Prostaglandin D2 receptor-mediated desensitization of the α isoform of the human thromboxane A2 receptor11Abbreviations: cAMP, cyclic adenosine 5′ monophosphate; [Ca2+]i, intracellular calcium; DP, PGD2 receptor; HA, hemagluttinin; HEK, human embryonic kidney; HEL, human erythroleukaemia; HBS, HEPES-buffered saline; IP, prostacyclin receptor; IP3, inositol 1,4,5 trisphosphate; PG, prostaglandin; PKA, protein kinase A; PKC, protein kinase C; RT–PCR, reverse transcriptase–polymerase chain reaction; TXA2, thromboxane A2; and TP, TXA2 receptor.

CoQ6 (coenzyme Q6) biosynthesis in yeast is a well-regulated process that requires the final conversion of the late intermediate DMQ6 (demethoxy-CoQ6) into CoQ6 in order to support respiratory metabolism in yeast. The gene CAT5/COQ7 encodes the Cat5/Coq7 protein that catalyses the hydroxylation step of DMQ6 conversion into CoQ6. In the present study, we demonstrated that yeast Coq7 recombinant protein purified in bacteria can be phosphorylated in vitro using commercial PKA (protein kinase A) or PKC (protein kinase C) at the predicted amino acids Ser20, Ser28 and Thr32. The total absence of phosphorylation in a Coq7p version containing alanine instead of these phospho-amino acids, the high extent of phosphorylation produced and the saturated conditions maintained in the phosphorylation assay indicate that probably no other putative amino acids are phosphorylated in Coq7p. Results from in vitro assays have been corroborated using phosphorylation assays performed in purified mitochondria without external or commercial kinases. Coq7p remains phosphorylated in fermentative conditions and becomes dephosphorylated when respiratory metabolism is induced. The substitution of phosphorylated residues to alanine dramatically increases CoQ6 levels (256%). Conversely, substitution with negatively charged residues decreases CoQ6 content (57%). These modifications produced in Coq7p also alter the ratio between DMQ6 and CoQ6 itself, indicating that the Coq7p phosphorylation state is a regulatory mechanism for CoQ6 synthesis.

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Involvement of AMP-activated protein kinase in thrombin-stimulated interleukin 6 synthesis in osteoblasts

Journal of Molecular Endocrinology ◽

10.1530/jme-11-0165 ◽

2012 ◽

Vol 49 (1) ◽

pp. 47-55 ◽

Cited By ~ 5

Author(s):

H Tokuda ◽

K Kato ◽

H Natsume ◽

A Kondo ◽

G Kuroyanagi ◽

...

Keyword(s):

Protein Kinase ◽

Map Kinase ◽

Interleukin 6 ◽

P38 Map Kinase ◽

Thrombin Time ◽

Rt Pcr ◽

Α Subunit ◽

Compound C ◽

Kinase C ◽

Amp Activated Protein Kinase

We previously demonstrated that thrombin stimulates synthesis of interleukin 6 (IL6), a potent bone resorptive agent, in part via p44/p42 MAP kinase and p38 MAP kinase but not through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) among the MAP kinase superfamily in osteoblast-like MC3T3-E1 cells. In this study, we investigated the involvement of AMP-activated protein kinase (AMPK), a regulator of energy metabolism, in thrombin-stimulated IL6 synthesis in MC3T3-E1 cells. The phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, SAPK/JNK, or AMPK was determined by western blot analysis. The release of IL6 was determined by the measurement of IL6 concentration in the conditioned medium using an ELISA kit. The expression ofIL6mRNA was determined by RT-PCR. Thrombin time dependently induced the phosphorylation of AMPK α-subunit (Thr-172). Compound C, an inhibitor of AMPK, dose-dependently suppressed the thrombin-stimulated IL6 release in the range between 0.3 and 10 μM. Compound C reduced thrombin-induced acetyl-CoA carboxylase phosphorylation. TheIL6mRNA expression induced by thrombin was markedly reduced by compound C. Downregulation of AMPK by siRNA suppressed the thrombin-stimulated IL6 release. The thrombin-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was inhibited by compound C, which failed to affect SAPK/JNK phosphorylation. These results strongly suggest that AMPK regulates thrombin-stimulated IL6 synthesis via p44/p42 MAP kinase and p38 MAP kinase in osteoblasts.

