Activation of tyrosine kinases during induction of mammary gland tumors in GR mice by ovarian hormones

In GR mice, the induction of proliferative processes in mammary tumours with ovarian hormones (estrone and progesterone) is accompanied by the activation of phosphorylation of plasma membrane, cytosolic and nuclear proteins by endogenous protein kinases. The hormones stimulate tyrosine kinases of tumour cells whose activity is as high as 14.9-17.9% of the total phosphorylation in plasma membranes and 9.5-10.4% in cell nuclei. The ovarian hormones stimulate tyrosine kinases of tumour cells which phosphorylate proteins with Mr of 110-230 and 15 kD (plasma membranes), 170, 52 and 13 kD (cytosol) and 32 kD (nuclei) which are resistant to alkaline hydrolysis. Apart from tyrosine kinases, the ovarian hormones also stimulate serine and threonine protein kinases which seems to be due to the activation of protein kinase C and other protein kinases.     

Protein Kinases in Zucchini (Characterization of Calcium-Requiring Plasma Membrane Kinases)

Using an in situ phosphorylation assay with zucchini (Cucurbita pepo L. cv Dark Green) seedling tissue, we have identified numerous polypeptides that are capable of acting as protein kinases. Total protein preparations from different organs contain different kinase profiles, but all are within the range of 55 to 70 kD. At least four kinases are associated with highly purified plasma membranes from etiolated zucchini hypocotyls. The major phosphorylated polypeptides from plasma membranes range in apparent molecular mass from 58 to 68 kD. The plasma membrane kinases are activated by micromolar concentrations of calcium and phosphorylate serine, and, to a lesser extent, threonine residues. These characteristics are similar to those of a soluble calcium-dependent protein kinase that has been purified to homogeneity from soybean suspension cultures. Three of the zucchini plasma membrane kinases share antigenic epitopes with the soluble soybean kinase. The presence of kinase activity at different apparent molecular masses may be indicative of separate kinases with similar characteristics. The zucchini hypocotyl protein kinases are not removed from plasma membrane vesicles by 0.5 M NaCl/5 mM ethylenediaminetetraacetate or by detergent concentrations below the critical micelle concentration of two types of detergent. This indicates that the plasma membrane protein kinases are tightly associated with the membrane in zucchini seedlings.     

The effect of estradiol on the activity of cAMP-dependent protein kinase in cells of estradiol-dependent tumors of the rat mammary gland. The role of a thermally stable protein inhibitor of cAMP-dependent protein kinase

Stimulation of rat mammary tumour growth by estradiol is due to the activation of the adenylate cyclase system and cAMP-dependent protein kinases. A single administration of estradiol to ovariectomized rats causes a rise in the cAMP-dependent protein kinase activity in cell nuclei within the first 4-6 hours after injection. This effect is probably due to the translocation of enzymes into nuclei and an increase of their synthesis. The high level of the cAMP-dependent protein kinase activity in cell nuclei was observed in actively growing intact mammary tumours, in contrast to regressing ones in ovariectomized animals. This phenomenon can be accounted for by the decrease in the content of a thermostable protein inhibitor of cAMP-dependent protein kinases rather than by the high level of cAMP.     

Specific phosphorylation of the beta subunit of eIF-2 factor from brain by three different protein kinases

The eukaryotic initiation factor 2 (eIF-2) from calf brain has been purified to homogeneity and free of endogenous kinase activity. Phosphorylation of eIF-2 factor has been examined with four different protein kinases. Casein kinase II, calcium/phospholipid-dependent protein kinase and cyclic AMP-dependent protein kinase from brain, phosphorylate the beta subunit of eIF-2, whilst hemin-controlled inhibitor phosphorylate the alpha subunit of the factor. According to the peptide maps obtained, the phosphorylation sites of the factor by the three beta kinases are specific and distinct. These data suggest a different regulation for the beta subunit through this modification.     

