scholarly journals Histone phosphatase and cyclic nucleotide-stimulated protein kinase from human lymphocytes

1972 ◽  
Vol 129 (5) ◽  
pp. 995-1002 ◽  
Author(s):  
A. W. Murray ◽  
M. Froscio ◽  
B. E. Kemp
Keyword(s):  
Molecular Weight ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Enzymic Activity ◽  
Lower Molecular Weight ◽  
Protein Kinase Activity ◽  
Cyclic Monophosphate ◽  
Lower Molecular Weight Species

1. Extracts of human peripheral blood lymphocytes contained a histone phosphatase that catalysed the release of Pi from phosphorylated whole thymus histone. 2. Stimulation of the phosphatase was obtained by concentrations of KCl and NaCl of up to 75mm, and by MgCl2; CaCl2 inhibited the enzymic activity. 3. In the absence of MgCl2, phosphoenol-pyruvate inhibited histone phosphatase activity; this inhibition could be partially reversed by adding MgCl2 to assays. 4. Lymphocyte extracts contained a protein kinase activity which was maximally stimulated by 1μm-cyclic AMP (adenosine 3′:5′-cyclic monophosphate) or by 0.1mm-cyclic GMP (guanosine 3′:5′-cyclic monophosphate). 5. Incubation of the enzyme with histone in the absence of ATP or MgCl2 resulted in the dissociation of the enzyme into a lower-molecular-weight species that was not stimulated by cyclic AMP. This effect could be prevented if ATP and MgCl2 were present in reaction mixtures before histone and enzyme were allowed to interact. 6. Cyclic AMP also dissociated the kinase into a lower-molecular-weight species. 7. In the presence of 1μm-AMP, half-maximal activities were obtained with 0.92mm-MgCl2, 6.0μm-ATP and 0.23mg of whole thymus histone/ml.

scholarly journals Phosphoproteins associated with cyclic nucleotide stimulation of ciliary motility in Paramecium

1990 ◽  
Vol 95 (2) ◽  
pp. 219-230
Author(s):  
N.M. Bonini ◽  
D.L. Nelson
Keyword(s):  
Molecular Weight ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Cyclic Gmp ◽  
Cyclic Nucleotide ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Permeabilized Cells ◽  
Ciliary Motility ◽  
Dependent Protein

Permeabilized, MgATP-reactivated cells of Paramecium (models) respond to cyclic AMP and cyclic GMP by increasing forward swimming speed. In association with the motile response, cyclic AMP and 8-bromo-cyclic GMP (8-Br-cyclic GMP) stimulated protein phosphorylation. Cyclic AMP addition to permeabilized cells reproducibly stimulated the phosphorylation of 10 proteins, ranging in molecular weight from 15 to 110K (K = 10(3) Mr). 8-Br-cyclic GMP, which selectively activates the cyclic GMP-dependent protein kinase of Paramecium, stimulated the phosphorylation of a subset of the proteins phosphorylated by cyclic AMP. Ca2+ addition caused backward swimming and stimulated the phosphorylation of four substrates, including a 25K target that may also be phosphorylated in response to cyclic nucleotide addition. Ba2+ and Sr2+ also induced backward swimming, but did not cause detectable phosphorylation. To identify ciliary targets of cyclic nucleotide-dependent protein kinase activity, permeabilized cells were deciliated following reactivation of motility with Mg-[gamma-32P]ATP in the presence or absence of cyclic nucleotide. Soluble proteins of the deciliation supernatant were enriched in 15 cyclic AMP-stimulated phosphoproteins, ranging in molecular weight from 15 to 95K. Most of the ciliary substrates were axonemal and could be released by high salt solution. A 29K protein that copurified in sucrose gradients with the 22S dynein, and a high molecular weight protein (greater than 300K) in the 19 S region were phosphorylated when cyclic AMP was added to permeabilized, motile cells. These data suggest that regulation of ciliary motility by cyclic AMP may include phosphorylation of dynein-associated proteins.

scholarly journals A study of 3′:5′-cyclic mononucleotide-dependent protein kinase from canine prostate glands

1974 ◽  
Vol 144 (2) ◽  
pp. 377-383 ◽  
Author(s):  
B K Tsang ◽  
R L Singhal
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Metal Ion ◽  
Maximum Velocity ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Cyclic Monophosphate ◽  
Diethylaminoethyl Cellulose ◽  
Dependent Protein ◽  
Stimulation Of

