scholarly journals Inhibition of thyrotropin-stimulated DNA synthesis by microinjection of inhibitors of cellular Ras and cyclic AMP-dependent protein kinase

1993 ◽  
Vol 13 (8) ◽  
pp. 4477-4484
Author(s):  
E Kupperman ◽  
W Wen ◽  
J L Meinkoth
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Dna Synthesis ◽  
Kinase Inhibitor ◽  
Dependent Protein Kinase ◽  
Thyroid Cells ◽  
Heat Stable ◽  
Heat Stable Protein ◽  
Dependent Protein ◽  
Rat Thyroid

Microinjection of a dominant interfering mutant of Ras (N17 Ras) caused a significant reduction in thyrotropin (thyroid-stimulating hormone [TSH])-stimulated DNA synthesis in rat thyroid cells. A similar reduction was observed following injection of the heat-stable protein kinase inhibitor of the cyclic AMP-dependent protein kinase. Coinjection of both inhibitors almost completely abolished TSH-induced DNA synthesis. In contrast to TSH, overexpression of cellular Ras protein did not stimulate the expression of a cyclic AMP response element-regulated reporter gene. Similarly, injection of N17 Ras had no effect on TSH-stimulated reporter gene expression. Moreover, overexpression of cellular Ras protein stimulated similar levels of DNA synthesis in the presence or absence of the heat-stable protein kinase inhibitor. Together, these results suggest that in Wistar rat thyroid cells, a full mitogenic response to TSH requires both Ras and cyclic APK-dependent protein kinase.

scholarly journals Inhibition of thyrotropin-stimulated DNA synthesis by microinjection of inhibitors of cellular Ras and cyclic AMP-dependent protein kinase.

1993 ◽  
Vol 13 (8) ◽  
pp. 4477-4484 ◽  
Author(s):  
E Kupperman ◽  
W Wen ◽  
J L Meinkoth
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Dna Synthesis ◽  
Kinase Inhibitor ◽  
Dependent Protein Kinase ◽  
Thyroid Cells ◽  
Heat Stable ◽  
Heat Stable Protein ◽  
Dependent Protein ◽  
Rat Thyroid

Microinjection of a dominant interfering mutant of Ras (N17 Ras) caused a significant reduction in thyrotropin (thyroid-stimulating hormone [TSH])-stimulated DNA synthesis in rat thyroid cells. A similar reduction was observed following injection of the heat-stable protein kinase inhibitor of the cyclic AMP-dependent protein kinase. Coinjection of both inhibitors almost completely abolished TSH-induced DNA synthesis. In contrast to TSH, overexpression of cellular Ras protein did not stimulate the expression of a cyclic AMP response element-regulated reporter gene. Similarly, injection of N17 Ras had no effect on TSH-stimulated reporter gene expression. Moreover, overexpression of cellular Ras protein stimulated similar levels of DNA synthesis in the presence or absence of the heat-stable protein kinase inhibitor. Together, these results suggest that in Wistar rat thyroid cells, a full mitogenic response to TSH requires both Ras and cyclic APK-dependent protein kinase.

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.

scholarly journals Activation of cyclic AMP-dependent kinase is required but may not be sufficient to mimic cyclic AMP-dependent DNA synthesis and thyroglobulin expression in dog thyroid cells.

1997 ◽  
Vol 17 (11) ◽  
pp. 6717-6726 ◽  
Author(s):  
S Dremier ◽  
V Pohl ◽  
C Poteet-Smith ◽  
P P Roger ◽  
J Corbin ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Dna Synthesis ◽  
Regulatory Subunit ◽  
Thyroid Cells ◽  
Morphological Effect ◽  
Heat Stable ◽  
Proliferation And Differentiation ◽  
C Subunit ◽  
Dependent Protein