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Use of a PCR-based method to characterize protein kinase C isoform expression in cardiac cells

AJP Cell Physiology ◽

10.1152/ajpcell.1993.264.5.c1350 ◽

1993 ◽

Vol 264 (5) ◽

pp. C1350-C1359 ◽

Cited By ~ 47

Author(s):

T. A. Kohout ◽

T. B. Rogers

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cardiac Cells ◽

Rt Pcr ◽

Kinase C ◽

Pkc Zeta ◽

Pcr Products ◽

Isoform Expression ◽

Novel Isoforms ◽

Single Reaction

Molecular cloning has identified at least nine unique isozymes of protein kinase C (PKC) designated alpha, beta I, beta II, gamma, delta, epsilon, zeta, and eta/L, with the recent addition of the theta-isoform. Previous attempts to characterize PKC isoform expression in heart have been limited by low levels of protein and perhaps by the presence of novel isoforms. Thus to critically examine the diversity of PKC expression in cardiac cells, we developed a reverse transcriptase-polymerase chain reaction (RT-PCR) approach that would amplify regions of the target cDNA of all the PKC isozymes in a single reaction. Degenerate oligonucleotide primers were designed to recognize sequences in the conserved regions of the PKC sequence motif: the cysteine-rich and the ATP-binding regions. Amplification of target PKC cDNA sequences resulted in PCR products with unique sizes and restriction digestion properties. The system was validated by identifying PCR products that correspond to all of the PKC isoform transcripts, except PKC-zeta, in a single reaction with cDNA derived from hippocampus. Cardiac cDNA was RT-PCR amplified, and the products were analyzed by a combination of restriction mapping and DNA sequencing that revealed the presence of only the alpha, delta, epsilon, and eta isoforms in adult rat cardiac myocytes and cultured neonatal ventricular myocytes. A unique nondegenerate primer pair was synthesized to recognize PKC-zeta cDNA. Results with these primers show that PKC-zeta is present in both cardiac myocyte preparations as well. The RT-PCR method developed here is an efficient approach that is broadly useful to examine PKC expression in many tissues, including the identification of potentially novel isoforms.(ABSTRACT TRUNCATED AT 250 WORDS)

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Involvement of protein kinase C in prostaglandin D2 synthesis by cultured astrocytes

Neurochemistry International ◽

10.1016/0197-0186(88)90077-0 ◽

1988 ◽

Vol 13 (4) ◽

pp. 475-480 ◽

Cited By ~ 9

Author(s):

Peter J. Gebicke-Haerter ◽

András Seregi ◽

Angelika Schobert ◽

Georg Hertting

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Prostaglandin D2 ◽

Kinase C ◽

Cultured Astrocytes

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Effect of thromboxane A2-receptor antagonist on bradykinin-induced bronchoconstriction in asthma

Journal of Applied Physiology ◽

10.1152/jappl.1996.80.6.1973 ◽

1996 ◽

Vol 80 (6) ◽

pp. 1973-1977 ◽

Cited By ~ 13

Author(s):

K. Rajakulasingam ◽

S. L. Johnston ◽

J. Ducey ◽

W. Ritter ◽

P. H. Howarth ◽

...

Keyword(s):

Receptor Antagonist ◽

Geometric Mean ◽

Thromboxane A2 ◽

Forced Expiratory Volume ◽

Significant Inhibition ◽

Prostaglandin D2 ◽

Double Blind ◽

Bronchial Responsiveness ◽

Significant Difference ◽

Txa2 Receptor

The role of the thromboxane A2 (TxA2) receptor in bradykinin-induced bronchial responses was investigated in this study by using a selective and potent TxA2-receptor antagonist BAY u 3405. Eleven asthmatic subjects were randomized to receive 50 mg of BAY u 3405 or matched placebo in a crossover and double-blind fashion. Ninety minutes after dosing, serum was taken for drug assay, and subjects underwent provocation with bradykinin or prostaglandin D2 (PGD2) to determine bronchial responsiveness [provocative concentration of agonist required to produce a 20% fall in forced expiratory volume in 1 s from the postdiluent baseline (PC20)]. Pretreatment with BAY u 3405 caused a twofold doubling-dilution reduction in bronchial reactivity to PGD2; the geometric mean PC20 values were 0.132 (0.015-0.871) and 0.034 (0.008-0.095) mg/ml, respectively, for active and placebo days (P = 0.001). There was, however, no significant difference in PC20 values for bradykinin between active and placebo treatment days. We have demonstrated that BAY u 3405 caused a significant inhibition of bronchconstriction induced by inhaled PGD2 but had no influence on bronchial responsiveness to inhaled bradykinin. This study suggests therefore that TxA2 receptors do not play a role in bradykinin-induced bronchoconstriction in asthma.

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