1. An adenosine 3′:5′-cyclic monophosphate (cyclic AMP)-dependent protein kinase, located predominantly in the cytosol, was studied in canine prostate. 2. The enzyme exhibited cyclic AMP-binding activity, and could be isolated by chromatography on diethylaminoethyl cellulose. 3. The enzyme was maximally stimulated (fourfold) by 1μm-cyclic AMP, and half-maximal activation of the enzyme was observed in presence of 50nm-cyclic AMP. 4. Equilibrium studies at pH5.0 indicated the presence of one major class of binding site for cyclic AMP, with an association constant of approx. 108m-1. 5. Stimulation of the enzyme was also observed with the 3′:5′-cyclic monophosphate derivatives of cytidine, inosine, guanosine and uridine as well as with dibutyryl cyclic AMP, but higher concentrations of these cyclic nucleotides were required to provide the same degree of activation as that seen with cyclic AMP. 6. Comparing α-casein, protamine and different histone subfractions as substrates, highest cyclic AMP stimulation was demonstrated with histones. 7. Although maximum velocity of the enzyme was enhanced approximately fivefold in presence of cyclic AMP, kinetic studies indicated that the apparent Km for histone (0.5mg/ml) remained the same whether determined in the presence or absence of the cyclic nucleotide. 8. In addition, cyclic AMP did not significantly change the apparent Km for ATP (1.2×10-5m). 9. The purified enzyme showed an absolute requirement for bivalent metal ion. Substitution of Mn2+for Mg2+decreased basal protein kinase activity as well as the stimulation noted with cyclic AMP. Similarly, the basal activity was lowered when Mg2+was replaced by Ca2+and cyclic AMP produced only little stimulation of the prostatic enzyme.

scholarly journals Multiple protein kinases from human lymphocytes. Identification enzymes phosphorylating exogenous histon and casein

1975 ◽  
Vol 145 (2) ◽  
pp. 241-249 ◽  
Author(s):  
B E Kemp ◽  
M Froscio ◽  
A Rogers ◽  
A W Murray
Keyword(s):  
Molecular Weight ◽  
Cyclic Amp ◽  
Kinase Inhibitor ◽  
Muscle Protein ◽  
Human Peripheral Blood ◽  
Human Lymphocytes ◽  
Casein Kinase ◽  
Rabbit Muscle ◽  
Sephadex Column ◽  
Histone Kinase

1. Cell-free lysates of human peripheral blood lymphocytes contained two casein kinase activities and two histone kinase activities, which could be separated by chromatography on DEAE-Sephadex. 2. Neither of the casein kinase activities were stimulated by cyclic AMP. The major activity was eluted from DEAE-Sephadex between 0.4 and 0.45M-KCl, had a molecular weight of approx. 130,000 (sucrose density gradients) and was stimulated by KCl (maximum 150mM). It also formed higher-molecular-weight aggregates when centrifuged in sucrose gradients containing 150mM-KCl. The minor activity was not retained by DEAE-Sephadex, had a molecular weight of approx. 50,000 and was not stimulated by KCl. 3. The major histone kinase activity was stimulated by cyclic AMP and was eluted from the DEAE-Sephadex column between 0.05 and 0.2M-KCl. The other activity was not stimulated by cyclic AMP and was insensitive to the rabbit muscle protein kinase inhibitor. 4. Evidence was obtained suggesting that the lymphocyte casein kinases were located primarily in the nuclei.

scholarly journals Protein phosphorylation in human peripheral blood lymphocytes. Subcellular distribution and partial characterization of adenosine 3′:5′-cyclic monophosphate-dependent protein kinase

1979 ◽  
Vol 182 (2) ◽  
pp. 525-536 ◽  
Author(s):  
D D Chaplin ◽  
H J Wedner ◽  
C W Parker
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Human Peripheral Blood ◽  
Deae Cellulose ◽  
Peripheral Blood Lymphocytes ◽  
Protein Kinase Activity ◽  
Dependent Protein Kinase ◽  
Human Peripheral Blood Lymphocytes ◽  
Dependent Protein

Cytoplasmic and membrane fractions prepared from human peripheral-blood lymphocytes both contained cyclic AMP-dependent protein kinase activity and endogenous protein kinase substrates. Protein kinase activity in the particulate fractions was not eluted with 0.25 M-NaCl, suggesting that it was not derived from non-specifically absorbed soluble cytoplasmic protein kinase. Nor was the particulate protein kinase activity eluted by treatment with cyclic AMP, suggesting that the catalytic subunit is membrane-bound and arguing against cyclic AMP-induced translocation of particulate activity. Cyclic AMP-dependent protein-phosphorylating activity in the cytoplasmic fraction was highly sensitive to inhibition by Mn2+, and was co-eluted from DEAE-cellulose primarily with type-I rabbit skeletal-muscle kinase. Cyclic AMP-dependent phosphorylating activity in the plasma-membrane fractions was stimulated at low [Mn2+] and inhibited only at high [Mn2+]. When solubilized with Nonidet P-40, plasma-membrane protein kinase was co-eluted from DEAE-cellulose with type-II rabbit muscle kinase. These differences, together with the strong association of the particulate kinases with the particulate fraction, suggest the possibility of compartmentalized protein phosphorylation in intact lymphocytes.

scholarly journals Preferential phosphorylation of the 150,000 molecular weight component of neurofilaments by a cyclic AMP-dependent, microtubule-associated protein kinase.

