Protein phosphorylation and the proliferation of chick embryo fibroblasts: analysis by in vitro phosphorylation using isolated plasma membranes and whole cell homogenates

1980 ◽  
Vol 58 (1) ◽  
pp. 30-39 ◽  
Author(s):  
Philip E. Branton
Keyword(s):  
Cell Proliferation ◽  
Plasma Membrane ◽  
Chick Embryo ◽  
Protein Phosphorylation ◽  
Plasma Membranes ◽  
Kinase Activity ◽  
Cell Protein ◽  
Protein Kinase Activity ◽  
Whole Cell

The relationship between plasma membrane and whole cell protein phosphorylation and chick embryo fibroblast proliferation was studied using an in vitro labeling technique employing [γ-32P]ATP and isolated plasma membranes or whole cell homogenates. Cultures containing proliferating cells in log phase or containing cells stimulated to proliferate by the addition of serum were compared with cultures in which proliferation was inhibited due to cell density. Cell proliferation was found to be associated with increased plasma membrane basal protein kinase activity, but decreased membrane and whole cell cyclic AMP-dependent kinase activity. The phosphorylation of a number of membrane and whole cell polypeptides differed between proliferating and density-inhibited cells, suggesting that these phosphoproteins may be involved in the regulatory events associated with cell proliferation. Of interest, however, was the fact that the phosphoprotein differences found with serum-stimulated and sparse, rapidly dividing cells were generally not the same. These observations suggest that the protein phosphorylation events associated with rapid proliferation of sparse, log-phase cells may differ from those involved in serum-activated proliferation of quiescent cells.

scholarly journals Evidence for modification of protein phosphorylation by cytokinins

1972 ◽  
Vol 130 (4) ◽  
pp. 901-911 ◽  
Author(s):  
R. K. Ralph ◽  
P. J. A. McCombs ◽  
G. Tener ◽  
S. J. Wojcik
Keyword(s):  
Protein Phosphorylation ◽  
Chinese Cabbage ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Secondary Phloem ◽  
Phloem Tissue ◽  
Cyclic Monophosphate ◽  
Cellular Processes ◽  
Ylacetic Acid

Kinetin stimulated phosphorylation of protein in floated Chinese-cabbage leaf discs, but inhibited protein phosphorylation in nuclei+chloroplast extracts from Chinese-cabbage or tobacco leaves. Kinetin also inhibited protein phosphorylation in isolated tobacco nuclei or nuclei from carrot secondary-phloem tissue. Purified Chinese-cabbage leaf ribosomes exhibited protein kinase activity which was inhibited by kinetin and zeatin. The ribosome-associated kinase responded to kinetin and zeatin differently from that associated with nuclei+chloroplast preparations. Protein phosphorylation in vitro was not affected by adenosine 3′:5′-cyclic monophosphate, indol-3-ylacetic acid or gibberellic acid. It was only inhibited by N9-unsubstituted purines, among which the known cytokinins were the most effective inhibitors. The results are discussed in relation to possible similarities between the effects of cytokinins in plant tissues and the effects of adenosine 3′:5′-cyclic monophosphate in animal tissues. Both compounds appear to modify the activity of protein kinases and both affect many different cellular processes.

PROTEIN KINASE ACTIVITY ASSOCIATED WITH THE FAT CELL PLASMA MEMBRANE

1981 ◽  
Vol 9 (2) ◽  
pp. 232P-232P
Author(s):  
G. J. Belsham ◽  
R. W. Brownsey ◽  
R. M. Denton
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Fat Cell ◽  
Cell Plasma Membrane ◽  
Cell Plasma

scholarly journals Novel Chemical Class of pUL97 Protein Kinase-Specific Inhibitors with Strong Anticytomegaloviral Activity

2004 ◽  
Vol 48 (11) ◽  
pp. 4154-4162 ◽  
Author(s):  
Thomas Herget ◽  
Martina Freitag ◽  
Monika Morbitzer ◽  
Regina Kupfer ◽  
Thomas Stamminger ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
Human Fibroblasts ◽  
Growth Factor Receptor ◽  
Protein Kinase Activity ◽  
Hcmv Replication

ABSTRACT Human cytomegalovirus (HCMV) is a major human pathogen frequently associated with life-threatening disease in immunosuppressed patients and newborns. The HCMV UL97-encoded protein kinase (pUL97) represents an important determinant of viral replication. Recent studies demonstrated that pUL97-specific kinase inhibitors are powerful tools for the control of HCMV replication. We present evidence that three related quinazoline compounds are potent inhibitors of the pUL97 kinase activity and block in vitro substrate phosphorylation, with 50% inhibitory concentrations (IC50s) between 30 and 170 nM. Replication of HCMV in primary human fibroblasts was suppressed with a high efficiency. The IC50s of these three quinazoline compounds (2.4 ± 0.4, 3.4 ± 0.6, and 3.9 ± 1.1 μM, respectively) were in the range of the IC50 of ganciclovir (1.2 ± 0.2 μM), as determined by the HCMV green fluorescent protein-based antiviral assay. Importantly, the quinazolines were demonstrated to have strong inhibitory effects against clinical HCMV isolates, including ganciclovir- and cidofovir-resistant virus variants. Moreover, in contrast to ganciclovir, the formation of resistance to the quinazolines was not observed. The mechanisms of action of these compounds were confirmed by kinetic analyses with infected cells. Quinazolines specifically inhibited viral early-late protein synthesis but had no effects at other stages of the replication cycle, such as viral entry, consistent with a blockage of the pUL97 function. In contrast to epithelial growth factor receptor inhibitors, quinazolines affected HCMV replication even when they were added hours after virus adsorption. Thus, our findings indicate that quinazolines are highly efficient inhibitors of HCMV replication in vitro by targeting pUL97 protein kinase activity.

