Induction of deoxypyrimidine kinase activity in human embryonic lung cells infected with varicella-zoster virus.

Deoxypyrimidine kinase (deoxythymidine [TdR] kinase and deoxycytidine kinase) activity was induced in human embryonic lung cells after infection with varicella-zoster virus (VZ virus). Increased enzyme activity was also produced by using cell-associated virus as inoculum instead of cell-free virus. Anti-VZ virus serum inhibited both the appearance of cytopathic effect and the induction of enzyme activity. The induced TdR kinase activity was more thermostable than that induced by herpes simplex virus type 1. Also, the TdR kinase activity of VZ virus-infected cells was inhibited by dTTP less than in mock-infected cells and more than in herpes simplex virus type 1-infected cells.     

Isolation of mutants of bacteriophage T4 unable to induce thymidine kinase activity. II. Location of the structural gene for thymidine kinase.

Amber mutants of bacteriophage T4 have been isolated that induce thymidine kinase activity only after infection of a strain of Escherichia coli carrying a suppressor mutation. The activity induced when one of these mutants infected this suppressor strain is much more heat sensitive than the activity induced by wild-type T4. This indicates that this amber mutation lies within the structural gene for thymidine kinase. This gene is between fI and v on the standard T4 genetic map. A mutant of tt4 that is unable to induce thymidine kinase activity incorporates only about one-eighth as much thymidine into its DNA as phage that do induce thymidine kinase. This contrasts to the findings that the total thymidine kinase activity in extracts prepared from cells infected with phage able to induce thymidine kinase in only twice as great as the activity in cells infected with the mutant unable to induce the enzyme.     

Characterization of new regulatory mutants of bacteriophage T4. II. New class of mutants.

Amber mutants of bacteriophage T4 have been isolated that induce thymidine kinase activity only after infection of a strain of Escherichia coli carrying a suppressor mutation. The activity induced when one of these mutants infected this suppressor strain is much more heat sensitive than the activity induced by wild-type T4. This indicates that this amber mutation lies within the structural gene for thymidine kinase. This gene is between fI and v on the standard T4 genetic map. A mutant of tt4 that is unable to induce thymidine kinase activity incorporates only about one-eighth as much thymidine into its DNA as phage that do induce thymidine kinase. This contrasts to the findings that the total thymidine kinase activity in extracts prepared from cells infected with phage able to induce thymidine kinase in only twice as great as the activity in cells infected with the mutant unable to induce the enzyme.     

The bcr-c-abl tyrosine kinase activity is extinguished by TPA in K562 leukemia cells

Tyrosine kinase activity is associated with the transforming potential of several oncogenes. Human chronic myeloid leukemia (CML) cells and cell lines have been shown to contain an active bcr-c-abl p210 tyrosine kinase as a consequence of the Philadelphia chromosomal translocation. In the present work the activity of the c-abl and c-src oncogene-encoded tyrosine kinase was investigated during phorbol diester (TPA) induced differentiation of the K562 CML cells. The high tyrosine kinase activity of p210bcr-c-abl is strongly reduced during the initial 24 h of TPA treatment. In contrast, the activity of the c-src tyrosine kinase is not changed. No change occurs in the expression of the c-abl-specific RNAs during this period. Following the reduction of bcr-c-abl kinase activity, cell proliferation is arrested and megakaryoblastic antigens appear on the cells. Sodium butyrate caused a slight decrease in growth rate and of bcr-c-abl kinase activity during erythroid differentiation whereas no changes in c-src or c-abl tyrosine kinase activities were seen in DMSO-treated control cells.     

Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase activity in the somatic cells of the seminiferous tubules. II. Effect of retinol

The effect of retinol on cyclic AMP dependent protein kinase activity of Sertoli cells and peritubular cells isolated from prepubertal rats has been investigated. Treatments longer than six hours induced a significant inhibition of type I protein kinase activity of Sertoli cells without appreciable variation of type II protein kinase. Short time treatments with the vitamin did not affect the Sertoli cell protein kinase activity. The vitamin A addition did not induce any appreciable variation of peritubular cell protein kinase activity.     

