src to the PSD, were also inhibited by GF. The inactive homologue of GF, bisindolylmaleimide V, had no effect on these parameters. The results are consistent with a role for PKC in the ischemia‐induced increase in tyrosine phosphorylation of the NMDAR, via a pathway involving Pyk2 and Src‐family kinases.     

Involvement of host cell tyrosine phosphorylation in the invasion of HEp-2 cells by Bartonella bacilliformis

We have provided evidence that exposure of human cells to protein kinase inhibitors results in decreased invasion of these cells by Bartonella bacilliformis in a dose-dependent manner. Preincubation of human laryngeal epithelial cells in the presence of genistein, a tyrosine protein kinase inhibitor, decreased the invasion of these cells by B. bacilliformis significantly. Further, exposure of normal human umbilical vein endothelial cells to staurosporine, a potent inhibitor of protein kinase C and some tyrosine protein kinases, resulted in a considerable reduction in the number of organisms internalized by these cells. Moreover, Bartonella infection of HEp-2 cells induced tyrosine phosphorylation of several Triton X-100 soluble proteins with approximate molecular masses of 243, 215 179, 172 (doublet), 160, 145 and 110 kDa that were absent or reduced in the presence of genistein in cells after 1 h of infection. Exposure of HEp-2 cell monolayers to anti-alpha 5 and anti-beta 1 chain integrin monoclonal antibodies resulted in a moderate decrease in the invasion of these cells, suggesting a possible role of alpha 5 beta 1 integrins in the uptake of Bartonella into nucleated cells.     

Src-Tyrosine kinases are major agents in mitochondrial tyrosine phosphorylation

Mitochondrial tyrosine phosphorylation is emerging as an important mechanism in regulating mitochondrial function. This article, aimed at identifying which kinases are the major agents in mitochondrial tyrosine phosphorylation, shows that this role should be attributed to Src family members. Indeed, various members of this family, for example, Fgr, Fyn, Lyn, c-Src, are constitutively present in the internal structure of mitochondria as well as Csk, a key enzyme in the regulation of the activity of this family. By means of different approaches, biochemical fractioning, Western blotting and immunogold analysis "in situ" of phosphotyrosine signaling, evidence is reported on the existence of a signal transduction pathway from plasma membrane to mitochondria, resulting in increasing Src-dependent mitochondrial tyrosine phosphorylation. The activation of Src kinases at mitochondrial level is associated with the proliferative status where several mitochondrial proteins are specifically tyrosine-phosphorylated.     

In vitro formation of different tubulin polymers from purified tubulin of Ehrlich ascites tumor cells

Preparations of cycled tubulin from Ehrlich ascites tumor cells contain several acessory proteins; once or twice cycled microtubule preparations are usually composed of fibers 10 nm in diameter, but lack vimentin. Highly purified tubulin consists of α- and β-tubulin and a minor component which was identified by peptide mapping as a second β-chain. This pure tubulin is able to form in vitro at low concentrations (1 mg protein/ml) fibers of about 10 nm width, and at higher concentrations (3.5 mg protein/ml) normal microtubules.     

Phosphorylation of Ser19 increases both Ser40 phosphorylation and enzyme activity of tyrosine hydroxylase in intact cells

We have previously shown that the phosphorylation of Ser19 in tyrosine hydroxylase can increase the rate of phosphorylation of Ser40 in tyrosine hydroxylase threefold in vitro. In this report we investigated the role of Ser19 on Ser40 phosphorylation in intact cells. Treatment of bovine chromaffin cells with anisomycin produced a twofold increase in Ser19 phosphorylation with no increase in Ser31 phosphorylation and only a small increase in Ser40 phosphorylation. Treatment of bovine chromaffin cells with forskolin produced a fourfold increase in Ser40 phosphorylation but no significant increase in either Ser19 or Ser31 phosphorylation. When chromaffin cells were first treated with anisomycin, the level of Ser40 phosphorylation after treatment by forskolin was 76% greater than the level of Ser40 phosphorylation in cells treated with forskolin alone. This potentiation of Ser40 phosphorylation by anisomycin could be completely blocked by the p38 MAP (mitogen-activated protein) kinase inhibitor SB 203580. The potentiation of Ser40 phosphorylation by anisomycin was not due to an increase in Ser40 kinase activity. Anisomycin treatment of chromaffin cells potentiated the forskolin-induced increase in tyrosine hydroxylase activity by 50%. This potentiation of activity was also blocked by SB 203580. These data provide the first evidence that the phosphorylation of Ser19 can potentiate the phosphorylation of Ser40 and subsequent activation of tyrosine hydroxylase in intact cells.     

