scholarly journals Respiratory-induced coenzyme Q biosynthesis is regulated by a phosphorylation cycle of Cat5p/Coq7p

2011 ◽  
Vol 440 (1) ◽  
pp. 107-114 ◽  
Author(s):  
Alejandro Martín-Montalvo ◽  
Isabel González-Mariscal ◽  
Sergio Padilla ◽  
Manuel Ballesteros ◽  
David L. Brautigan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
Regulatory Mechanism ◽  
Coenzyme Q ◽  
In Vitro Assays ◽  
Respiratory Metabolism ◽  
Total Absence ◽  
Kinase C ◽  
Pkc Protein Kinase C

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.

scholarly journals AH/PH domain-mediated interaction between Akt molecules and its potential role in Akt regulation.

1995 ◽  
Vol 15 (4) ◽  
pp. 2304-2310 ◽  
Author(s):  
K Datta ◽  
T F Franke ◽  
T O Chan ◽  
A Makris ◽  
S I Yang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
Catalytic Domain ◽  
Protein Complexes ◽  
Ph Domain ◽  
Terminal Portion ◽  
Protein Protein Interaction ◽  
Kinase C ◽  
Nih 3T3

The cytoplasmic serine-threonine protein kinase coded for by the c-akt proto-oncogene features a protein kinase C-like catalytic domain and a unique NH2-terminal domain (AH domain). The AH domain is a member of a domain superfamily whose prototype was observed in pleckstrin (pleckstrin homology, or PH, domain). In this communication, we present evidence that the AH/PH domain is a domain of protein-protein interaction which mediates the formation of Akt protein complexes. The interaction between c-akt AH/PH domains is highly specific, as determined by the failure of this domain to bind AKT2. The AH/PH domain-mediated interactions depend on the integrity of the entire domain. Akt molecules with deletions of the NH2-terminal portion (amino acids 11 to 60) and AH/PH constructs with deletions of the C-terminal portion of this domain (amino acids 107 to 147) fail to interact with c-akt. To determine the significance of these findings, we carried out in vitro kinase assays using Akt immunoprecipitates from serum-starved and serum-starved, platelet-derived growth factor-stimulated NIH 3T3 cells. Addition of maltose-binding protein-AH/PH fusion recombinant protein, which is expected to bind Akt, to the immunoprecipitates from serum-starved cells induced the activation of the Akt kinase.

In situ determination of [Ca2+]i threshold for translocation of the alpha-protein kinase C isoform

1994 ◽  
Vol 266 (6) ◽  
pp. C1544-C1551 ◽  
Author(s):  
R. A. Khalil ◽  
C. Lajoie ◽  
K. G. Morgan
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Second Messengers ◽  
In Vitro Assays ◽  
Intact Cells ◽  
Kinase C ◽  
Biochemical Assays

Because of inherent difficulties in maintaining physiological conditions in biochemical assays, the intracellular free Ca2+ concentration ([Ca2+]i) required for activation of protein kinase C (PKC) in intact cells remains unclear. In the present study, [Ca2+]i was measured in freshly isolated vascular smooth muscle cells loaded with fura 2 while, in parallel, the distribution of the Ca(2+)-dependent alpha-PKC isoform was monitored using digital imaging microscopy. The [Ca2+]i alpha-PKC translocation threshold was determined by changing extracellular free Ca2+ concentration in steps while monitoring [Ca2+]i. In the absence of agonists, increasing [Ca2+]i caused < 25% of maximal translocation. In the presence of phenylephrine, maximum translocation occurred at [Ca2+]i > or = 198 nM. Phenylephrine augmented translocation of alpha-PKC primarily by increasing the slope of the [Ca2+]i-PKC translocation relationship. These results indicate that the [Ca2+]i threshold of alpha-PKC translocation in situ is less than that reported in most in vitro assays and are consistent with an effect of agonist-induced generation of other second messengers that cause cooperative interactions leading to translocation.

scholarly journals N-Acetylcysteine and α-cyano-4-hydroxycinnamic acid alter protein kinase C (PKC)-δ and PKC-ζ and diminish dysmorphogenesis in rat embryos cultured with high glucose in vitro

2007 ◽  
Vol 192 (1) ◽  
pp. 207-214 ◽  
Author(s):  
Mattias Gäreskog ◽  
Parri Wentzel
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
High Glucose ◽  
Culture Medium ◽  
Hydroxycinnamic Acid ◽  
Oxygen Species ◽  
Rat Embryos ◽  
Protein Kinase C Inhibitors ◽  
Kinase C

Malformations and growth disturbances are two- to threefold more common in infants of diabetic mothers than in offspring of non-diabetic pregnancy. Several suggestions have emerged to explain the reasons for diabetic embryopathy, including enhanced mitochondrial production of reactive oxygen species leading to altered activation of protein kinase C. This study aimed to evaluate the effect of α-cyano-4-hydroxycinnamic acid (CHC) and N-acetylcysteine (NAC) addition on morphology and activity of protein kinase C-δ and protein kinase C-ζ in rat embryos exposed to a high glucose concentration in vitro. Day 9 embryos from normal rats were cultured in 10 or 30 mM glucose concentrations with or without supplementation of CHC, NAC, or protein kinase C inhibitors specific for protein kinase C-δ and protein kinase C-ζ. Embryos were evaluated for malformations, crown rump length, and somite number. Protein kinase C-δ and protein kinase C-ζ activities were estimated by western blot by separating membranous and cytosolic fractions of the embryo. We found increased malformations and growth retardation in embryos cultured in high versus low glucose concentrations. These abnormalities were diminished when CHC and NAC or specific protein kinase C-inhibitors were added to the culture medium. The activities of embryonic protein kinase C-δ and protein kinase C-ζ were increased in the high glucose environment after 24-h culture, but were normalized by the addition of CHC and NAC as well as respective inhibitor to the culture medium. These findings suggest that mitochondrial overproduction of reactive oxygen species is involved in diabetic embryopathy. Furthermore, such overproduction may affect embryonic development, at least partly, by enhancing the activities of protein kinase C-δ and protein kinase C-ζ.

Sign in / Sign up

Export Citation Format

Share Document