Phosphorylation of proteins in the kidney papillary zone by endogenous cAMP-dependent protein kinases

Using SDS-PAAG electrophoresis with subsequent autoradiography, several proteins from plasma membranes and cell cytosol of rat kidney papillary zone were identified as substrates for endogenous cAMP-dependent protein kinases. The cAMP-dependent phosphorylation of plasma membrane proteins was made possible only after the destruction of membrane vesicles. Plasma membrane and cytosol fractions were found to contain a 58 kDa protein whose properties are similar to those of the regulatory subunit of cAMP-dependent protein kinase of the second type. It was shown also that the content of endogenous substrates of cAMP-dependent protein kinases in cell cytosol is higher than that in plasma membranes.     

MARCKS protein is a key molecule regulating mucin secretion by human airway epithelial cells in vitro

Hypersecretion of airway mucin characterizes numerous respiratory diseases. Although diverse pathological stimuli can provoke exocytotic release of mucin from secretory cells of the airway epithelium, mechanisms involved remain obscure. This report describes a new paradigm for the intracellular signaling mechanism regulating airway mucin secretion. Direct evidence is provided that the myristoylated alanine-rich C kinase substrate (MARCKS) is a central regulatory molecule linking secretagogue stimulation at the cell surface to mucin granule release by differentiated normal human bronchial epithelial cells in vitro. Down-regulation of MARCKS expression or disruption of MARCKS function in these cells inhibits the secretory response to subsequent stimulation. The intracellular mechanism controlling this secretory process involves cooperative action of two separate protein kinases, protein kinase C and cGMP-dependent protein kinase. Upon stimulation, activated protein kinase C phosphorylates MARCKS, causing translocation of MARCKS from the plasma membrane to the cytoplasm, where it is then dephosphorylated by a protein phosphatase 2A that is activated by cGMP-dependent protein kinase, and associates with both actin and myosin. Dephosphorylated cytoplasmic MARCKS would also be free to interact with mucin granule membranes and thus could link granules to the contractile cytoskeleton, mediating their movement to the cell periphery and subsequent exocytosis. These findings suggest several novel intracellular targets for pharmacological intervention in disorders involving aberrant secretion of respiratory mucin and may relate to other lesions involving exocytosis of membrane-bound granules in various cells and tissues.     

Identification of immune-related protein kinases from mosquitoes (Aedes aegypti)

Protein kinases are known to be involved in signal transduction for numerous physiological events. However, little is known about the roles of protein kinases in insect immunity. A fragment around 150 bp was amplified by polymerase chain reaction using cDNA templates from bacterial inoculated mosquitoes and primers corresponding to the conserved domain of protein kinases. Based on sequence analysis, 11 groups of protein kinases were characterized including 3 nonreceptor tyrosine kinases, 3 receptor tyrosine kinases, 3 serine/threonine kinases, and 2 novel protein kinases. The most abundant kinase obtained in this study reveals a high degree of similarity to human cholinesterase-related cell division controller (CHED) protein kinase. The expression of this mosquito CHED-like kinase is not detectable in normal female mosquitoes, but induced only after bacterial inoculation and trauma. A mosquito protein kinase was demonstrated to share homology with a plant Tousled gene, but has not yet been characterized in the animal system. In addition, analysis of the sequences of several protein kinases cloned from mosquitoes suggests that they might be involved in the regulation of cellular or humoral immunity.     

Assembly of an A kinase-anchoring protein-beta(2)-adrenergic receptor complex facilitates receptor phosphorylation and signaling

Phosphorylation of G-protein-coupled receptors by second-messenger-stimulated kinases is central to the process of receptor desensitization [1-3]. Phosphorylation of the beta(2)-adrenergic receptor (beta(2)-AR) by protein kinase A (PKA), in addition to uncoupling adenylate cyclase activation, is obligatory for receptor-mediated activation of mitogen-activated protein kinase (MAP kinase) cascades [4] [5]. Although mechanisms for linking G-protein-coupled receptor kinases to the activated receptor are well established, analogous mechanisms for targeting second messenger kinases to the beta(2)-AR at the plasma membrane have not been elucidated. Here we show that the A-kinase-anchoring protein, AKAP79/150, co-precipitates with the beta(2)-AR in cell and tissue extracts, nucleating a signaling complex that includes PKA, protein kinase C (PKC) and protein phosphatase PP2B. The anchoring protein directly and constitutively interacts with the beta(2)-AR and promotes receptor phosphorylation following agonist stimulation. Functional studies show that PKA anchoring is required to enhance beta(2)-AR phosphorylation and to facilitate downstream activation of the MAP kinase pathway. This defines a role for AKAP79/150 in the recruitment of second-messenger-regulated signaling enzymes to a G-protein-coupled receptor.     

