Investigations on myelinogenesis in vitro: II. The occurrence and regulation of protein kinases by thyroid hormone in primary cultures of cells dissociated from embryonic mouse brain

1987 ◽  
Vol 7 (2) ◽  
pp. 159-165 ◽  
Author(s):  
G. Shanker ◽  
R. A. Pieringer
Keyword(s):  
Thyroid Hormone ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Mouse Brain ◽  
Primary Cultures ◽  
Calf Serum ◽  
Phosphatidyl Serine ◽  
Protein Kinase Activity ◽  
Embryonic Mouse

The occurrence and regulation by thyroid hormone of four protein kinases (cyclic AMP independent and dependent, calcium/calmodulin stimulated, and calcium/phosphatidyl serine stimulated protein kinases) was studied in primary cultures of cells dissociated from embryonic mouse brain. Serum from a thyroidectomized calf, which contained low levels of L-3,5,3′-triiodothyronine, T3 (<25 ng/100 ml), and thyroxine, T4 (<1 μg/100 ml) was used in the culture medium in place of normal calf-serum (T3, 130 ng/100 ml; T4 5.9 μg/100 ml) to render the cultures responsive to exogenously added T3. Cultures grown in hypothyroid calf-serum containing medium had less cAMP dependent and independent protein kinase activity than control cultures grown in normal calf-serum containing medium. However, this activity was restorable to a considerable degree if the cultures grown in hypothyroid calf serum containing medium were supplemented with L-3,5,3′-triiodothyronine (T3). The presence of calcium/calmodulin stimulated protein kinase was also distinctly observed. In comparison, the activity of calcium/phosphatidyl serine stimulated protein kinase was less than the other protein kinases.

Investigations on myelinogenesis in vitro: I. The occurrence of endogenous protein kinase and its role in the phosphorylation of myelin basic proteins in “Myelin-like membranes” isolated from cerebral cell cultures

1987 ◽  
Vol 7 (2) ◽  
pp. 151-157
Author(s):  
G. Shanker ◽  
R. A. Pieringer
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Membrane Fraction ◽  
Primary Cultures ◽  
Embryonic Mouse ◽  
Basic Proteins ◽  
Endogenous Protein ◽  
Dependent Protein ◽  
Endogenous Proteins

The presence of a protein kinase capable of phosphorylating endogenous as well as exogenously added myelin basic proteins has been demonstrated in a myelin-like membrane fraction isolated from reaggregating and surface adhering, primary cultures of cells dissociated from embryonic mouse brain. Only the large and small components of myelin basic proteins were found to be phosphorylated when myelin-like membrane fraction was incubated with [γ-32P]ATP. The protein kinase endogenous to the myelin-like membrane fraction was mainly of the cyclic AMP independent type. There was very little cyclic AMP dependent or cyclic GMP dependent protein kinase activities in this myelin-like fraction. Although the myelin basic proteins were the only endogenous proteins phosphorylated, protein kinase of the myelin-like membrane was capable of catalyzing the phosphorylation of exogenous substrates, such as histones.

Coordinate regulation of pp90rsk and a distinct protein-serine/threonine kinase activity that phosphorylates recombinant pp90rsk in vitro

1991 ◽  
Vol 11 (4) ◽  
pp. 1868-1874
Author(s):  
J Chung ◽  
R H Chen ◽  
J Blenis
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Kinase Activity ◽  
Cultured Cells ◽  
Protein Kinase Activity ◽  
Kinase C ◽  
Dependent Protein