Thyrotropin (TSH), via a cyclic AMP (cAMP)-dependent pathway, induces cytoplasmic retractions, proliferation, and differentiation expression in dog thyroid cells. The role of cAMP-dependent protein kinase (PKA) in the induction of these events was assessed by microinjection into living cells. Microinjection of the heat-stable inhibitor of PKA (PKI) inhibited the effects of TSH, demonstrating that activation of PKA was required in this process. Overexpression of the catalytic (C) subunit of PKA brought about by microinjection of the expression plasmid pC alpha ev or of purified C subunit itself was sufficient to mimic the cAMP-dependent cytoplasmic changes and thyroperoxidase mRNA expression but not to induce DNA synthesis and thyroglobulin (Tg) expression. The cAMP-dependent morphological effect was not observed when C subunit was coinjected with the regulatory subunit (RI or RII subunit) of PKA. To mimic the cAMP-induced PKA dissociation into free C and R subunits, the C subunit was coinjected with the regulation-deficient truncated RI subunit (RIdelta1-95) or with wild-type RI or native RII subunits, followed by incubation with TSH at a concentration too low to stimulate the cAMP-dependent events by itself. Although the cAMP-dependent morphology changes were still observed, neither DNA synthesis nor Tg expression was stimulated in these cells. Taken together, these data suggest that in addition to PKA activation, another cAMP-dependent mechanism could exist and play an important role in the transduction of the cAMP signal in thyroid cells.

scholarly journals Parallel regulation of cyclic amp-dependent protein kinase and phosphoprotein phosphatase in rat thyroid

FEBS Letters ◽  
1979 ◽  
Vol 99 (1) ◽  
pp. 167-171 ◽  
Author(s):  
Shankar Huprikar ◽  
Michael Lang ◽  
Yochanan Friedman ◽  
Gerald Burke
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Dependent Protein Kinase ◽  
Phosphoprotein Phosphatase ◽  
Dependent Protein ◽  
Rat Thyroid

scholarly journals Protein kinase activities in rat pancreatic islets of Langerhans

1979 ◽  
Vol 180 (1) ◽  
pp. 219-229 ◽  
Author(s):  
M C Sugden ◽  
S J Ashcroft ◽  
P H Sugden
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Islets Of Langerhans ◽  
Kinase Inhibitor ◽  
Protein Kinase Inhibitor ◽  
Dependent Protein Kinase ◽  
Rat Islets ◽  
Dependent Protein ◽  
Rat Islets Of Langerhans ◽  
Independent Protein

1. Protein kinase activities in homogenates of rat islets of Langerhans were studied. 2. On incubation of homogenates with [gamma-32P]ATP, incorporation of 32P into protein occurred: this phosphorylation was neither increased by cyclic AMP nor decreased by the cyclic AMP-dependent protein kinase inhibitor described by Ashby & Walsh [(1972) J. Biol. Chem. 247, 6637–6642]. 3. On incubation of homogenates with [gamma-32P]ATP and histone as exogenous substrate for phosphorylation, incorporation of 32P into protein was stimulated by cyclic AMP (approx. 2.5-fold) and was inhibited by the cyclic AMP-dependent protein kinase inhibitor. In contrast, when casein was used as exogenous substrate, incorporation of 32P into protein was not stimulated by cyclic AMP, nor was it inhibited by the cyclic AMP-dependent protein kinase inhibitor. 4. DEAE-cellulose ion-exchange chromatography resolved four peaks of protein kinase activity. One species was the free catalytic subunit of cyclic AMP-dependent protein kinase, two species corresponded to ‘Type I’ and ‘Type II’ cyclic AMP-dependent protein kinase holoenzymes [see Corbin, Keely & Park (1975) J. Biol. Chem. 250, 218–225], and the fourth species was a cyclic AMP-independent protein kinase. 5. Determination of physical and kinetic properties of the protein kinases showed that the properties of the cyclic AMP-dependent activities were similar to those described in other tissues and were clearly distinct from those of the cyclic AMP-independent protein kinase. 6. The cyclic AMP-independent protein kinase had an s20.w of 5.2S, phosphorylated a serine residue(s) in casein and was not inhibited by the cyclic AMP-dependent protein kinase inhibitor. 7. These studies demonstrate the existence in rat islets of Langerhans of multiple forms of cyclic AMP-dependent protein kinase and also the presence of a cyclic AMP-independent protein kinase distinct from the free catalytic subunit of cyclic AMP-dependent protein kinase. The presence of the cyclic AMP-independent protein kinase may account for the observed characteristics of 32P incorporation into endogenous protein in homogenates of rat islets.

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