Lipoproteins and mitogen-activated protein kinase signaling: a role in atherogenesis?

PURPOSE OF REVIEW: Lipoproteins play a critical role in the development of atherosclerosis, which might result partly from their capacity to induce specific intracellular signaling pathways. The goal of this review is to summarize the signaling properties of lipoproteins, in particular, their capacity to induce activation of mitogen-activated protein kinase pathways and the resulting modulation of cellular responses in blood vessel cells. RECENT FINDINGS: Lipoproteins activate the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways in all blood vessel cell types. This may require lipoprotein docking to scavenger receptor B1, allowing transfer of cholesterol and sphingosine-1-phosphate to plasma membranes. Subsequent propagation of the signals probably requires the stimulation of G protein-coupled receptors, followed by the transactivation of receptor tyrosine kinases. Lipoprotein-induced extracellular signal-regulated kinase activity favors cell proliferation, whereas lipoprotein-induced p38 mitogen-activated protein kinase activity leads to cell hyperplasia and promotes cell migration. Some signaling pathways and cellular effects induced by lipoproteins have been observed in atherosclerotic plaques and therefore represent potential targets for the development of anti-atherosclerotic drugs. SUMMARY: The main blood vessel cell types have the capacity to activate protein kinase pathways in the presence of lipoproteins. This induces cell proliferation, hyperplasia and migration, known to be dysregulated in atherosclerotic lesions.     

Translocation of protein kinase C from cytosol into cell membranes after treatment with estradiol and enzyme activation in target cells

The effect of 17 beta-estradiol on protein kinase C in target cells was studied. It was shown that 10-15 min after injection of ovariectomized animals with estradiol (10 micrograms intraperitoneally) protein kinase C is translocated from the cytosol into the cell membranes of estradiol-dependent mammary gland tumours. A similar effect of estradiol on protein kinase C is observed in uterine tissue. On the contrast, in hormone-independent rat mammary gland tumours estradiol causes no redistribution of protein kinase C between the cytosol and cell membranes. No protein kinase C accumulation in the membranes of hormone-dependent mammary gland tumours is observed 30 min after estradiol injection. However, this period is characterized by the appearance of protein kinase whose activity is not stimulated by Ca2+ or phosphatidylserine and which is eluted from DEAE-cellulose with 0.2 M NaCl. This protein kinase presumably corresponds to the M-fragment, i.e., the catalytic part of protein kinase C formed as a result of protein kinase C proteolysis on the membranes. It seems likely that estradiol, similar to growth factor peptides, realizes its stimulating effect on cell division primarily at the expense of coupling of its membrane receptors with the protein kinase C activation system.     

蛋白激酶在微血管通透性中的作用

微血管内皮细胞层是一层半选择通透性屏障,可以调节血液中的液体、溶质和血浆蛋白进入组织间隙。在炎症刺激作用下,可通过旁细胞途径和跨细胞途径引起内皮通透性上升。旁细胞通路主要由内皮细胞间的紧密连接、黏附连接和细胞与外基质的黏着斑组成。炎症介质,如脂多糖和肿瘤坏死因子α可激活多种蛋白激酶。活化的蛋白激酶主要包括Rho相关的卷曲蛋白激酶、肌球蛋白轻链激酶、蛋白激酶C、酪氨酸激酶和丝裂原活化蛋白激酶等,参与引发内皮屏障生化和结构改变,旁细胞通路开放,导致通透性上升。该文对上述蛋白激酶在微血管通透性中作用机制的研究进展进行综述。     