1981 ◽  
Vol 90 (3) ◽  
pp. 755-760 ◽  
Author(s):  
J F Leterrier ◽  
R K Liem ◽  
M L Shelanski
Keyword(s):  
Molecular Weight ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Nerve Root ◽  
Kinase Activity ◽  
Bovine Brain ◽  
Protein Kinase Activity ◽  
Histone Phosphorylation ◽  
Microtubule Associated Proteins ◽  
Associated Proteins

Highly purified preparations of bovine brain and rabbit nerve root neurofilaments were found to be lacking in protein kinase activity when either histone FIIA or the neurofilaments themselves were used as acceptors. There was no augmentation of activity in the presence of cyclic AMP. Addition of microtubule proteins prepared by cycles of assembly and disassembly resulted in phosphorylation of histone, phosphorylation of tubulin and the microtubule-associated proteins, and phosphorylation of neurofilament subunits. The phosphorylation of neurofilaments was predominantly in the 150,000-dalton species and was completely cyclic AMP dependent.

scholarly journals Evidence for Ecto-Protein Kinase Activity That Phosphorylates Kemptide in a Cyclic AMP-dependent Mode

1989 ◽  
Vol 264 (24) ◽  
pp. 14549-14555 ◽  
Author(s):  
D Kübler ◽  
W Pyerin ◽  
O Bill ◽  
A Hotz ◽  
J Sonka ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Protein Kinase Activity

Regulation of protein kinase by vasopressin in renal medulla in situ

1977 ◽  
Vol 232 (1) ◽  
pp. F50-F57
Author(s):  
T. P. Dousa ◽  
L. D. Barnes
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Renal Medulla ◽  
Protein Kinase Activity ◽  
Kinase Activation ◽  
Heat Stable ◽  
Protein Kinase Activation ◽  
Heat Stable Protein

Results of this study demonstrate that vasopressin activates protein kinase in intact renal medullary cells as detected by measurement of the (-cyclic AMP/+cyclic AMP) protein kinase activity ratios in freshly prepared tissue extracts (40,000 X g supernates) from bovine renal medullary slices. The activation of protein kinase was specific for vasopressin since parathyroid hormone, histamine, angiotensin II, or the inactive analog of vasopressin did not activate protein kinase. There was a direct correlation between the extent of protein kinase activation and the elevation in tissue levels of cyclic AMP elicited by increasing doses of vasopressin or with an increase in incubation time. The elevation of tissue cyclic AMP level and maximum activation of protein kinase reached maximum level at a vasopressin concentration of about 2 X 10(-9) M. Incubation of slices with vasopressin caused a dose-dependent decrease in the cyclic AMP-dependent protein kinase activity in the 40,000 X g supernate of homogenate from the renal medullary slices. This effect of vasopressin was specific for protein kinase since activity of lactate dehydrogenase or a specific [3H]colchicine-binding activity was not affected, and the decrease in the protein kinase was not due to the accumulation of a heat-stable protein kinase inhibitor. There was an increase in protein kinase was not due to the accumulation of a heat-stable protein kinase inhibitor. There was an increase in protein kinase activity extracted from 40,000 X g pellets of homogenate prepared from slices exposed to vasopressin. Results thus provide evidence that cyclic AMP-mediated protein kinase activation in the intact cells is an integral part of cellular response of the mammalian renal medulla to vasopressin.

Granulocyte-macrophage colony-stimulating factor preferentially activates the 94-kD STAT5A and an 80-kD STAT5A isoform in human peripheral blood monocytes