Structural and functional modification of pp60c-src associated with polyoma middle tumor antigen from infected or transformed cells

1985 ◽  
Vol 5 (10) ◽  
pp. 2647-2652
Author(s):  
C A Cartwright ◽  
M A Hutchinson ◽  
W Eckhart
Keyword(s):  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Tumor Antigen ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Amino Terminal ◽  
Exogenous Substrate ◽  
And Function ◽  
Amino Terminal Domain

The polyoma middle tumor antigen (MTAg) associates with the src proto-oncogene product pp60c-src in infected or transformed rodent cells. The tyrosine protein kinase activity of pp60c-src, as measured by in vitro phosphorylation of pp60c-src itself or the exogenous substrate enolase, was increased 10- to 20-fold in cells transformed or infected with transformation-competent polyoma virus compared with controls. pp60c-src associated with MTAg and precipitated with polyoma antitumor serum had a novel site(s) of in vitro tyrosine phosphorylation within its amino-terminal domain. These observations suggest that association of MTAg with pp60c-src alters the accessibility of pp60c-src tyrosine residues for phosphorylation in vitro and increases pp60c-src protein kinase activity. Several transformation-defective mutants of MTAg did not cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro or enhance its protein kinase activity, suggesting that these properties correlate with the transforming ability of MTAg. However, one transformation-defective MTAg mutant, dl1015, did cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro and did enhance its protein kinase activity. This suggests that properties of MTAg, in addition to modifying the structure and function of pp60c-src, may be important for transformation.

Mitotic arrest caused by the amino terminus of Xenopus cyclin B2

1993 ◽  
Vol 13 (3) ◽  
pp. 1480-1488
Author(s):  
H M van der Velden ◽  
M J Lohka
Keyword(s):  
Sea Urchin ◽  
Kinase Activity ◽  
Histone H1 ◽  
Amino Terminus ◽  
Protein Kinase Activity ◽  
Truncated Protein ◽  
Amino Terminal ◽  
Egg Extracts ◽  
The Stability

Progression through mitosis requires the inactivation of the protein kinase activity of the p34cdc2-cyclin complex by a mechanism involving the degradation of cyclin. We have examined the stability in Xenopus egg extracts of radiolabeled Xenopus or sea urchin B-type cyclins synthesized in reticulocyte lysates. Xenopus cyclin B2 and sea urchin cyclin B were stable in metaphase extracts from unfertilized eggs but were specifically degraded following addition of Ca2+ to the extracts. The degradation of either cyclin was inhibited by the addition of an excess of unlabeled Xenopus cyclin B2 but not by the addition of a number of control proteins. A truncated protein containing only the amino terminus of Xenopus cyclin B2, including sequences known to be essential for cyclin degradation in other species, also inhibited cyclin degradation, even though the truncated protein was stable in extracts following Ca2+ addition. The addition of the truncated protein did not stimulate histone H1 kinase activity in extracts but prevented the loss of H1 kinase activity that normally follows Ca2+ addition to metaphase extracts. When the amino-terminal fragment was added to extracts capable of several cell cycles in vitro, progression through the first mitosis was inhibited and elevated histone H1 kinase activity was maintained. These results indicate that although the amino terminus of cyclin does not contain all of the information necessary for cyclin destruction, it is capable of interacting with components of the cyclin destruction pathway and thereby preventing the degradation of full-length cyclins.

Correlation between phosphorylation of a 34,000-molecular-weight protein, pp60src-associated kinase activity, and tumorigenicity in transformed and revertant vole cells

1984 ◽  
Vol 4 (1) ◽  
pp. 212-215
Author(s):  
J F Nawrocki ◽  
A F Lau ◽  
A J Faras
Keyword(s):  
Molecular Weight ◽  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Cell Types ◽  
Cell Protein ◽  
Protein Kinase Activity ◽  
Rous Sarcoma ◽  
Tumorigenic Potential ◽  
Sarcoma Virus ◽  
Weight Protein

The phosphorylation of a 34,000-molecular-weight (34K) cell protein, purported to be a substrate of the avian retrovirus pp60src-associated protein kinase activity, was compared in three types of Rous sarcoma virus-infected vole cells: fully transformed cells, partial revertants which are morphologically normal in appearance but retain their tumorigenic potential, and full revertants which are similar to normal vole cells in all parameters including a lack of tumorigenicity. Although similar amounts of 34K protein are present in all three cell types, phosphorylation of the 34K protein was significantly reduced in the full revertant cell type. The reduced phosphorylation occurred at the tyrosine residue.

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.

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