Serine/threonine protein kinases and apoptosis

Over the past decade, our understanding of apoptosis, or programmed cell death, has increased greatly, with the identification of some of the major components of the apoptotic programme and the processes regulating their activation. Although apoptosis is an intrinsic process present in all cells, it can be regulated by extrinsic factors, including hormones, growth factors, cell surface receptors, and cellular stress. The actions of both pro- and antiapoptotic factors are often affected by modulation of the phosphorylation status of key elements of the apoptotic process. This minireview will focus on the role of protein kinases in apoptosis. Apoptosis is a multistep process and protein kinases have been implicated both in the upstream induction phase of apoptosis and in the downstream execution stage, as the direct targets for caspases. Due to the space constraints of this review it is not possible to discuss all of the kinases involved in the apoptotic process and we have focused here on the role of the serine/threonine protein kinases. The kinases of this family that have been suggested to play a role in apoptosis are the mitogen-activated protein kinase (MAPK) family, specifically p42/44 ERK, p38 MAPK and c-Jun N-terminal kinase (JNK), cyclic AMP-dependent protein kinase (PKA), protein kinase B (PKB), or Akt and protein kinase C (PKC). We have also considered briefly the potential for the regulation of these kinases by tyrosine protein kinases, such as c-abl.     

An Epstein-Barr virus transformation-associated membrane protein interacts with src family tyrosine kinases.

In latently infected growth-transformed human lymphocytes, Epstein-Barr virus (EBV) encodes two integral plasma membrane proteins: LMP1, which constitutively induces B-lymphocyte activation and intercellular adhesion, and LMP2A, which associates with LMP1 and is a tyrosine kinase substrate. We now demonstrate that LMP2A associates with src family protein tyrosine kinases, particularly lyn kinase, in nonionic detergent extracts of transfected B lymphoma cells or in extracts of EBV-transformed B lymphocytes. The LMP2A and tyrosine kinase association is stable in nonionic detergents and includes a 70-kDa cell protein which is also an in vitro or in vivo kinase substrate. This LMP2A association with B-lymphocyte src family tyrosine kinases is likely to be an important pathway in EBV's effects on cell growth.     

Aurora B expression in post-puberal testicular germ cell tumours

Aurora/Ipl1-related kinases are a conserved family of proteins that are essential for the regulation of chromosome segregation and cytokinesis during mitosis. Aberrant expression and activity of these kinases occur in a wide range of human tumours and have been implicated in mechanisms leading to mitotic spindle aberrations, aneuploidy, and genomic instability. Previous studies of our group have shown that Aurora B expression is restricted to specific germinal cells. In this study, we have evaluated by immunohistochemical analysis Aurora B expression in post-puberal testicular germ cell tumours (22 seminomas, 2 teratomas, 15 embryonal carcinomas, 5 mixed germinal tumours with a prominent yolk sac tumour component and 1 choriocarcinoma). The Aurora B protein expression was detected in all intratubular germ cell tumours, seminomas and embryonal carcinomas analysed but not in teratomas and yolk sac carcinomas. The immunohistochemical data were further confirmed by Western blot analysis. In addition, the kinase Aurora B was vigorously expressed in GC-1 cells line derived from murine spermatogonia. The block of Aurora B function induced by a pharmacological inhibitor significantly reduced the growth of GC-1 cells suggesting that Aurora B is a potential therapeutic target. J. Cell. Physiol. 221: 435–439, 2009. © 2009 Wiley-Liss, Inc.     

Plasma membrane protein kinase activity in normal and Rous sarcoma virus-transformed chick embryo fibroblasts.

A preliminary study has been carried out to investigate the effect of Rous sarcoma virus transformation on plasma membrane protein kinase activity in chick embryo fibroblasts. Enzyme activity was measured using an in vitro phosphorylation method employing [gamma-32P]ATP with isolated plasma membranes serving as the source of both protein kinase and protein substrate. In general, the enzymatic properties observed were similar to those of other known protein kinases. However, for maximal activity a marked dependence on high Mg2+ concentrations was noted. Evidence was obtained which showed that cyclic nucleotide-dependent protein kinases were present in membranes from normal cells, but none could be measured in preparations from transformed cells. In addition, transformation appeared to result in a slight increase in basal plasma membrane protein kinase activity.     