Blood ◽  
1996 ◽  
Vol 88 (4) ◽  
pp. 1206-1214 ◽  
Author(s):  
RL Rosen ◽  
KD Winestock ◽  
G Chen ◽  
X Liu ◽  
L Hennighausen ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Human Peripheral Blood ◽  
Janus Kinase ◽  
Lower Molecular Weight ◽  
Colony Stimulating Factor ◽  
Enhancer Element ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces immediate effects in monocytes by activation of the Janus kinase (JAK2) and STAT transcription factor (STAT5) pathway. Recent studies have identified homologues of STAT5, STAT5A, and STAT5B, as well as lower molecular weight variants of STAT5. To define the activation of the STAT5 homologues and lower molecular weight variant in human monocytes and monocytes differentiated into macrophages by culture in macrophage- CSF (M-CSF), we measured the GM-CSF induced tyrosine phosphorylation of STAT5A, STAT5B, and any lower molecular weight STAT5 isoforms. Freshly isolated monocytes expressed 94-kD STAT5A, 92-kD STAT5B, and an 80-kD STAT5A molecule. Whereas 94-kD STAT5A was clearly tyrosine phosphorylated and bound to the enhancer element, the gamma response region (GRR), of the Fc gamma RI gene, substantially less tyrosine phosphorylated STAT5B bound to the immobilized GRR element. Macrophages lost their ability to express the 80-kD STAT5A protein, but retained their ability to activate STAT5A. STAT5A-STAT5A homodimers and STAT5A- STAT5B heterodimers formed in response to GM-CSF. Therefore, activation of STAT5A predominates compared to STAT5B when assayed by direct immunoprecipitation and by evaluation of bound STATs to immobilized GRR. Selective activation of STAT5 homologues in addition to generation of lower molecular isoforms may provide specificity and control to genes expressed in response to cytokines such as GM-CSF.

scholarly journals Expression of the mammalian c-fes protein in hematopoietic cells and identification of a distinct fes-related protein.

1985 ◽  
Vol 5 (10) ◽  
pp. 2543-2551 ◽  
Author(s):  
I MacDonald ◽  
J Levy ◽  
T Pawson
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Human Peripheral Blood ◽  
Hematopoietic Cells ◽  
Specific Protein ◽  
Protein Kinase Activity ◽  
Sarcoma Virus ◽  
Human And Mouse ◽  
Specific Protein Kinase

The avian c-fps and mammalian c-fes proto-oncogenes are cognate cellular sequences. Antiserum raised against the P140gag-fps transforming protein of Fujinami avian sarcoma virus specifically recognized a 92,000-Mr protein in human and mouse hematopoietic cells which was closely related in structure to Snyder-Theilen feline sarcoma virus P87gag-fes. This polypeptide was apparently the product of the human c-fes gene and was therefore designated p92c-fes. Human p92c-fes was associated with a tyrosine-specific protein kinase activity in vitro and was capable of both autophosphorylation and phosphorylation of enolase as an exogenous protein substrate. The synthesis of human and mouse p92c-fes was largely, though not entirely, confined to myeloid cells. p92c-fes was expressed to relatively high levels in a multipotential murine myeloid cell line, in more mature human and mouse granulocyte-macrophage progenitors, and in differentiated macrophage like cells as well as in the mononuclear fraction of normal and leukemic human peripheral blood. p92c-fes was not found in erythroid cells, with the exception of a human erythroleukemia line which retains the capacity to differentiate into macrophage like cells. These results suggest a normal role for the p92c-fes tyrosine kinase in hematopoiesis, particularly in granulocyte-macrophage differentiation. In addition, a distinct 94,000-Mr polypeptide, antigenically related to p92c-fes, was identified in a number of hematopoietic and nonhematopoietic human and mouse cells and was also found to be associated with a tyrosine-specific protein kinase activity.

scholarly journals The role of protein kinase activation in the control of steroidogenesis by adrenocorticotrophic hormone in the adrenal cortex

1973 ◽  
Vol 136 (4) ◽  
pp. 993-998 ◽  
Author(s):  
Malcolm C. Richardson ◽  
Dennis Schulster
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Adrenal Cortex ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Adrenocorticotrophic Hormone ◽  
Dependent Protein Kinase ◽  
Kinase Activation ◽  
Protein Kinase Activation ◽  
Dependent Protein

A method has been developed for investigation of the effect of adrenocorticotrophic hormone (ACTH) on the state of activation of a cyclic AMP-dependent protein kinase within cells of the adrenal cortex. Enzyme activity was measured in terms of the quantity of32P transferred from [γ-32P]ATP to histone under conditions in which bound cyclic AMP did not dissociate from the regulatory subunit of the protein kinase ACTH (1×10-2i.u./ml) caused a rapid and complete activation of the cyclic AMP-dependent protein kinase activity within 2min of hormone addition to the isolated cells. In response to a range of ACTH concentrations a sigmoid log dose–response curve for protein kinase activation was obtained, with half-maximal stimulation attained at about 1×10-3i.u./ml. However, some low doses of ACTH that elicited a marked (but submaximal) steroidogenic response failed to cause a clear stimulation of protein kinase activity in isolated adrenal cells. Theophylline (2mm) potentiated the effect of ACTH on protein kinase activity. The results implicate an important role for protein kinase in ACTH action on the adrenocortical cell.

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