Protein kinase assays that use recombinant pp90rsk as a substrate were developed in an attempt to identify growth-regulated enzymes responsible for the phosphorylation and activation of pp90rsk S6 phosphotransferase activity. With this assay we have ientified a pp60v-src-, growth factor-, phorbol ester-, and vanadate-regulated serine/threonine protein kinase activity that is not related to two other cofactor-independent, growth-regulated protein kinases, pp70-S6 protein kinase and pp90rsk. The pp90rsk-protein kinase activity (referred to as rsk-kinase) is also not related to cofactor-dependent signal transducing protein kinases such as the cyclic AMP-dependent protein kinases, members of the protein kinase C family, or other Ca2(+)-dependent protein kinases. In vitro, partially purified rsk-kinase phosphorylates several of the sites (serine and threonine) that are phosphorylated in growth-stimulated cultured cells. A detailed examination of the mitogen-regulated activation kinetics of rsk-kinase and pp90rsk activities demonstrated that they are coordinately regulated. In addition, protein kinase C is not absolutely required for epidermal and fibroblast growth factor-stimulated activation of rsk-kinase, whereas, like pp90rsk, platelet-derived growth factor- and vanadate-stimulated rsk-kinase activity exhibits a greater dependence on protein kinase C-mediated signal transduction. The characterization and future purification of the rsk-kinase(s) will improve our understanding of the early signaling events regulating cell growth.

scholarly journals Coordinate regulation of pp90rsk and a distinct protein-serine/threonine kinase activity that phosphorylates recombinant pp90rsk in vitro.

1991 ◽  
Vol 11 (4) ◽  
pp. 1868-1874 ◽  
Author(s):  
J Chung ◽  
R H Chen ◽  
J Blenis
Keyword(s):  
Protein Kinase C ◽  
Growth Factor ◽  
Protein Kinase ◽  
Protein Kinases ◽  
Kinase Activity ◽  
Cultured Cells ◽  
Protein Kinase Activity ◽  
Kinase C ◽  
Dependent Protein

Protein kinase assays that use recombinant pp90rsk as a substrate were developed in an attempt to identify growth-regulated enzymes responsible for the phosphorylation and activation of pp90rsk S6 phosphotransferase activity. With this assay we have ientified a pp60v-src-, growth factor-, phorbol ester-, and vanadate-regulated serine/threonine protein kinase activity that is not related to two other cofactor-independent, growth-regulated protein kinases, pp70-S6 protein kinase and pp90rsk. The pp90rsk-protein kinase activity (referred to as rsk-kinase) is also not related to cofactor-dependent signal transducing protein kinases such as the cyclic AMP-dependent protein kinases, members of the protein kinase C family, or other Ca2(+)-dependent protein kinases. In vitro, partially purified rsk-kinase phosphorylates several of the sites (serine and threonine) that are phosphorylated in growth-stimulated cultured cells. A detailed examination of the mitogen-regulated activation kinetics of rsk-kinase and pp90rsk activities demonstrated that they are coordinately regulated. In addition, protein kinase C is not absolutely required for epidermal and fibroblast growth factor-stimulated activation of rsk-kinase, whereas, like pp90rsk, platelet-derived growth factor- and vanadate-stimulated rsk-kinase activity exhibits a greater dependence on protein kinase C-mediated signal transduction. The characterization and future purification of the rsk-kinase(s) will improve our understanding of the early signaling events regulating cell growth.

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.

Mutational analysis of the Saccharomyces cerevisiae SNF1 protein kinase and evidence for functional interaction with the SNF4 protein

1989 ◽  
Vol 9 (11) ◽  
pp. 5034-5044
Author(s):  
J L Celenza ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Mutational Analysis ◽  
Gene Dosage ◽  
Regulatory System ◽  
Kinase Domain ◽  
Regulatory Function ◽  
Protein Kinase Activity ◽  
Glucose Limitation

The SNF1 gene of Saccharomyces cerevisiae encodes a protein-serine/threonine kinase that is required for derepression of gene expression in response to glucose limitation. We present evidence that the protein kinase activity is essential for SNF1 function: substitution of Arg for Lys in the putative ATP-binding site results in a mutant phenotype. A polyhistidine tract near the N terminus was found to be dispensable. Deletion of the large region C terminal to the kinase domain only partially impaired SNF1 function, causing expression of invertase to be somewhat reduced but still glucose repressible. The function of the SNF4 gene, another component of the regulatory system, was required for maximal in vitro activity of the SNF1 protein kinase. Increased SNF1 gene dosage partially alleviated the requirement for SNF4. C-terminal deletions of SNF1 also reduced dependence on SNF4. Our findings suggest that SNF4 acts as a positive effector of the kinase but does not serve a regulatory function in signaling glucose availability.

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