Chondrogenesis induced by actin cytoskeleton disruption is regulated via protein kinase C-dependent p38 mitogen-activated protein kinase signaling

Disruption of the actin cytoskeleton in subconfluent mesenchymal cells induces chondrogenic differentiation via protein kinase C (PKC) alpha signaling. In this study, we investigated the role of p38 mitogen-activated protein (MAP) kinase in the chondrogenic differentiation of mesenchymal cells that is induced by depolymerization of the actin cytoskeleton. Treatment of mesenchymal cells derived from chick embryonic limb buds with cytochalasin D (CD) disrupted the actin cytoskeleton with concomitant chondrogenic differentiation. The chondrogenesis was accompanied by an increase in p38 MAP kinase activity and inhibition of p38 MAP kinase with SB203580 blocked chondrogenesis. Together these results suggest an essential role for p38 MAP kinase in chondrogenesis. In addition, inhibition of p38 MAP kinase did not alter CD-induced increased expression and activity of PKC alpha, whereas down-regulation of PKC by prolonged exposure of cells to phorbol ester inhibited CD-induced p38 MAP kinase activation. Our results therefore suggest that PKC is involved in the regulation of chondrogenesis induced by disruption of the actin cytoskeleton via a p38 MAP kinase signaling pathway.     

Phospholipase D prevents apoptosis in v-Src-transformed rat fibroblasts and MDA-MB-231 breast cancer cells

Phospholipase D (PLD) activity is elevated in response to mitogenic and oncogenic signals. PLD also cooperates with overexpressed tyrosine kinases to transform rat fibroblasts. 3Y1 rat fibroblasts overexpressing the tyrosine kinase c-Src undergo apoptosis in response to serum withdrawal. We report here that elevated expression of either PLD1 or PLD2 in these cells prevents apoptosis induced by serum withdrawal. 3Y1 cells transformed by the activated tyrosine kinase v-Src have elevated PLD activity and are resistant to apoptosis induced by serum withdrawal. However, if PLD activity is blocked, the v-Src-transformed cells underwent apoptosis. MDA-MB-231 cells are a human breast cancer cell line with substantially elevated levels of PLD activity. Inhibiting PLD activity in these cells similarly rendered them sensitive to the apoptotic insult of serum withdrawal. These data indicate that elevated PLD activity generates a survival signal(s) allowing cells to overcome default apoptosis programs.     

Thymidine kinase with altered substrate specificity of acyclovir resistant varicella-zoster virus

The acyclovir resistant mutant of varicella-zoster virus ACV-R (A 8) induced the same level of thymidine kinase activity in infected cells as the parent Kawaguchi strain. However, it induced less deoxycytidine kinase activity and did not induce phosphorylating activity for the nucleotide analogue, 9-(2 hydroxy-ethoxymethyl)-guanine-(acyclovir). Another acyclovir resistant mutant, ACV-R (A 4), which is cross-resistant to phosphonoacetate and is thought to be a viral DNA polymerase mutant, induced the same level of phosphorylating activities for thymidine, deoxycytidine and acyclovir as the parent strain. The altered substrate specificity of thymidine kinase induced by ACV-R (A 8) is concluded to confer resistance to acyclovir on ACV-R (A 8).     

Deoxycytidine kinase from rabbit kidney cells infected with herpes simplex virus type 1 or 2.

When rabbit kidney cells were infected with herpes simplex virus type 1 (strain Seibert) or herpes simplex virus type 2 (strain 316D), deoxycytidine kinase (CdR kinase) activity, assayed at 38 degrees, increased 5- to 15-fold relative to controls. The CdR kinase activity induced by type 2 virus was more thermolabile than the enzyme activity induced by type 1 virus. When CdR kinase activity was assayed at various temperatures between 0.5 and 38 degrees, maximum activity for type 1 enzyme was obtained at 16 degrees while maximum activities for host and type 2 enzymes were obtained at 38 degrees. Both type 1 and type 2 induced CdR kinase activities eluted at the same positions as deoxythymidine kinase activities on a Sephadex G-100 column. The estimated mol wt for HSV-1 (Seibert) and HSV-2 (316D) induced CdR kinases are 67,000 and 60,000, respectively.     