Effect of brassinolide on tyrosine phosphorylation of pea leaf proteins

Brassinosteroid-induced phosphorylation of tyrosine residues in proteins was studied. Proteins of crude extract of pea leaves were analyzed by one- and two-dimensional electrophoresis followed by Western blotting with monoclonal antibodies PY20 to phosphotyrosine proteins. One- and two-dimensional electrophoresis revealed 7 and 13 tyrosine-phosphorylated proteins, respectively. Brassinolide increased the phosphorylation level of most of these proteins. With inhibitors of tyrosine protein phosphatases, such as phenylarsine oxide and orthovanadate, the level of tyrosine phosphorylation of these proteins increased.     

声化学激活血卟啉诱导艾氏腹水肿瘤细胞凋亡

本实验采用频率为2．0MHz，声强分别为1．0w／cm^2、1．5w／cm^2、2．0w／cm^2等不同参数，研究超声激活血卟啉对艾氏腹水肿瘤细胞的杀伤作用和诱导肿瘤细胞凋亡现象。通过扫描电镜、透射电镜以及荧光显微镜观察受损后细胞形态结构的变化，主要表现为细胞微绒毛的减少，胞膜结构和通透性的改变，细胞器的受损以及核物质的分解、丢失；同时发现处理后的肿瘤细胞有核物质凝集、趋边排列以及凋亡小体的形成等细胞凋亡特征。研究中首次发现声化学激活血卟啉在对艾氏腹水肿瘤细胞杀伤的同时，也能诱导艾氏腹水肿瘤细胞发生凋亡，提示在声动力疗法中并存着对癌细胞的直接杀伤和通过诱导癌细胞凋亡的两种抗癌途径。     

TULA proteins regulate activity of the protein tyrosine kinase Syk

TULA belongs to a two-member family: TULA (STS-2) is a lymphoid protein, whereas STS-1/TULA-2 is expressed ubiquitously. TULA proteins were implicated in the regulation of signaling mediated by protein tyrosine kinases (PTKs). The initial experiments did not fully reveal the molecular mechanism of these effects, but suggested that both TULA proteins act in a similar fashion. It was shown recently that STS-1/TULA-2 dephosphorylates PTKs. In this study, we analyzed the effects of TULA proteins on Syk, a PTK playing an important role in lymphoid signaling. First, we have shown that TULA-2 decreases tyrosine phosphorylation of Syk in vivo and in vitro and that the intact phosphatase domain of TULA-2 is essential for this effect. We have also shown that TULA-2 exhibits a certain degree of substrate specificity. Our results also indicate that inactivated TULA-2 increases tyrosine phosphorylation of Syk in cells co-transfected to overexpress these proteins, thus acting as a dominant-negative form that suppresses dephosphorylation of Syk caused by endogenous TULA-2. Furthermore, we have demonstrated that phosphatase activity of TULA is negligible as compared to that of TULA-2 and that this finding correlates with an increase in Syk tyrosine phosphorylation in cells overexpressing TULA. This result is consistent with the dominant-negative effect of inactivated TULA-2, arguing that TULA acts in this system as a negative regulator of TULA-2-dependent dephosphorylation. To summarize, our findings indicate that TULA proteins may exert opposite effects on PTK-mediated signaling and suggest that a regulatory mechanism based on this feature may exist.