Immunological and molecular characterization of the cAMP-dependent protein kinases in AtT20 cells

The properties of the cAMP-dependent protein kinases in AtT20 mouse pituitary tumor cells were characterized by a combination of immunological and biochemical techniques. Ninety per cent of the total cAMP-dependent protein kinase was in the 40,000 X g supernatant fraction. Protein kinases I and II were immunoprecipitated with specific antisera directed against their regulatory subunits. The immunoprecipitated kinases bound [3H]cAMP and were catalytically active when incubated with [gamma-32P]ATP-Mg and protamine or histone H2B. Immunoprecipitated protein kinases I and II bound [3H]cAMP with apparent Kb values of 1.5 and 15 nM, respectively. Regulatory subunit concentrations in AtT20 cells were measured by immunoprecipitation of [3H]cAMP-R complexes. R-I and R-II levels were 2.7 and 3.0 pmol of [3H]cAMP binding activity per mg of cytosolic protein, respectively, however, the ratio of protein kinase II to protein kinase I was 2.5 indicating the presence of a significant amount of free R-I. This was confirmed by DEAE-cellulose chromatography and the isolation of immunoreactive R-I devoid of protein kinase activity. A significant amount of R-I also coeluted with protein kinase II when AtT20 cell extracts were subjected to DEAE-cellulose chromatography. In quantitative immunoprecipitation experiments, 0.1 microliter of anti-brain R-II serum complexed up to 0.5 pmol of the [3H]cAMP-binding activity of protein kinase II prepared from bovine and rat brain, and AtT20 cells while 2 microliter of anti-brain R-II serum was required to precipitate an equal amount of protein kinase II from bovine skeletal muscle showing that the protein kinase II in AtT20 cells contained the neural-specific R-II subunit.     

The dissociation of chicken erythrocyte deoxyribonucleoprotein and some properties of its partial nucleoproteins

Histones were completely dissociated from their native complex with DNA in 2.0m-sodium chloride. Histone fractions IIb, V and I were dissociated in 1.2m-sodium chloride, fractions V and I in 0.7m-sodium chloride and fraction I in 0.45m-sodium chloride. Repeated extraction of partial dRNP (deoxyribonucleoprotein) preparations with sodium chloride of the same concentration as that from which they were prepared resulted in release of histones that previously had remained associated with the DNA of the complex. Gradual removal of histones from dRNP was paralleled by an improvement in solubility, a decrease in wavelength of the u.v.-absorption minimum, and a fall in sedimentation coefficient of the remaining partial dRNP. X-ray diffraction patterns of partial dRNP preparations showed that removal of histone fractions I and V from dRNP did not destroy the super-coil structure of the dRNP, but further removal of histones did. Infrared spectra of partial dRNP preparations showed that in native dRNP histone fraction I was present in the form of extended, isolated polypeptide chains, and that the other histone fractions probably contain a helical component that lies roughly parallel to the polynucleotide chains in the double helix and an extended polypeptide component that is more nearly parallel to the DNA helix axis. An analysis of the sedimentation of partial dRNP preparations on sucrose gradients showed that native dRNP consists of DNA molecules each complexed with histone fractions of all types.     

High level of tyrosine protein kinase in a murine lymphoma cell line induced by Moloney leukemia virus.

Several sarcoma-inducing viruses encode protein kinases that phosphorylate tyrosine residues. Such enzymatic activities can be detected within the detergent-insoluble matrix of transformed fibroblasts. We have analysed the protein kinase activities in two murine lymphoma cell lines ( MBL2 and LSTRA) induced by Moloney murine leukemia virus (Mo-MuLV). After incubation of the detergent-insoluble matrix of these cells with [gamma-32P]ATP, several alkali-resistant phosphoproteins, including a very heavily labelled 55 000 mol. wt. protein ( p55 ), have been detected in LSTRA, reflecting the activity of a protein kinase specific to this cell line. This protein kinase activity shares some of the distinctive properties of the protein kinases of transforming viruses, i.e., specificity for tyrosine residues, association with membranous and/or cytoskeletal structures, and inhibition by a synthetic peptide derived from the phosphorylation site of pp60src. In view of the absence of a transforming gene in MoMuLV , it is likely that the high level of protein kinase detected in the LSTRA cell line arises from the expression of a cellular gene.