Differential redistribution of protein kinase C in human aldosteronoma cells and adjacent adrenal cells stimulated with ACTH and angiotensin II

We have examined protein kinase C activity and hormone secretion in aldosteronoma cells derived from adrenocortical glomerulosa cells and in adjacent adrenal cells containing adrenocortical fasciculata-reticularis cells. When aldosteronoma cells were stimulated with ACTH or angiotensin II, protein kinase C activity gradually decreased in cytosol whereas it increased in membrane. Coincident with the changes of protein kinase C activity, there was enhancement of secretion of aldosterone. On the other hand, incubation of adjacent adrenal fasciculata-reticularis cells with ACTH induced cortisol secretion and an increase in cytosolic protein kinase C activity, accompanied by a decrease in the enzyme activity in membrane. Upon stimulation with angiotensin II, adjacent adrenal fasciculata-reticularis cells did not secrete cortisol and no significant changes of protein kinase C activities were observed in either cytosolic or membrane fractions. These results indicate that both ACTH and angiotensin II stimulate aldosterone secretion and cause translocation of protein kinase C from cytosol to membranes in aldosteronoma cells, whereas, in fasciculata-reticularis cells, only ACTH stimulates cortisol secretion and this is associated with translocation of protein kinase C in the opposite direction, viz., from membrane to cytosol.     

Differential effect of hemin-controlled eIF-2 alpha kinases from mouse erythroleukemia cells on protein synthesis

Cultured mouse erythroleukemia (MEL) cells can be induced to erythroid differentiation by a variety of chemical agents. This differentiation process is marked by the onset of globin mRNA and hemoglobin synthesis. In rabbit reticulocytes, globin synthesis is regulated by a hemin-controlled translational inhibitor (HCI) which acts via phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2). From both uninduced and induced MEL cells, hemin-controlled eIF-2 alpha kinases have been partially purified. They resemble HCI with respect to their chromatographic behaviour and their sensitivity towards physiological concentrations of hemin (5-10 microM). Further purification on phosphocellulose, however, reveals that the eIF-2 alpha kinase from uninduced MEL cells is chromatographically distinct from HCI, whilst the eIF-2 alpha kinase activity from induced MEL cells represents a mixture of the former and the HCI-type eIF-2 alpha kinase. The latter inhibits protein synthesis in a fractionated system from rabbit reticulocytes which is free of, but sensitive to, HCI, whereas the eIF-2 alpha kinase from uninduced MEL cells does not show any inhibitory activity. This observation is supported by the finding that induced MEL cells respond in vivo to iron depletion with a shut-off of protein synthesis (as do rabbit reticulocytes), whilst uninduced MEL cells do not.     

Integrins mediate the inhibitory effect of focal adhesion on angiotensin II-induced p44/42 mitogen-activated protein (MAP) kinase activity in human mesangial cells.

Previously, we reported that the formation of focal adhesion accelerated by accumulation of extracellular matrices may inhibit the angiotensin II-stimulated proliferation of human mesangial cells (HMCs). The process is regulated by p44/42 MAP kinase activity through the mediation of paxillin and GTPase activating proteins. In this report, we investigated the effect of integrin molecules on the angiotensin II-induced p44/42 MAP kinase activation in non-adherent HMCs. The results demonstrated that incubation of cells with both antibody to integrin beta(1) chain (K20) and GRGDS peptide induced integrin clustering, paxillin aggregation, and marked suppression of angiotensin II-induced p44/42 MAP kinase activation. On the other hand, incubation of cells with K20 alone induced integrin clustering without paxillin aggregation and the suppressive effect on angiotensin II-stimulated p44/42 MAP kinase activity. Our results strongly suggest the pivotal role of integrins in the inhibitory effect of focal adhesion on p44/42 MAP kinase activity, the checking system against angiotensin II-induced MAP kinase overactivation.     

Possible involvement of p38 MAP kinase in prostaglandin E1-induced ALP activity in osteoblast-like cells

Prostaglandins are now recognized to be important regulators for both bone formation and resorption. Among them, prostaglandin E(1) (PGE(1)) has been reported to stimulate cAMP accumulation and to induce alkaline phosphatase (ALP) activity, a marker of differentiation, in osteoblast-like cells. Recently, we have shown that p38 mitogen-activated protein (MAP) kinase pathway regulates ALP activity in response to activation of Gi protein-coupled receptors in mouse osteoblast-like MC3T3-E1 cells (Suzuki et al., Endocrinology 140 (1999) 3177). In the present study, we investigated whether p38 MAP kinase is involved in ALP activation by PGE(1) in MC3T3-E1 osteoblast-like cells. PGE(1) dose-dependently enhanced ALP activities in the concentration range between 1 nM and 1 microM in MC3T3-E1 cells. SB203580, a specific inhibitor of p38 MAP kinase, blocked the increase in ALP activity induced by PGE(1). Further analysis with western blotting suggested that PGE(1) induced an increase in tyrosine (Tyr) phosphorylation of p38 MAP kinase. Both Bt(2)cAMP, a permeable analogue of cAMP, and forskolin, which directly activates adenylate cyclase, also induced an increase in Tyr phosphorylation of p38 MAP kinase. H-89, a potent inhibitor of protein kinase A (PKA), significantly suppressed PGE(1)-induced Tyr phosphorylation of p38 MAP kinase. The results of this study suggest that PGE(1) stimulates p38 MAP kinase through the activation of PKA, resulting in the enhancement of ALP activity.     

Phosphofructokinase and pyruvate kinase in mouse embryonal carcinoma P19 cells in relation to growth and differentiation

Two key enzymes of glycolysis, phosphofructokinase and pyruvate kinase, were studied in embryonal carcinoma cells (P19 EC cells) and three differentiated derivatives in relation to growth rate and differentiation state. The growth rates of P19 EC cells and its differentiated derivatives are positively correlated with both the specific activity of phosphofructokinase and the expression of the L-subunit of this enzyme. The specific activity of pyruvate kinase and its isozyme composition is not correlated with growth rate but seems to be correlated with the differentiation state of these cells. The decrease in specific activity of pyruvate kinase during differentiation of P19 EC cells induced by retinoic acid or dimethylsulfoxide preceded the shift from K- to M-type pyruvate kinase. In contrast to aggregates that were treated with dimethylsulfoxide, the specific activity of pyruvate kinase was reduced after aggregation in the presence of retinoic acid. Only after plating dimethylsulfoxide-treated aggregates again in the presence of dimethylsulfoxide, was a decrease in specific activity obtained. Both retinoic acid and dimethylsulfoxide are able to induce a K- to -M shift of pyruvate kinase.     

Epstein-Barr virus induces a unique pyrimidine deoxynucleoside kinase activity in superinfected and virus-producer B cell lines

Epstein-Barr (EB) virus induces a new pyrimidine deoxynucleoside kinase [thymidine kinase (dTk)] activity in Raji B lymphocyte cells after superinfection. This dTk activity is also present in small amounts in the HR-1 virus-producer cell line and in larger amounts in the B95-8 virus-producer line. The dTk activity induced by EB virus coelutes from DEAE-cellulose columns with deoxycytidine kinase (dCk) activity and elutes as a broad peak well separated from the large peaks of cellular dTk and dCk activities. This EB virus-induced pyrimidine deoxynucleoside kinase activity from HR-1 cells differs from cellular kinases in most basic biochemical properties but shares certain properties with the herpes simplex virus dTk.     

Suppression of protein tyrosine kinase activity of the epidermal growth factor receptor by epidermal growth factor

Epidermal growth factor (EGF) receptor protein kinase activity, estimated by the use of peptide substrates, was reduced by as much as 70% after the treatment of intact A431 human carcinoma cells with EGF. The apparent decrease in protein kinase activity was observed after immunoprecipitation of the receptor or after purification of the receptor by lectin chromatography. By the use of [35S]methionine, it was determined that the total amount of receptor obtained was the same whether or not cells were treated with EGF. EGF stimulated the purified receptor protein kinase activity in vitro; however, the EGF-stimulated activity of receptor from EGF-treated cells continued to be reduced by as much at 70% compared to the EGF-stimulated activity from untreated cells. The reduction in receptor protein kinase activity induced by EGF may represent a feedback mechanism by which responsiveness to the growth factor is regulated.     

Relocation of a Ca2+-dependent protein kinase activity during pollen tube reorientation

Pollen tube reorientation is a dynamic cellular event that is crucial for successful fertilization. We have shown previously that pollen tube orientation is regulated by cytosolic free calcium ([Ca2+]c). In this paper, we studied the activity of a Ca2+-dependent protein kinase during reorientation. The kinase activity was assayed in living cells by using confocal ratio imaging of BODIPY FL bisindolylmaleimide. We found that growing pollen tubes exhibited higher protein kinase activity in the apical region, whereas nongrowing cells showed uniform distribution. Modification of growth direction by diffusion of inhibitors/activators from a micropipette showed the spatial redistribution of kinase activity to predict the new growth orientation. Localized increases in [Ca2+]c induced by photolysis of caged Ca2+ that led to reorientation also increased kinase activity. Molecular and immunological assays suggest that this kinase may show some functional homology with protein kinase C. We suggest that the tip-localized gradient of kinase activity promotes Ca2+-mediated exocytosis and may act to regulate Ca2+ channel activity.